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Differential Effects of Grape Seed Extract against Human Colorectal Cancer Cell Lines: The Intricate Role of Death Receptors and Mitochondria
Abstract
Failure of anti-cancer therapy in colorectal cancer (CRC) cells involves resistance to death mechanisms. We investigated grape seed extract (GSE) ability to target CRC cells and delineated the mechanisms involved in GSE-induced CRC cell death. GSE selectively induced apoptotic death in human CRC cells; efficacy increased as the metastatic potential of the cancer cells increased. Oxidative stress, loss of mitochondrial membrane potential, modulation of pro- and anti-apoptotic proteins, and involvement of both caspase-dependent / independent apoptotic pathways contributed to GSE-induced CRC cell death. GSE intervention may serve as a multi-targeted CRC therapeutics, capable of inducing selective cancer cell death.
... Multiple studies have demonstrated the anti-CRC activity of a grape seed extract (GSE). Derry and coworkers (33) examined the influence of a commercial standardized GSE, consisting of 89.3% procyanidins, 6.6% monomeric flavonols, 2.24% proteins, 1.06% moisture, and 0.8% ash on the viability of SW480, SW620, and HCT116 cells. In case of SW480 (stage II CRC), the authors observed 37-60% dead cells after treatment with 25-100 μg/mL GSE. ...
... It is worth emphasizing that the GSE at 25-100 μg/mL had no impact or only a slight impact (8% of dead cells) on the viability of the NCM460 normal human colon epithelial cell line. (33) The commercial standardized GSE and prepared GSE (procyanidins, catechins) were also active against LoVo, HT-29, Caco-2, and HCT-8 colon cancer cell lines. (34,35) They caused the death of 8-55% LoVo and 4-33% HT-29 and significantly reduced the growth of Caco-2 and HCT-8 cells. ...
... Further, the commercial standardized GSE managed to arrest cycle of SW480 cells in the G 2 /M phase, SW620 in G 1 , HCT116 in G 1 and G 2 , and LoVo and HT-29 in G 1 by the induction of p21 and/or p27 in the cells. (10,33,34,(36)(37)(38) Additionally, the commercial standardized GSE and prepared GSE (procyanidins, catechins) proved to be efficient in inducing apoptosis in colon cancer cell lines. A 1.5-to 3-fold increase was observed in apoptotic SW480 cells, 11-fold for SW620 and HCT116, 3-to 4-fold for LoVo, and 4to 9-fold increase for HT-29 over controls. ...
The consumption of vegetables and fruits, particularly apples, pomegranates , grapes, and berries, is associated with a decreased risk of developing cancer, especially colorectal cancer (CRC). This may be attributable to the presence of phytochemical constituents, such as polyphenols. This review summarizes the current state of knowledge concerning the chemopreventive and anti-CRC potential of polyphe-nol-rich apple, pomegranate, grape, and berry extracts with a focus on in vitro, in vivo, and clinical evidence. The extracts demonstrate antiproliferative, proapoptotic, antiangiogenic, antiinvasive, and anti-metastatic activities toward colon cancer cell lines and in animal models and markedly influence preneoplastic lesions and malignan-cies in humans.
... Accordingly, in this study, we investigated the potential of grape seed extract (GSE) to inhibit adipocytes-driven pro-tumorigenic signaling on both human CRC and colon CSCs in the context of CRC prevention and control under obese conditions. GSE, a well-defined chemical entity containing procyanidins [20,22,23], has shown strong preventive and therapeutic efficacy in various CRC in vitro and rodent models [19,20,[23][24][25][26][27][28][29][30][31][32] including azoxymethane (AOM)-induced aberrant crypt foci in Fisher 344 rats [31], AOM-induced colon tumorigenesis in A/J mice [26], spontaneous intestinal tumors in APC min/+ mice [32] and CRC cell xenografts in nude mice [29], together with a decrease in proliferative and an increase in apoptotic indices [26,29,31,32]. Also importantly, a series of animal studies have shown that GSE partially alleviates the high fat diet-induced obesity, decreases the weight of fat pads, and suppresses the body weight increase together with an improvement in associated metabolic abnormalities [33][34][35][36]. ...
... Standardized preparation of GSE [22][23][24] was a gift from Kikkoman Corp. (Nado City, Japan) and was dissolved in DMSO for cell culture use. Antibodies used were: CD44 total (Cat # sc-65410), C/EBP-α (Cat # 2295), C/EBP-β (Cat # 3082), FAS (Cat # 3180) and SREBP-1c (Cat # 366), and PPAR-γ (Cat # sc-7273) from Santa Cruz Biotechnology; BrdU-FITC (Cat # ab74545) and OCT-4 (Cat # ab18976) from Abcam; CD44-FITC (Cat # 555478, BD Pharmingen); EpCAM-PE (Cat # 347211BD Biosciences); β-catenin (Cat # 9582), Snail-1 (Cat # 3895), E-cadherin (Cat # 3195), and Perilipin (Cat # 9349) from Cell Signaling Technology. ...
... Adipocyte-conditioned media was obtained by subjecting specific adipocytes to serum free media for 48 h, and then using the cell clarified, generated sterile media, in subsequent in vitro experiments in place of regular media. Total cellular lysates, determination of protein concentrations and western blotting followed by ECL detection were done as described previously [23,24]. ...
With global rise in obesity, it is imperative that we identify obesity-driven factors that increase growth and progression of colorectal cancer (CRC), and also discover and develop agents with anti-CRC efficacy under obese conditions. Here in, we investigated grape seed extract (GSE), a well-defined agent with both preventive and anti-CRC efficacy, for its potential to impair pro-tumorigenic signaling of adipocytes on CRC/colon cancer stem cells (CSCs) and associated molecular mechanisms, to control CRC under obese conditions. GSE treatment significantly decreased the growth and invasion promoting effects of both mouse and human adipocytes on CRC cells. Moreover, GSE exerted a direct inhibitory effect, as well as it strongly reduced the growth promoting signals of adipocytes, on colon CSCs. These GSE effects were associated with a decrease in both mRNA and protein levels of various CSC-associated molecules. Notably, GSE effects on adipocytes were not due to changes in lipid content, but by inducing the 'browning' of adipocytes as evidenced by an increase in UCP-1 mRNA level and mitochondriogenesis. Together, these findings, for the first time, suggest the ability of GSE to induce 'brown remodeling' of white adipocytes, which causes functional modification of adipocytes thus impairing their pro-tumorigenic signals on colon CSCs/CRC cells.
... Examining drug-protein interactions, prior to pre-clinical efficacy studies, allows scientist to effectively screen for the best small molecule candidates and to further predict any associated toxicity with the drug administration [7][8][9]. Amongst the various natural agents screened, grape seed extract (GSE) is one such non-toxic chemopreventive agent which has demonstrated anticancer efficacy in various pre-clinical in vitro and in vivo models of prostate, lung, breast, bladder and colon cancers [4,[10][11][12][13][14][15][16][17][18][19][20][21]. GSE contains proanthocyanidins [a mix of dimers, trimers and other oligomers (procyanidins) of catechin and epicatechin and their gallate derivatives], which are also widely distributed throughout the plant kingdom and are present in high quantities within the seeds of the grapes [12,[22][23][24]. ...
... The composition of the standardized GSE preparation (Kikkoman Corp., Nado City, Japan) is listed as: 89.3% procyanidins, 6.6% monomeric flavanols, 2.24% moisture content, 1.06% of protein, and 0.8% of ash [10,11,25]. Dimethyl Sulfoxide (DMSO), oligomycin, antimycin A, 2-deoxyglucose (2-DG), carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) were from Sigma Chemical Co. ...
... To identify potential protein targets of GSE we utilized DARTS (Fig. 1) [7][8][9]; GSE treatment was done at 60-100 μg/ml doses for 3 h in a panel of human CRC cell lines; these cell lines were chosen based on phenotypic and genetic variations, so as to cover different clinical stages of CRC, viz., SW480 (stage II CRC), SW620 (stage III CRC) and HCT116 (stage IV CRC) cells [10]. The selection of doses was based on our previous study documenting the differential efficacy of GSE in different human CRC cell lines [10]. ...
Various natural agents, including grape seed extract (GSE), have shown considerable chemopreventive and anti-cancer efficacy against different cancers in pre-clinical studies; however, their specific protein targets are largely unknown and thus, their clinical usefulness is marred by limited scientific evidences about their direct cellular targets. Accordingly, herein, employing, for the first time, the recently developed drug affinity responsive target stability (DARTS) technique, we aimed to profile the potential protein targets of GSE in human colorectal cancer (CRC) cells. Unlike other methods, which can cause chemical alteration of the drug components to allow for detection, this approach relies on the fact that a drug bound protein may become less susceptible to proteolysis and hence the enriched proteins can be detected by Mass Spectroscopy methods. Our results, utilizing the DARTS technique followed by examination of the spectral output by LC/MS and the MASCOT data, revealed that GSE targets endoplasmic reticulum (ER) stress response proteins resulting in overall down regulation of proteins involved in translation and that GSE also causes oxidative protein modifications, specifically on methionine amino acids residues on its protein targets. Corroborating these findings, mechanistic studies revealed that GSE indeed caused ER stress and strongly inhibited PI3k-Akt-mTOR pathway for its biological effects in CRC cells. Furthermore, bioenergetics studies indicated that GSE also interferes with glycolysis and mitochondrial metabolism in CRC cells. Together, the present study identifying GSE molecular targets in CRC cells, combined with its efficacy in vast pre-clinical CRC models, further supports its usefulness for CRC prevention and treatment.
... GSE 0.25 and 0.5% (p/p) for two weeks A/J mice Decrease in the size of tumors induced by azoxymethane. [44] The GSE exhibits antiproliferative and pro-apoptotic activity. ...
... Ortiz et al. [43] showed that administering a concentration of 200 _g/ml of GSE for 24 hours induced apoptosis close to 50% and 40% in Caco-2 and HT29 cells, respectively. In turn, Derry et al. [44] used GSEs (in low concentrations of up to 30 _g/ml) in SW480, SW620, and HT116 cells. After 12 hours of treatment, they observed that the apoptosis percentage increased in a dose-dependent manner, thus inducing the death of up to 60% of the cell population. ...
In the pursuit of potent antioxidant sources for potential health benefits, significant attention has been directed toward polyphenols. This research is especially relevant in the context of colorectal cancer (CRC), a widespread and deadly malignancy. The exploration of preventive strategies has led to a growing interest in grapes and their derivatives, such as juices and wines, which offer various consumption options based on grape type. This investigation encompasses a comprehensive analysis of multiple grape components: skin, pulp, seed, pomace, wine, juice, and extracts. These assessments serve as a critical reference for exploring functional ingredients from grapes and their potential as chemopreventive agents against colon cancer. This scientific literature review aims to provide an in-depth overview of advancements from 2012 to 2023. A systematic review reveals varying polyphenol concentrations in different grape components and their correlation with antioxidant capacities. In addition, the chemical profiles of these matrices are analyzed concerning their antioxidant properties, antiproliferative effects, and cytotoxicity, validated through in vitro and in vivo models simulating CRC scenarios. This research seeks to demonstrate grapes’ potential, in their diverse forms, as promising functional food options.
... These lifestyle variables are well understood for encouraging the formation of excess oxygen and nitrogen reactive species in the liver, which can lead to oxidative stress. Despite the absence of clinical research, preclinical tests imply that natural antioxidants from products like grape leaves block or reduce the prevalence of liver disorders induced by oxidative pathways (Derry et al., 2013) [46] . Animals have been tested for morphological and biochemical changes produced by hepatotoxic chemicals, as well as the protective impact of grape leaf extracts. ...
... These lifestyle variables are well understood for encouraging the formation of excess oxygen and nitrogen reactive species in the liver, which can lead to oxidative stress. Despite the absence of clinical research, preclinical tests imply that natural antioxidants from products like grape leaves block or reduce the prevalence of liver disorders induced by oxidative pathways (Derry et al., 2013) [46] . Animals have been tested for morphological and biochemical changes produced by hepatotoxic chemicals, as well as the protective impact of grape leaf extracts. ...
For centuries, grape leaves, especially red grape leaves, have been employed in herbal medicines and are still in use today. While grape leaves have long been used to cure a variety of diseases, further research is still needed to determine their true effectiveness. Due to high polyphenol content (as enlisted in table 4), grape leaves are frequently used in food fortification due to their health advantages. As a result, a complete review of grape leaves extract polyphenols and their health advantages in functional foods are required. As a result, we highlighted how grape leaves are a rich source of nutrients for general health and wellness, as well as having a complex chemical composition that appears to contribute to their therapeutic potential, particularly antioxidant activity. Grape leaves also offer anti-inflammatory, reproductive, cardiovascular, diabetic, and anti-cholesterol qualities, among other physiological advantages. The bioactive polyphenols, health benefits, and biological activity of grape leaves have all been reviewed.
... Antitumor activity of GSE is also were observed in skin cancer (9). In colorectal cancer, it was suggested that GSE affects mitochondrial membrane potential, pro-and anti-apoptotic proteins, and both caspase-dependent/independent apoptotic pathways (10). Despite known anticancer effects of GSE in some type of cancers, the precise mechanism of its function in ovarian cancer has not yet been defined. ...
... Agraval et al. suggested that in prostatic cancer GSE induces apoptosis via activation of caspases in companion with the destruction of mitochondrial membrane and releasing cytochrome C (37). In another study it was shown that anticancer effects of GSE in colon cancer are associated with differential modulation of pro-and antiapoptotic proteins (10). ...
Background and purpose:
Ovarian cancer is the deadliest cancer in women. The main challenge in the inhibition of ovarian cancer cells is chemo-resistance. Seeking to overcome this issue, several strategies have been suggested, including the administration of natural products. Grape seed extract (GSE) is a good source of polyphenols and its anticancer effects have been reported by many studies. In this study we aimed to evaluate the effects of GSE on OVCAR-3, a chemo-resistant ovarian cancer line.
Experimental approach:
OVCAR-3 cells were treated with GSE (71 μg/mL) for 24 and 48 h. Cell viability and cell apoptosis were measured by MTT and flow cytometry. The real-time polymerase chain reaction was used to determine the expression of genes involved in the cell cycle (PTEN, DACT1, AKT, MTOR, GSK3B, C-MYC, CCND1, and CDK4) and apoptosis (BAX, BCl2, CASP3, 8 and 9). The expression of CASP3 protein was evaluated by the CASP3 assay.
Findings / results:
The results showed that treatment of OVCAR-3 cells with GSE, increased the expression level of PTEN and DACT1 tumor suppressor genes, as well as apoptotic genes, CASP3, 8, and 9 (P < 0.001). Also, the induction of tumor suppressor genes expression was associated with an increase in the expression of BAX/BCL2 gene ratio as pro- and anti-apoptotic genes. The expression of the genes involved in the cell cycle, CCND1 and CDK4, was inhibited (P < 0.001). The results indicated that GSE induced cell apoptosis in a time-dependent manner (P < 0.001). Also, the GSE treatment resulted in the CASP3 protein expression (P < 0.001).
Conclusion and implications:
According to the results of this study, GSE may exert anti-tumorigenic effects on chemo-resistant OVCAR-3 ovarian cancer cells which might be mediated by the expression of tumor suppressor genes that interact with cell signaling pathways, cell cycle, and cell apoptosis. Hence, the consumption of GSE extract during chemotherapy may overcome part of chemo-resistance in ovarian cancer.
... Previous in vitro studies have shown that grape extracts can act differently on proliferation and apoptotic pathways [43,44]. These different biological effects of GSEs could depend both on the type of cancer cell and on the different polyphenolic content of grape extracts [43,45,46]. ...
... Previous in vitro studies have shown that grape extracts can act differently on proliferation and apoptotic pathways [43,44]. These different biological effects of GSEs could depend both on the type of cancer cell and on the different polyphenolic content of grape extracts [43,45,46]. In fact, it is known that there are cell lines more sensitive to treatment with polyphenols than others in relation to cellular differentiation degree. ...
Grapes contain many flavonoid and non-flavonoid compounds with anticancer effects. In this work we fully characterized the polyphenolic profile of two grape skin extracts (GSEs), Autumn Royal and Egnatia, and assessed their effects on Polyunsaturated Fatty Acid (PUFA) membrane levels of Caco2 and SW480 human colon cancer cell lines. Gene expression of 15-lipoxygenase-1 (15-LOX-1), and peroxisome proliferator-activated receptor gamma (PPAR-γ), as well as cell morphology, were evaluated. The polyphenolic composition was analyzed by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) analysis. PUFA levels were evaluated by gas chromatography, and gene expression levels of 15-LOX-1 and PPAR-γ were analyzed by real-time Polymerase Chain Reaction (PCR). Morphological cell changes caused by GSEs were identified by field emission scanning electron microscope (FE-SEM) and photomicrograph examination. We detected a different profile of flavonoid and non-flavonoid compounds in Autumn Royal and Egnatia GSEs. Cultured cells showed an increase of total PUFA levels mainly after treatment with Autumn Royal grape, and were richer in flavonoids when compared with the Egnatia variety. Both GSEs were able to affect 15-LOX-1 and PPAR-γ gene expression and cell morphology. Our results highlighted a new antitumor mechanism of GSEs that involves membrane PUFAs and their downstream pathways.
... Those effects have been recorded in several cancer lines amended with GSE by ours and other groups [64, 103, [109][110][111], and noticed also when using single polyphenolic molecules [112][113][114]. It is worth noting that the pro-oxidant effect is a very early event (occurring after 5-30 minutes after GSE supplementation), and it happens well before the subsequent onset of apoptosis and cell cycle inhibition. ...
... Indeed, we have shown that apoptosis inducing factor (AIF), known to induce apoptosis via a caspase-independent mechanism, increases early in GSE-treated samples and anticipate caspase-dependent apoptosis [137]. Those results have been further confirmed [109]. Furthermore, both caspase-dependent and caspase-independent apoptosis has been documented in prostate cancer cells after GSE treatment. ...
... Those effects have been recorded in several cancer lines amended with GSE by ours and other groups [64, 103, [109][110][111], and noticed also when using single polyphenolic molecules [112][113][114]. It is worth noting that the pro-oxidant effect is a very early event (occurring after 5-30 minutes after GSE supplementation), and it happens well before the subsequent onset of apoptosis and cell cycle inhibition. ...
... Indeed, we have shown that apoptosis inducing factor (AIF), known to induce apoptosis via a caspase-independent mechanism, increases early in GSE-treated samples and anticipate caspase-dependent apoptosis [137]. Those results have been further confirmed [109]. Furthermore, both caspase-dependent and caspase-independent apoptosis has been documented in prostate cancer cells after GSE treatment. ...
Grape seed extract (GSE) is a complex mixture of several compounds, mostly represented by polyphenols and phenolic acids. Their consumption is safe and is recognized to exert several and meaningful health benefits. In particular, grape-related anti-tumoral activity encompasses a wide array of biological mechanisms and cellular targets, eventually leading to inhibition of cell growth and to enhanced apoptosis in several cancer cell lines, including lung, colon, breast, bladder, leukemia and prostate tumors. Those effects are likely modulated at the molecular level through selectively modulating the redox balance and displaying anti-oxidant as well as pro-oxidant actions, according to the specific context. GSE-related anti-cancer activity mostly relies on the induced increase in reactive oxygen species, followed by the orchestrated down- and up-regulation of several key-molecular pathways, including MAPK kinases, PI3K/Akt, NF-kB, cytoskeleton proteins and metalloproteinases. Promising results obtained in vitro as well as on animal studies suggest that GSE may have a great relevance as source of potential new pharmacological molecules, and could represent an important opportunity for clinical research.
... Furthermore, GSE inhibited aberrant β-catenin expression and downstream proteins, cyclin D1, and c-myc (Velmurugan et al., 2010b). Moreover, mechanistic studies of GSE, at various stages of CRC development, identified apoptosis induction as the major factor in the chemopreventive efficacy of GSE against CRC; specifically it induces caspase-3, -8, -9 resulting in the cleavage of PARP, DNA fragmentation, and PCD (Derry et al., 2012). The apoptotic effect was specific to CRC cells, with minimal effect on normal colon epithelial cells; furthermore, this effect was attenuated with antioxidant treatment, indicating ROS as a potential upstream stimulus in GSE-induced CRC cell death (Derry et al., 2012). ...
... Moreover, mechanistic studies of GSE, at various stages of CRC development, identified apoptosis induction as the major factor in the chemopreventive efficacy of GSE against CRC; specifically it induces caspase-3, -8, -9 resulting in the cleavage of PARP, DNA fragmentation, and PCD (Derry et al., 2012). The apoptotic effect was specific to CRC cells, with minimal effect on normal colon epithelial cells; furthermore, this effect was attenuated with antioxidant treatment, indicating ROS as a potential upstream stimulus in GSE-induced CRC cell death (Derry et al., 2012). In addition, GSE has been shown to modulate p21 levels; resulting in decreased proliferation, leading to cell cycle arrest, and further downstream pathway activation, including that of ERK1/2 (Kaur et al., 2006(Kaur et al., , 2011. ...
One in four deaths in the United States is cancer-related, and colorectal cancer (CRC) is the second leading cause of cancer-associated deaths. Screening strategies are utilized but have not reduced disease incidence or mortality. In this regard, there is an interest in cancer preventive strategies focusing on lifestyle intervention, where specific etiologic factors involved in cancer initiation, promotion, and progression could be targeted. For example, exposure to dietary carcinogens, such as nitrosamines and polycyclic aromatic hydrocarbons influences colon carcinogenesis. Furthermore, dietary deficiencies could alter sensitivity to genetic damage and influence carcinogen metabolism contributing to CRC. High alcohol consumption increases the risk of mutations including the fact that acetaldehyde, an ethanol metabolite, is classified as a group 1 carcinogen. Tobacco smoke exposure is also a risk factor for cancer development; approximately 20% of CRCs are associated with smoking. Additionally, obese patients have a higher risk of cancer development, which is further supported by the fact that physical activity decreases CRC risk by 55%. Similarly, chronic inflammatory conditions also increase the risk of CRC development. Moreover, the circadian clock alters digestion and regulates other biochemical, physiological, and behavioral processes that could influence CRC. Taken together, colon carcinogenesis involves a number of etiological factors, and therefore, to create effective preventive strategies, molecular targets need to be identified and beleaguered prior to disease progression. With this in mind, the following is a comprehensive review identifying downstream target proteins of the above lifestyle risk factors, which are modulated during colon carcinogenesis and could be targeted for CRC prevention by novel agents including phytochemicals.
... For example, an up-regulation of cleaved caspase-3, cleaved PARP, phospho-p53, and total Bad (p < 0.05) in SW480 cells treated with 8 % v/v ABJ was found, suggesting pro-apoptotic effects in this cell line . Commercial grape seed extracts (25-100 μg/mL) containing procyanidins and monomeric flavonols found a significant increase of cytochrome C release, cleaved caspase-9, and cleaved caspase-3 on SW480, SW620, and HCT116 colorectal cancer cell lines, compared to the untreated cells (Derry et al., 2013). Activation of TRAIL pathways is notorious since CRC is particularly resistant to this activation (Deng & Shah, 2020). ...
Anthocyanins are polyphenols contained in berries. There is evidence that act as possible modulators in cell death mechanisms like apoptosis. Cancer is a relevant scenario where modulate apoptosis can be relevant. For this reason, in this study it was evaluated the binding molecular affinity with apoptotic proteins of some anthocyanins from Vaccinium meridionale Swartz berry to identify potential antitumoral biological compounds. Cyanidin 3-glucoside, cyanidin 3-galactoside, and delphinidin 3-glucoside structures were downloaded from PubChem and crystalographic structures of apoptosis proteins were obtained from Protein Data Bank. Docking experiments in silico were implemented with structures prepared. Cyanidin 3-glucoside and cyanidin 3-galactoside showed the best affinity with FADD/fas protein while delphinidin 3-glucoside with procaspase 3 protein. These results of affinity suggest a potential proapoptotic activity related with these anthocyanins.
... Its medical importance lies in its skin protection, antioxidant, antibacterial, anticancer, anti-inflammatory and antidiabetic activities, as well as its hepatoprotective, cardioprotective and neuroprotective effects [15]. Studies have shown that grape seed extracts (GSE) possess potential anticancer activity against oral squamous cell carcinoma (KB cells) [16], human colorectal cancer (CRC cell) [17], and chemo-resistant ovarian cancer (OVCAR-3 cells) [18]. Additionally, grape stem extracts showed anticancer activity against colon (HT29), breast (MCF-7 and MDA-MB-23), renal (786-0 and Caki-1) and thyroid (K1) cancer cells [19]. ...
Vitis vinifera commonly known as grapevine is one of the most important fruit crops worldwide. Its cultivation started more than 7000 years ago in the Near East, and over the millennia was followed by the development of thousands of cultivars that were further selected and characterized for specific purposes. Its important pharmacological value and its richness in phytoconstituents were the triggers to perform this project. Seven extracts were prepared from five different V. vinifera varieties (V. vinifera ‘Black Pearl’ (BP), V. vinifera ‘Red Glob’ (RG), V. vinifera ‘Crimson’ (CR), V. vinifera ‘Beitamouni’ (BE) and V. vinifera ‘Superior’ (SU)) by separating the pulp from the seeds, followed by methanolic extraction. The phytochemical analysis showed that red colored grapes (RE, BP and CR), the seeds from V. vinifera ‘Black Pearl’ and V. vinifera ‘Red Globe’ contain higher amounts of primary and secondary metabolites such as polyphenols, anthocyanins and reducing sugars. In addition to their richness in phytoconstituents, these varieties/seeds possess an important antioxidant activity. The results of the cell viability assays showed that the red varieties have a potential anticancer activity against Capan-2 pancreatic cancer and MDA-MB231(TNBC) breast cancer cell lines, with the greatest promise when combined with the seeds.
... [73] Grape [74] Grape Seed Extract SW480 and HCT116 cells 0-00 μg/mL Apoptosis induction due to loss of mitochondrial membrane potential, oxidative stress, modulation of pro-and antiapoptotic proteins, and modulation of caspasedependent/independent apoptotic pathways. [77] Grape Seed Extract from 3 different cultivars: ...
Inflammatory Bowel Diseases (IBD) are inflammatory conditions characterized by bowel disruption and the involvement of the immune system and can be associated with the development of colorectal cancer. Conventional therapeutic approaches are based on administering drugs capable of modulating the inflammatory response; however, many patients are irresponsive to conventional therapies. Grapes are largely used in producing wine, juice, jam, and other products. However, the waste from the production process represents an environmental problem. On the other hand, grape by-products are rich in bioactive compounds such as proanthocyanidins, anthocyanins, phenolic acids, stilbenes, and flavonols. These compounds can help in the prevention or in treatment of several conditions such as IBD and colorectal cancer. Grape by-products can promote remarkable effects in reducing pro-inflammatory, pro-oxidative, and proliferative actions in IBD and CRC both in vivo and in vitro. These effects are related to the improvement of epithelial integrity, reduction of Disease Activity Index, mucosal barrier reinforcement, induction of strong chemopreventive actions, reduction of tumor incidence, and cell proliferation.
... Nassiri-Asl and Hosseinzadeh (2016) performed a bibliographic review about the bioactive properties of grape waste; among them, anticancer effects and apoptosis induced in colon cancer strains were assigned to the grape seed of Italia and Palieri varieties (Dinicola et al. 2012). Furthermore, Derry et al. (2013) performed tests with grape seed extracts to induce apoptosis in colorectal cancer strains (SW480, SW620, and HCT116) and measured cytochrome C release; in addition to mitochondrial activity loss in carcinogenic cells, mediated cell death efficacy was found to be specific against colorectal cancer strain cells as it exhibited no toxicity in normal colon epithelial cells. ...
Reduction of food waste by extensively using raw sources builds good impact on the climate, environment, and food security. Fruits and vegetables are the commodities of horticultural crops that are commonly used. Thus, the waste generated during processing can be proclaimed as a valuable by-product if extensive technological interventions are used to improve the value of successive goods to balance the cost of processing. Fruit and vegetable wastes have great potential for bioconversion into valuable products with biotechnological, biocontrol, bioenergy, and industrial applications. These wastes could serve as a source of edible coating, fat mimetics, fortified probiotics, natural pigment, and volatile compounds in the food industry. Additionally, these wastes can also be used for synthesizing green carbon dots, biochar, and biosorbents that have environmental applications. The utilization of these fruit and vegetable wastes for generating valuable products is a foundation step towards sustainable development. The current chapter discusses the valuable products that can be obtained by bioconversion of waste of fruits and vegetables by enlisting their application in both food and environmental sectors.
... Nassiri-Asl and Hosseinzadeh (2016) performed a bibliographic review about the bioactive properties of grape waste; among them, anticancer effects and apoptosis induced in colon cancer strains were assigned to the grape seed of Italia and Palieri varieties (Dinicola et al. 2012). Furthermore, Derry et al. (2013) performed tests with grape seed extracts to induce apoptosis in colorectal cancer strains (SW480, SW620, and HCT116) and measured cytochrome C release; in addition to mitochondrial activity loss in carcinogenic cells, mediated cell death efficacy was found to be specific against colorectal cancer strain cells as it exhibited no toxicity in normal colon epithelial cells. ...
Agricultural residues, including fruit and vegetable residues, which usually contain starch or cellulose, are used for bioconversion into value-added products. Bioconversion provides several advantages, such as high value considering their material and energy recovery, decrease in landfill areas, lower cost of the technology, and the farmers’ income. Bioprocessing is of great importance for horticultural waste management to satisfy the needs of developing countries. Fermentation (solid-state and submerged) is considered an important tool to increase the nutritional value (providing more proteins, amino acids, and other biomolecules) of SCP and aquafeed meal ingredients. This chapter investigates the role of fruit and vegetable wastes in the production of single-cell protein (SCP) and aquafeed meal.
... For example, an up-regulation of cleaved caspase-3, cleaved PARP, phospho-p53, and total Bad (p < 0.05) in SW480 cells treated with 8 % v/v ABJ was found, suggesting pro-apoptotic effects in this cell line . Commercial grape seed extracts (25-100 μg/mL) containing procyanidins and monomeric flavonols found a significant increase of cytochrome C release, cleaved caspase-9, and cleaved caspase-3 on SW480, SW620, and HCT116 colorectal cancer cell lines, compared to the untreated cells (Derry et al., 2013). Activation of TRAIL pathways is notorious since CRC is particularly resistant to this activation (Deng & Shah, 2020). ...
Colorectal cancer (CRC) can be either prevented or alleviated using conventional drugs combined with natural treatments. Andean berry (AB, Vaccinium meridionale Sw.) is an underutilized berry with promising anti-inflammatory and antiproliferative effects that could be used to alleviate CRC markers in combination with Aspirin, a well-known CRC preventive drug. This research aimed to evaluate the impact of Aspirin, AB juice (ABJ), and their mixture on colorectal cancer in vitro and in vivo. The treatments (ABJ: 0, 10, 20, and 30 % v/v; Aspirin: 0, 10, 15, and 20 mM; and their combination) were assessed on SW480 cells to test their antiproliferative and pro-apoptotic effect. To evaluate their chemopreventive and chemoprotective effect in vivo, azoxymethane (AOM, 15 mg/kg BW) was used as a chemical inductor of early-stage colon cancer. Balb/c mice (8 weeks’ age) were randomly assigned to five groups (n = 6 mice/group): control (no treatment), positive control (AOM-treated mice), AOM + Aspirin (20 mM: 25 mg/kg BW), AOM + ABJ (30 % v/v), and AOM + Aspirin + ABJ (Aspirin: 25 mg/kg BW; ABJ: 30 % v/v). ABJ contained phenolic compounds such as 3,4-dihydroxybenzoic and gallic acids, morin, and rutin. The mixture showed a strongest antiproliferative effect than their counterparts (+10.39–46.23 %). Except for Aspirin (20 mM), the cells were not able to proliferate based on the cloning efficiency test. The mixture was the most effective treatment arresting the cell cycle and increasing G2/M cell population (p < 0.01). Aspirin and ABJ showed mainly intrinsic and extrinsic-mediated apoptotic processes, while the mixture decreased most pro-apoptotic (cytochrome C, DR4, DR5, TNFRSF1A, Bax, and Bad) and anti-apoptotic proteins (Hsp70, Hsp32, and XIAP) compared to the untreated cells. In silico simulations highlighted the interaction between rutin and catalase as the strongest affinity (−10.30 Kcal/mol). ABJ and the mixture decreased aberrant crypt foci in vivo compared to AOM-only treated mice and protected the colonic and liver architecture, this was latter used as a secondary indicator of AOM-metabolic activity. The chemopreventive approach was more effective, related to a prior regulation of cancer-protective mechanisms in vivo, alleviating the AOM-induced damage. The results indicated that Aspirin and ABJ mixtures exhibit antiproliferative and pro-apoptotic effects in SW480 cells inducing mechanisms linked to extrinsic (TNF and TRAIL-mediated apoptosis) and intrinsic (Bax and cytochrome C modulation) pathways. At in vivo levels, the treatments displayed defensive effects against the AOM-induced damage as observed by macroscopic measurements. However, more in vitro, and in vivo approaches are required to completely fulfill the pro-apoptotic, anti-proliferative, and chemopreventive/chemoprotective effects of ABJ.
... Moreover, GSE can increase the abundance of beneficial bacteria such as Lactobacillus and Akkermansia, as well as inhibit the growth of pathogens such as Clostridium histolyticum and Helicobacter Pylori (González-Quilen et al., 2020). Dietary GSE could induce cell cycle arrest and apoptosis in cell cultures (Derry, Raina, Agarwal, & Agarwal, 2013). In animal models, dietarysupplementation with GSE prevents the formation of abnormal crypt foci in the colon of rats (Velmurugan, Singh, Agarwal, & Agarwal, 2010). ...
2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) is a common carcinogen produced in thermally processed protein-rich foods. This study explored the protective effects of grape seed extract (GSE) against colonic injury induced by short-term exposure to PhIP and the underlying mechanisms involved. Wistar rats were randomly divided into four groups: control, GSE only (GSE), PhIP (PhIP), and GSE prevention (GPhIP). In the GPhIP group, rats were fed a diet supplemented with GSE for 2 weeks before administering PhIP. GSE significantly ameliorated PhIP-induced oxidative stress and colonic DNA damage. Moreover, GSE effectively maintained homeostasis in intestinal flora, especially by preventing PhIP-mediated reduction in Lactobacillus abundance. Fecal metabolome and colonic transcriptome analyses revealed that GSE remarkably ameliorated PhIP-induced colonic injury by regulating lipid metabolic pathways. Notably, nuclear factor-κB signaling pathway involved in the process of prevention. Therefore, GSE is recommended as an effective dietary supplement to prevent the harm of PhIP in vivo.
... It can be explained by five times greater resistance to drug treatment of this cell line compared to other colon adenocarcinoma cell line-HCT116 [52]. Other researchers established that grape seed extract containing about 89% of proanthocyanins, was also more active in the HCT116 cell line (30 µg/mL of extract killed about 60% of cells already after 48 h) [53]. Interestingly, grape seed extract showed lower activity against melanoma cancer cell line A431 (cell viability was reduced by only~20% at 50 and 100 µg/mL concentrations, while in our experiment L4 and F4 fractions reduced melanoma IGR-39 cell viability by 50% at 50-100 µg/mL concentrations after 72 h of incubation [54]. ...
Lingonberry leaves and fruits are associated with a range of potential bioactivities related to their phenolic content and composition, but the identification of major biological activity markers remains limited. The present study aimed at the isolation of lingonberry phenolic fractions and biological activity evaluation of them. Crude dry extracts of lingonberry leaves and fruits were fractionated by chromatography using Sephadex LH-20 and analyzed by validated HPLC-PDA method. For each fraction, the anticancer activity against human clear cell renal cell carcinoma (CaKi-1), human colon adenocarcinoma (HT-29), and human malignant melanoma (IGR39) cell lines was determined using MTT assay, and the radical scavenging, reducing, and chelating activities were investigated using ABTS, FRAP, and FIC assays, respectively. Further, 28 phenolics were identified and quantified in the crude extract of lingonberry leaves and 37 in the extract of fruits. These compounds, during fractionation steps, were selectively eluted into active fractions, enriched with different groups of phenolics—monophenols, anthocyanins, phenolic acids, catechins, flavonols, or proanthocyanidins. Fractions of lingonberry leaves and fruits, obtained by the last fractionation step, proved to be the most active against tested cancer cell lines and possessed the greatest antioxidant activity. In this perspective, the predominant compounds of these fractions—polymeric and mainly A-type dimeric proanthocyanidins—also quercetin can be considered to be anticancer and antioxidant activity markers of lingonberries.
... The administration of grape seed extract was not only effective against colorectal cancer but also safe to healthy cells of the body. [7] Chemical constituents: Numerous studies have demonstrated that certain nutritive and nonnutritive phytochemicals with potential cancer-preventive or antitumor activity can be isolated from grape seeds. Of these compounds, proanthocyanidins are worthy of mention. ...
... In SW480, having a lower grade of differentiation, we observed a lower sensitivity to treatment with both GSEs. However, different behavior of grape extracts on antiproliferative and proapoptotic processes has been previously demonstrated in different human CRC cell lines [39][40][41][42]. In particular, Dinicola et al. [43] have demonstrated that different polyphenols might act additively and/or synergistically to exert total antiproliferative action of the grape extract. ...
The polyphenolic compounds present in grape extracts have chemopreventive and anticancer properties. Here, we studied the ability of two grape skin extracts (GSEs), Autumn Royal and Egnatia, to influence the cell motility and membrane fluidity regulated by the enzyme Stearoyl-CoA desaturase-1 (SCD1) which increases with the cancer aggressiveness. Caco2 and SW480 human colon cancer cell lines were treated with increasing concentrations of GSEs to evaluate cell proliferation and motility. SCD1 levels were evaluated in both treated cell lines, by membrane lipidomic analysis conducted by gas chromatography. The expression levels of SCD1 and other factors involved in the reorganization of the cytoskeleton and focal adhesions were assessed by Real-time PCR, Western Blotting, and Immunofluorescence staining. High-performance liquid chromatography (HPLC) analyses were performed to determine the phenolic composition in the GSEs, finding them more expressed in Autumn Royal than in Egnatia. Both treatments reduced the levels of SCD1, phospho-Rac1/Cdc42/Rac1/Cdc42 ratio, Cofilin, Vimentin, and phospho-Paxillin especially in Caco2 compared to SW480, showing a different behavior of the two cell lines to these natural compounds. Our findings show that GSEs block the cell migration and membrane fluidity through a new mechanism of action involving structural cellular components.
... Moreover, treatment of HCT116, SW620, and SW480 colorectal cancer cell lines with Grape seed extract resulted in induced cleavage of caspases−3,−8,−9, and PARP. In addition, cytochrome-c was released from the mitochondria of all these three cell lines, suggesting the involvement of both pathways in the apoptotic death (43). To better understand the mechanism of IV induced cell death, additional studies are required. ...
Colorectal cancer (CRC) is the second most common cancer in females and the third in males worldwide. Conventional therapy of CRC is limited by severe side effects and by the development of resistance. Therefore, additional therapies are needed in order to combat the problem of selectivity and drug resistance in CRC patients. Inula viscosa (IV) is a well-known medicinal perennial herb in traditional medicine. It is used for different therapeutic purposes, such as; topical anti-inflammatic, diuretic, hemostatic, antiseptic, antiphlogistic, and in the treatment of diabetes. Several studies attempted to reveal the anti-cancer activity of different extracts prepared by different organic solvents from different parts of the IV plant. The aim of the present study is to examine the potential beneficial effects of IV leaf aqueous extract on the growth of colon cancer cells in vitro and in vivo. The results indicated that exposure of colorectal cancer cells to IV extract, significantly reduced cell viability in a dose and time dependent manner. Moreover, treatment of cells with 300 μg/ml of IV extract induced apoptosis, as it was detected by Annexin V/FITC/PI, TUNEL assay, and the activation of caspases. In vivo studies revealed that treatment with 150 or 300 mg/kg IV extract inhibited tumor growth in mice transplanted with MC38 cells. Tumors' weight and volume were significantly (P < 0.001) reduced when compared to untreated-control group. Staining of the paraffin section of tumors revealed that IV treatment inhibited cell proliferation and induced apoptosis. Additionally, no side effects such as; weight loss, behavior changes, ruffled fur or changes in kidney, and liver functions were observed. These results may indicate that active doses of IV extract are not toxic. Further studies are needed in order to identify the structure of the active compounds. Results from this study may contribute to the development of new and efficient strategies for treatment of human colon cancer.
... Extract of grape seeds showed inhibition of colorectal cancer cell growth. The extract of grape seed is also useful against colorectal cancer (Derry et al. 2013). The phytochemicals isolated from seeds of grapes have both nonnutritive and nutritive values that showed significant antitumor property. ...
Cancer is a main health challenge for the world due to unavailability of standard treatments and severe side effects of chemotherapy. It is the second major cause of casualty worldwide after cardiac disorder. Medicinal plants are used for the treatment of various diseases since ancient time. Due to toxicity of allopathic medicines, peoples are coming back toward the use of natural medicines for the treatment of diseases. Approximately 38% of Americans are using alternative medicine or herbal medicines and spend around $34 billion dollars yearly on it. Constituents isolated from plants showed a crucial role in the development of useful anticancer agent. These include etoposide, vincristine and vinblastine from Catharantus roseus, camptothecin from Camptotheca acuminata, podophyllotoxin from Podophyllum peltatum, etc. The aim of the chapter is to describe various anticancer plants and their possible mechanisms of action in the prevention of cancer.
... In addition it may be cause damages in the tissue of the thyroid gland (4,7). Grape seed extract GSE has a major role in free radicals resistance that led to oxidative stress, as well as protecting cells from the toxic effects of drugs (8). ...
The aim of the current study is to identify the role of grape seed extract in thyroid gland and lipid profile disorders induced by Carbimazole 30 mg/kg.b.w. The experience including 40 male rats randomly divided into five groups each group consist of 8 animals, grape seed extract used by dose 150mg/kg daily for the duration of the 30-day study period. Results showed that the animals treated with carbimazole had a significant decrease(P<0.05) in the concentration of thyroid hormones and increase TSH compared with their normal range level in the control group and the rest of the experimental groups, While groups in which grape seed extract(GSE) interferes with drug showed improvement in thyroid hormones level closer to normal range in control group. Also the results showed significant increase (P<0.05) in TC, TG HDL, vLDL and significant decrease in LDL in carbimazole group, in addition to damage in thyroid tissue. On the other hand animals treated by both grape seed extract (GSE) and drug showed significant improvement(P<0.05) in TC, TG, HDL, vLDL, LDL and thyroid tissue compared with groups treated with drug only. It was concluded from this study that grape seeds extract had protective role against damage caused by carbimazole.
... In Colo205, unlike that happens in HT-29/Lovo, p-Erk1/2 was increased by oxaliplatin at late stage and not affected by the addition of GSEs, consistently with an oxaliplatin-induced apoptosis which is not dependent by mitochondria [19]. As GSEs has been reported to affect the intrinsic and/or the extrinsic pathways of apoptosis [35,36], we evaluated the effects on: i) the activation of caspase-8 as a crucial event in death receptors apoptosis induction, ii) the truncation of BID, to assess the involvement of mitochondrial apoptosis after the death-receptors activated one, and in addition, we performed a comprehensive analysis of apoptosis by using the Human Apoptosis Array kit which allows to track simultaneously the expression/activities of several proteins involved in apoptosis. The results demonstrated that the addition of GSEs prevented the activation of the mitochondrial apoptosis and reduced the activation of the death receptors-dependent apoptosis in both Colo205 and HT-29/Lovo cells, through mechanisms that we extensively reported above. ...
Grape seed extracts are commonly utilized as dietary supplements for their antioxidant properties, even from cancer patients. However, whether these natural extracts interfere with chemotherapeutics utilized in colon cancer treatment is still poorly investigated. The cytotoxicity of extracts from Italia and Palieri cultivars either alone or in combination with oxaliplatin was evaluated in colon cancer cells. Grape seed extracts displayed anti-proliferative activity depending on the concentration utilized through apoptosis induction. In combination, they affected the activation of Erk1/2 and counteracted the intrinsic and the extrinsic pathway of apoptosis, the DNA damage and the generation of ROS induced by oxaliplatin. Noteworthy grape seed extracts strongly enhanced the uptake of oxaliplatin into all cells, by affecting the cell transport system of platinum. The addition of these natural extracts to oxaliplatin strongly reduced the cellular response to oxaliplatin and allowed a huge accumulation of platinum into cells. Here, we shed light on the chemical biology underlying the combination of grape seed extracts and oxaliplatin, demonstrating that they might be detrimental to oxaliplatin effectiveness in colon cancer therapy.
... 12 In this context, a large number of preclinical and clinical studies have shown a broad spectrum of pharmacological and therapeutic benefits of GSE against oxidative stress, degenerative disease like cardiovascular dysfunctions and various types of cancers. 5,6,[11][12][13][14] Given that the protective effects of GSE on oxidative stress, cardiovascular diseases and neoplasm is dependent on its free radical scavenging capability and its antioxidant impacts and since the DOX-induced cardiotoxicity is mainly mediated through free radical production, natural antioxidants like GSE may offer an effective and safe means to counteract some of the problems and bolstering the antioxidant defense systems against cardiovascular diseases via neutralizing harmful free radicals. Therefore, the aim of the present study was to determine the ability of GSE to reduce the DOXinduced cardiotoxicity in a rat model. ...
Purpose: The aim of the present study was to determine the ability of grape seed extract
(GSE) as a powerful antioxidant in preventing adverse effect of doxorubicin (DOX) on
heart function.
Methods: Male rats were divided into three groups: control, DOX (2 mg/kg/48h, for 12
days) and GSE (100 mg/kg/24h, for 16 days) plus DOX. Left ventricular (LV) function and
hemodynamic parameters were assessed using echocardiography, electrocardiography and a
Millar pressure catheter. Histopathological analysis and in vitro antitumor activity were also
evaluated.
Results: DOX induced heart damage in rats through decreasing the left ventricular systolic
and diastolic pressures, rate of rise/decrease of LV pressure, ejection fraction, fractional
shortening and contractility index as demonstrated by echocardiography,
electrocardiography and hemodynamic parameters relative to control group. Our data
demonstrated that GSE treatment markedly attenuated DOX-induced toxicity, structural
changes in myocardium and improved ventricular function. Additionally, GSE did not
intervene with the antitumor effect of DOX.
Conclusion: Collectively, the results suggest that GSE is potentially protective against
DOX-induced toxicity in rat heart and maybe increase therapeutic index of DOX in human
cancer treatment.
... El extracto de semilla de uva se demuestra eficaz contra el cáncer colorrectal (98). ...
Review of the most known and used medicinal plants in our cultural environment that traditionally have been studied in cell lines with animals and patients, who have shown their potential anti-tumor and may be an opportunity for reasonable use by patients.
... Grape seed extract selectively induced apoptosis in different colorectal cancer cell lines (SW480, SW620 and HCT116) accompanying the release of cytochrome c in the cytoplasm of the cancer cells and the loss of mitochondrial membrane potential (Derry et al., 2013). ...
Vitis vinifera fruit (grape) contains various phenolic compounds, flavonoids and stilbenes. In recent years, active constituents found in the fruits, seeds, stems, skin and pomaces of grapes have been identified and some have been studied. In this review, we summarize the active constituents of different parts of V. vinifera and their pharmacological effects including skin protection, antioxidant, antibacterial, anticancer, antiinflammatory and antidiabetic activities, as well as hepatoprotective, cardioprotective and neuroprotective effects in experimental studies published after our 2009 review. Clinical and toxicity studies have also been examined. Copyright © 2016 John Wiley & Sons, Ltd.
... And these could also be probable mechanisms involved in the prooxidant activity of GSE and for its cytotoxicity specifically against HNSCC cells. Importantly, GSE has shown no or only marginal cytotoxicity against normal human cells (normal human epidermal keratinocytes NHEK cells and normal colon epithelial NCM460 cells) [19,48]. ...
Head and neck squamous cell carcinoma (HNSCC) is a major killer worldwide and innovative measures are urgently warranted to lower the morbidity and mortality caused by this malignancy. Aberrant redox and metabolic status in HNSCC cells offer a unique opportunity to specifically target cancer cells. Therefore, we investigated the efficacy of grape seed extract (GSE) to target the redox and bioenergetic alterations in HNSCC cells. GSE treatment decreased the mitochondrial electron transport chain complex III activity, increased the mitochondrial superoxide levels and depleted the levels of cellular antioxidant (glutathione), thus resulting in the loss of mitochondrial membrane potential in human HNSCC Detroit 562 and FaDu cells. Polyethylene glycol-SOD addition reversed the GSE-mediated apoptosis without restoring complex III activity. Along with redox changes, GSE inhibited the extracellular acidification rate (representing glycolysis) and oxygen consumption rate (indicating oxidative phosphorylation) leading to metabolic stress in HNSCC cells. Molecular studies revealed that GSE activated AMP-activated protein kinase (AMPK), and suppressed Akt/mTOR/4E-BP1/S6K signaling in both Detroit 562 and FaDu cells. Interestingly, GSE increased the autophagic load specifically in FaDu cells, and autophagy inhibition significantly augmented the apoptosis in these cells. Consistent with in vitro results, in vivo analyses also showed that GSE feeding in nude mice activated AMPK and induced-autophagy in FaDu xenograft tumor tissues. Overall, these findings are innovative as we for the first time showed that GSE targets ETC complex III and induces oxidative and metabolic stress, thereby, causing autophagy and apoptotic death in HNSCC cells. © 2014 Wiley Periodicals, Inc.
... Additionally, GSE reduces expression of iNOS and COX-2, decreasing oxidative cellular stress [37]. Derry et al. [38] performed in vitro studies on CRC cell lines, and showed that GSE induces their apoptosis, mainly due to activation of casapses 3, 8, and 9 and also generation of ROS. Moreover the proapoptotic function of GSE was limited only to cancer cells and there was no effect in normal colonocytes. ...
... Another important source of phytochemicals, in particular proanthocyanidins, is grape seed (GS) extract. Data indicate that GS extract has strong growth inhibitory and apoptosis-inducing effects in CRC cell lines (18,19) and xenografts (20). Fish oil is a source of omega-3 (ω-3) polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA). ...
Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. The development of novel anti-CRC agents able to overcome drug resistance and/or off-target toxicity is of pivotal importance. The mammalian target of rapamycin (mTOR) plays a critical role in CRC, regulating protein translation and controlling cell growth, proliferation, metabolism and survival. The aim of this study was to explore the effect of a combination of three natural compounds, eicosapentaenoic acid-free fatty acid (EPA-FFA), epigallocatechin-3-gallate (EGCG) and proanthocyanidins (grape seed [GS] extract) at low cytotoxic concentrations on CRC cells and test their activity on mTOR and translational regulation. The CRC cell lines HCT116 and SW480 were treated for 24h with combinations of EPA-FFA (0-150 µM), EGCG (0-175 µM) and GS extract (0-15 µM) to evaluate the effect on cell viability. The low cytotoxic combination of EPA-FFA 150 µM, EGCG 175 µM and GS extract 15 µM completely inhibited the mTOR signaling in HCT116 and SW480 cells, reaching an effect stronger than or comparable to that of the mTOR inhibitor Rapamycin in HCT116 or SW480 cells, respectively. Moreover, the treatment led to changes of protein translation of ribosomal proteins, c-Myc and cyclin D1. In addition, we found a reduction of clonal capability in both cell lines, with block of cell cycle in G0G1 and induction of apoptosis. Our data suggest that the low cytotoxic combination of EPA-FFA, EGCG and GS extract, tested for the first time here, inhibits mTOR signaling and thus could be considered for CRC treatment. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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... Additionally, GSE reduces expression of iNOS and COX-2, decreasing oxidative cellular stress [37]. Derry et al. [38] performed in vitro studies on CRC cell lines, and showed that GSE induces their apoptosis, mainly due to activation of casapses 3, 8, and 9 and also generation of ROS. Moreover the proapoptotic function of GSE was limited only to cancer cells and there was no effect in normal colonocytes. ...
Many studies suggest that Western lifestyle and dietary factors may be responsible for the high incidence of colorectal cancer in industrialized countries. Consumption of high amounts of red and processed meat and low intake of fiber and multiple protective phytochemicals found in fruits, vegetables, and whole grains might be responsible for the high incidence of this neoplasm in the Western world. Additionally, obesity, lack of physical activity, tobacco and alcohol use, sleep deprivation, and other factors have been proven to further increase the risk of colorectal cancer. Identifying and understanding the mechanisms through which they impact colon carcinogenesis is needed for the introduction of protective lifestyle recommendations.
... Results of the present study, however, for the first time, identified that GSE causes a strong growth inhibition of human bladder cancer cell lines T24 and HTB9, which is accompanied by a strong and significant apoptotic death. Importantly, growth inhibition was not observed in normal cells (NHEK and NCM460) even at higher doses of GSE (Shrotriya et al., 2012;Derry et al., 2012). Interestingly, the apoptotic death events in bladder cancer cells were preceded by vacuolar appearance in the cytoplasm. ...
In present study, we evaluated grape seed extract (GSE) efficacy against bladder cancer and associated mechanism in two different bladder cancer cell lines T24 and HTB9. A significant inhibitory effect of GSE on cancer cell viability was observed, which was due to apoptotic cell death. Cell death events were preceded by vacuolar appearance in cytoplasm, which under electron microscopy was confirmed as swollen mitochondrial organelle and autophagosomes. Through detailed in vitro studies, we established that GSE generated oxidative stress that initiating an apoptotic response as indicated by the reversal of GSE-mediated apoptosis when the cells were pre-treated with antioxidants prior to GSE. However, parallel to a strong apoptotic cell death event, GSE also caused a pro-survival autophagic event as evidenced by tracking the dynamics of LC3-II within the cells. Since the pro-death apoptotic response was stronger than the pro-survival autophagy induction within the cells, cell eventually succumbed to cellular death after GSE exposure. Together, the findings in the present study are both novel and highly significant in establishing, for the first time, that GSE-mediated oxidative stress causes a strong programmed cell death in human bladder cancer cells, suggesting and advocating the effectiveness of this non-toxic agent against this deadly malignancy.
Traditionally, grape is one of the most efficient medicinal plant species due to its diverse uses as grape juice, wine, and supplementary food products. Grape seed extract (GSE) and its secondary metabolites have shown anticancer activity in breast, lung, prostate, bladder, and colorectal cancers. Polyphenols are the chief active constituents of grape. In several in vitro and preclinical studies, polyphenols have shown significant promising anticarcinogenic and chemopreventive effects and their underlying mechanism have also been demonstrated. However, due to a lack of clinical trials or studies, these polyphenols are in the infancy stage. Consequently, more extensive studies are required for cancer of precise sites or organs with standardized grape seed extract and its products to establish the dose regimen and to conclude the bioactive pharmacologically achievable phytoconstituents.
Gastrointestinal cancer (GIC), including gastric cancer and colorectal cancer, is a common malignant tumor originating from gastrointestinal epithelial cells. Although the pathogenesis of GIC remains unclear, aberrant lipid metabolism has emerged as a hallmark of cancer. Several enzymes, proteins, and transcription factors are involved in lipid metabolism reprogramming in GIC, and their abnormal expression can promote lipid synthesis and accumulation of lipid droplets through numerous mechanisms, thereby affecting the growth, proliferation, and metastasis of GIC cells. Studies show that some natural compounds, including flavonoids, alkaloids, and saponins, can inhibit the de novo synthesis of lipids in GIC, reduce the level of lipid accumulation, and subsequently, inhibit the occurrence and development of GIC by regulating Sterol regulatory element-binding protein 1 (SREBP-1), adenosine monophosphate-activated protein kinase (AMPK), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), phosphatidylinositol-3-kinase/Akt and the mammalian target of rapamycin PI3K/Akt/mTOR, amongst other targets and pathways. Therefore, targeting tumor lipid metabolism is the focus of anti-gastrointestinal tumor therapy. Although most natural products require further high-quality studies to firmly establish their clinical efficacy, we review the potential of natural products in the treatment of GIC and summarize the application prospect of lipid metabolism as a new target for the treatment of GIC, hoping to provide a reference for drug development for gastrointestinal tumors.
Orange and grape crops play a significant role in the Brazilian economy. Industrial processing generates large amounts of fruit wastes, which emerge as sources of enzymes and other bioactive compounds of interest in several studies due to their different biotechnological applications. The method adopted to obtain biomolecules plays an essential role in reducing costs with bioprocessing; thus, both isolated (direct use) and fermented (indirect use) waste types are seen as sustainable alternatives to chemical technologies. High commercial value enzymes such as phytases, lipases, and proteases are widely used in catalytic processes since they have versatile biochemical properties and broad applications. Their antioxidant, anti-inflammatory, antimicrobial, antidiabetic, and cell modulation actions enhanced the biological potential of orange and grape waste. Therefore, this chapter emphasizes enzyme obtainment and the biomolecules mentioned above from orange and grape waste.KeywordsCitrus sinensis L. Osbeck
Vitis vinifera
V. labrusca
PhytasesLipasesProteasesAntioxidantAntimicrobialAntidiabeticAnti-inflammatoryCellular modulation activity
MicroRNAs are classified as small non-coding RNAs that regulate gene expression mainly through targeting the 3′UTR region of mRNAs. A great number of miRNAs play important role in the regulation of signaling pathways in normal and cancer cells. Here, we predicted hsa-miR-5195-3p (miR-5195-3p) as a potential regulator of TGFβ signaling and investigated its effect on TGFB-R1, TGFB-R2, SMAD2, SMAD3 and SMAD4 transcripts which are key players of TGFβ/SMAD signaling pathway. Overexpression of miR-5195 in HCT116 cells resulted in a significant reduction of TGFB-R1, SMAD2, SMAD3, and SMAD4 at the mRNA level which was confirmed using RT-qPCR. Consistently, western blot analysis confirmed that miR-5195 overexpression in HCT116 cells resulted in downregulation of TGFBR1 at the protein level. Furthermore, dual luciferase analysis verified the direct interaction of miR-5195 with TGFB-R1 and SMAD4 3′UTR sequences in SW480 cells. Additionally, flow cytometry analysis confirmed that miR-5195 overexpression significantly increased the sub-G1 and decreased the G-1 cell populations in both SW480 and HCT116 cell lines. Finally, miR-5195 overexpression significantly downregulated c-MYC and cyclin D1 but upregulated p21 genes. Overall, our results indicated that miR-5195 modulates TGFβ signaling pathway and affects the cell cycle progression through targeting TGFB-R1, TGFB-R2, SMAD2, SMAD3, SMAD4 transcripts.
Transforming growth factor-β (TGFβ) signaling acts as suppressor and inducer of tumor progression during early and late stages of cancer, respectively. Some miRNAs have shown regulatory effect on TGFβ signaling and here, we have used combination of bioinformatics and experimental tools to show that hsa-miR-5590-3p is a regulator of multiple genes expression in TGFβ signaling pathway. Consistent to the bioinformatics predictions, hsa-miR- 5590-3p had a negative correlation of expression with TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4 genes, detected by RT-qPCR. Then, dual luciferase assay supported direct interaction between hsa-miR-5590-3p and TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4 -3′UTR sequences. Consistently, TGFβ-R1 protein level was reduced following the overexpression of hsa-miR-5590-3p, detected by Western analysis. Also, hsa-miR-5590-3p overexpression brought about downregulation of TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4 expression in HCT-116 cells, detected by RT-qPCR, followed by cell cycle arrest in sub-G1 phase, detected by flow cytometry. RT-qPCR results indicated that hsa-miR-5590-3p is significantly downregulated in breast tumor tissues (late stage) compared to their normal pairs. Altogether, data introduces hsa-miR-5590-3p as negative regulator of TGFβ/SMAD signaling pathway which acts through downregulation of TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4 transcripts. Therefore, it can be tested as therapy target in cancers in which TGFβ/SMAD pathway is deregulated.
Transforming growth factor β (TGFβ) signaling pathway which is regulated by factors such as microRNAs (miRNAs) has pivotal roles in various cellular processes. Here, we intended to verify bioinformatics predicted regulatory effect of hsa-miR-5582-3P against TGFβ/SMAD signaling pathway components. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis indicated a negative correlation of expression between hsa-miR-5582-3P against TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 putative target genes in all of tested cell lines. Also, hsa-miR-5582-3P was significantly downregulated in glioma, breast, and ovarian tumor tissues compared with their normal pairs, detected by RT-qPCR. Then dual luciferase assay supported direct interaction between this miRNA and TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4, 3' untranslated region sequences. Western blot analysis confirmed negative effect of hsa-miR-5582-3P overexpression on at least TGFβ-R1 expression. Consistently, hsa-miR-5582-3P overexpression brought about downregulation of TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 expression in HCT-116 cell line, followed by cell cycle arrest in sub-G1 phase, detected by flow cytometry. Altogether, our data suggest that hsa-miR-5582-3P reduces the TGFβ/SMAD signaling pathway through downregulation of TGFβ-R1, TGFβ-R2, SMAD3, and SMAD4 transcripts. These data introduce hsa-miR-5582-3P as a potential tumor suppressors-miR and a therapy candidate to be tested in cancers in which TGFβ/SMAD is deregulated.
The consumption of vegetables and fruits, particularly apples, pomegranates, grapes and berries, is associated with a decreased risk of developing cancer, especially colorectal cancer (CRC). This may be attributable to the presence of phytochemical constituents, such as polyphenols. This review summarizes the current state of knowledge concerning the chemopreventive and anti-CRC potential of polyphenol-rich apple, pomegranate, grape and berry extracts with a focus on in vitro, in vivo and clinical evidence. The extracts demonstrate antiproliferative, proapoptotic, antiangiogenic, antiinvasive and antimetastatic activities toward colon cancer cell lines and in animal models, and markedly influence preneoplastic lesions and malignancies in humans.
This study evaluates the anti-proliferative and anti-genotoxic actions of powdered red wine pomace seasonings (Sk-S:seedless, W-S:whole, Sd-S:seeds). In vitro gastrointestinal digested and colonic fermented fractions of the seasonings were used as cell treatments. Phenolic acids from Sk-S showed the highest bioaccessibility in the small intestine whereas polyphenols contained in Sd-S might be the most fermentable in the colon. Dietary fibre from Sk-S was the best substrate for short chain fatty acids production by gut microbiota. Colon cancerous (HT-29) cell viability was inhibited by 50% (IC50 values) at treatment concentrations ranging from 845 (Sk-S) to 1085 (Sd-S) μg/mL prior digestion, but all digested fractions exhibited similar anti-proliferative activities (mean IC50=814 μg/mL). Oxidative DNA damage in cells was also attenuated by the treatments (200 μg/mL, 24-h preincubation), with all colonic fermented fractions displaying similar genoprotective action. These results suggest the potential of red wine pomace seasonings as chemopreventive agents in colorectal cancer.
The seeds of grapevine (Vitis vinifera) are a byproduct of wine production. To examine the potential value of grape seeds, grape seeds from seven sources were subjected to fingerprinting using direct analysis in real time coupled with time-of-flight mass spectrometry combined with chemometrics. Firstly, we listed all reported components (56 components) from grape seeds and calculated the precise m/z values of the deprotonated ions [M-H](-) . Secondly, the experimental conditions were systematically optimized based on the peak areas of total ion chromatograms of the samples. Thirdly, the seven grape seed samples were examined using the optimized method. Information about 20 grape seed components was utilized to represent characteristic fingerprints. Finally, hierarchical clustering analysis and principal component analysis were performed to analyze the data. Grape seeds from seven different sources were classified into two clusters; hierarchical clustering analysis and principal component analysis yielded similar results. The results of this study lay the foundation for appropriate utilization and exploitation of grape seed samples. Due to the absence of complicated sample preparation methods and chromatographic separation, the method developed in this study represents one of the simplest and least time-consuming methods for grape seed fingerprinting. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Our increasing knowledge about health and disease suggests that vegetarian diets rich in fruits and vegetables have a significant impact in prevention and therapy of multiple types of cancer. Phytochemicals from both dietary and nondietary origins inhibit cancer growth and progression of human cancers by interacting at the cellular and molecular levels. The mitochondrion is the key organelle that provides energy to cells for their growth and survival. In addition, mitochondria play an important role in cancer cell apoptosis and may also regulate autophagy. Cancer cells have high proliferative and less apoptotic activities; therefore, agents that induce apoptosis selectively in cancer cells are desired for the treatment of cancer. Some dietary phytochemicals target mitochondria causing destruction of mitochondrial membranes leading to the discharge of proapoptotic mitochondrial protein, which initiates apoptotic cell death in cancer. Although the role of autophagy is debated as a mechanism of cell death or cell survival, many phytochemicals modulate autophagy in cancer cells. Therefore, selective targeting of cell-death pathways by phytochemicals in cancer cells could be an attractive strategy for the prevention and treatment of cancer.
Studies were carried out to determine whether the combination of IL-1 + M-CSF, similar to the effect of these cytokines on neutropenia, was able to reduce the duration of thrombocytopenia in the 5-fluorouracil (5-FU)-myelosuppressed mouse. In addition, comparisons were made between the in vivo effects of IL-1 + M-CSF and other "thrombopoietic" cytokines (e.g., IL-3, IL-6, and GM-CSF) that demonstrate some form of megakaryocytopoietic activity in vitro. Of the five cytokines studied, only IL-1 and IL-6, by themselves, were able to effect thrombopoietic recovery in the myelosuppressed mouse. IL-1, either when acting alone or interacting synergistically with M-CSF, was able to reduce significantly the period of thrombocytopenia, but the effects of IL-6 were restricted to enhancing platelet production during the period of rebound thrombocytopenia without altering the kinetics of thrombopoietic recovery. Moreover, none of the cytokine combinations studied were found to interact to reduce further the duration of thrombocytopenia beyond that observed with IL-1 + M-CSF. Nonetheless, IL-3, IL-6, and, to a lesser extent, GM-CSF were each able to interact with IL-1 + M-CSF to extend further the period of enhanced platelet production in the animal. However, scheduling studies suggested that these thrombopoietic cytokines interacted in sequence, rather than in concert, with IL-1 + M-CSF to enhance platelet production during thrombopoietic recovery. Furthermore, the data presented are consistent with the hypothesis that IL-1 + M-CSF initially acts on a multilineage, 5-FU-resistant target cell and that IL-6 (and possibly IL-3 and GM-CSF) serves as a secondary cytokine further to enhance platelet production during rebound thrombopoiesis in the 5-FU-treated mouse.
Grape seed extract has been proven to exert anticancer effects on different tumors. These effects are mainly ascribed to catechin and procyanidin content. Analytical studies demonstrated that grape seed extract composition is complex and it is likely other components could exert biological activities. Using cell count and flow cytometry assays, we evaluated the cytostatic and apoptotic effects produced by three different grape seed extracts from Italia, Palieri and Red Globe cultivars, on Caco2 and HCT-8 colon cancer cells. These effects were compared to those induced by epigallocatechin and procyanidins, alone or in association, on the same cell lines. All the extracts induced growth inhibition and apoptosis in Caco2 and HCT-8 cells, along the intrinsic apoptotic pathway. On both cell lines, growth inhibition induced by Italia and Palieri grape seed extracts was significantly higher than that it has been recorded with epigallocatechin, procyanidins and their association. In Caco2 cells, the extract from Red Globe cultivar was less effective in inducing growth inhibition than procyanidins alone and in association with epigallocatechin, whereas, in HCT-8 cells, only the association of epigallocatechin and procyanidins triggers a significant proliferation decrease. On both cell lines, apoptosis induced by Italia, Palieri and Red Globe grape seed extracts was considerably higher than has been recorded with epigallocatechin, procyanidins and their association. These data support the hypothesis by which other compounds, present in the grape seed extracts, are likely to enhance the anticancer effects.
As the therapeutic options for the treatment of colorectal cancer have expanded over the past 20 years, so has the complexity of decision making. The goals of treatment in the palliative, adjuvant and neoadjuvant settings vary and it is not only the efficacy of drugs that influence treatment decisions. Age, performance status, the presence of significant comorbidities and the different treatment regimens and strategies provide medical oncologists with an array of options to attempt to maximize patients' quality of life and longevity.
We assessed Ki-ras mutations by single-strand conformation polymorphism followed by DNA sequencing, p53 expression by immunohistochemistry, ploidy status, and S-phase fraction in 66 stage II and 163 stage III colon cancer patients enrolled on a randomized trial of surgery followed by observation or adjuvant levamisole or 5-fluorouracil (5FU) plus levamisole (Intergroup Trial 0035) to see whether these factors were independently associated with survival or with differential effects of adjuvant therapy. A Cox proportional hazards survival model was used to describe marker effects and therapy by marker interactions, with adjustment for the clinical covariates affecting survival. A Bonferroni adjustment was used to account for multiple testing. Mutation of the Ki-ras gene was found in 41% of the cancers and was associated with a poor prognosis in stage II but not stage III. In stage II, 7-year survival was 86% versus 58% in those with wild type versus Ki-ras mutations. After adjustment for treatment and clinical variables, the hazard ratio (HR) for death was 4.5; 95% confidence interval (CI), 1.7-12.1 (P = 0.012). p53 overexpression was found in 63% of cancers and was associated with a favorable survival in stage III but not stage II. Seven-year survival in stage III was 56% with p53 overexpression versus 43% with no p53 expression (HR, 2.2; 95% CI, 1.3-3.6; P = 0.012). Aneuploidy was more common in stage III than in stage II (66 versus 47%; P = 0.009) but was not independently related to survival in either group. The proliferative rate was greater in aneuploid than in diploid cancers but was not related to survival. There was no benefit of adjuvant therapy in stage II nor in any of the stage II subgroups defined by mutational status. In stage III, adjuvant therapy with 5FU plus levamisole improved 7-year survival in patients with wild-type Ki-ras (76 versus 44%; HR, 0.4; 95% CI, 0.2-0.8) and in those without p53 overexpression (64 versus 26%; HR, 0.3; 95% CI, 0.1-0.7). Adjuvant therapy did not benefit those with Ki-ras mutations or p53 overexpression. The effects of adjuvant therapy did not differ according to ploidy status or proliferative rate. Ki-ras mutation is a significant risk factor for death in stage II, and the absence of p53 expression is a significant risk factor for death in stage III colon cancer after adjustment for treatment and clinical covariates. Exploratory analyses suggest that patients with stage III colon cancer with wild-type Ki-ras or no p53 expression benefit from adjuvant 5FU plus levamisole, whereas those with Ki-ras mutations or p53 overexpression do not. An independent study will be required to determine whether response to adjuvant therapy in colon cancer depends on mutational status.
Chemopreventive effects and associated mechanisms of grape seed extract (GSE) against intestinal/colon cancer development are largely unknown. Herein, we investigated GSE efficacy against intestinal tumorigenesis in APC(min/+) mice. Female APC(min/+) mice were fed control or 0.5% GSE (wt/wt) mixed AIN-76A diet for 6 weeks. At the end of the experiment, GSE feeding decreased the total number of intestinal polyps by 40%. The decrease in polyp formation in the small intestine was 42%, which was mostly in its middle (51%) and distal (49%) portions compared with the proximal one. GSE also decreased polyp growth where the number of polyps of 1 to 2 mm in size decreased by 42% and greater than 2 mm in size by 71%, without any significant change in polyps less than 1 mm in size. Immunohistochemical analyses of small intestinal tissue samples revealed a decrease (80%-86%) in cell proliferation and an increase (four- to eight-fold) in apoptosis. GSE feeding also showed decreased protein levels of cyclooxygenase-2 (COX-2) (56%-64%), inducible nitric oxide synthase (iNOS) (58%-60%), and beta-catenin (43%-59%) but an increased Cip1/p21-positive cells (1.9- to 2.6-fold). GSE also decreased cyclin D1 and c-Myc protein levels in small intestine. Together, these findings show the chemopreventive potential of GSE against intestinal polyp formation and growth in APC(min/+) mice, which was accompanied with reduced cell proliferation and increased apoptosis together with down-regulation in COX-2, iNOS, beta-catenin, cyclin D1, and c-Myc expression, but increased Cip1/p21. In conclusion, the present study suggests potential usefulness of GSE for the chemoprevention of human intestinal/colorectal cancer.
The present study explored the bioavailability and brain deposition of a grape seed polyphenolic extract (GSPE) previously found to attenuate cognitive deterioration in a mouse model of Alzheimer's disease (AD). Plasma pharmacokinetic response of major GSPE phenolic components was measured following intragastric gavage of 50, 100, and 150 mg GSPE per kg body weight. Liquid chromatography-mass spectrometry (LC-MS) analysis identified gallic acid (GA), catechin (C), and epicatechin (EC) in plasma of rats gavaged acutely with GSPE. Additionally, 4-methylgallic acid (4-OMeGA), 3'-methylcatechin (3'-OMeC), and 3'-methylepicatechin (3'-OMeEC) were identified as circulating metabolites of GSPE phenolic constituents. Cmax for individual GSPE constituents and their metabolites increased in a dose-dependent fashion (with increasing GSPE oral dose). Repeated daily exposure to GSPE was found to significantly increase bioavailability (defined as plasma AUC0-8h) of GA, C, and EC by 198, 253, and 282% relative to animals receiving only a single acute GSPE dose. EC and C were not detectable in brain tissues of rats receiving a single GSPE dose but reached levels of 290.7 ± 45.9 and 576.7 ± 227.7 pg/g in brain tissues from rats administered GSPE for 10 days. This study suggests that brain deposition of GA, C, and EC is affected by repeated dosing of GSPE.
APO-1 (Fas/CD95), a member of the tumor necrosis factor receptor superfamily, induces apoptosis upon receptor oligomerization. In a search to identify intracellular signaling molecules coupling to oligomerized APO-1, several cytotoxicity-dependent APO-1-associated proteins (CAP) were immunoprecipitated from the apoptosis-sensitive human leukemic T cell line HUT78 and the lymphoblastoid B cell line SKW6.4. CAP1-3 (27-29 kDa) and CAP4 (55 kDa), instantly detectable after the crosslinking of APO-1, were associated only with aggregated (the signaling form of APO-1) and not with monomeric APO-1. CAP1 and CAP2 were identified as serine phosphorylated MORT1/FADD. The association of CAP1-4 with APO-1 was not observed with C-terminally truncated non-signaling APO-1. In addition, CAP1 and CAP2 did not associate with an APO-1 cytoplasmic tail carrying the lprcg amino acid replacement. Moreover, no APO-1-CAP association was found in the APO-1+, anti-APO-1-resistant pre-B cell line Boe. Our data suggest that in vivo CAP1-4 are the APO-1 apoptosis-transducing molecules.
A number of apoptosis-inducing agents used in cancer therapy (etoposide, doxorubicin, 1-beta-D-arabinofuranosylcytosine), as well as the proapoptotic second messenger ceramide, induce a disruption of the mitochondrial transmembrane potential (delta psi m) that precedes nuclear DNA fragmentation. This effect has been observed in tumor cell lines of T-lymphoid, B-lymphoid, and myelomonocytic origin in vitro. Circulating tumor cells from patients receiving chemotherapy in vivo also demonstrate a delta psi m disruption after in vitro culture that precedes nuclear apoptosis. Transfection-enforced hyperexpression of the proto-oncogenes bcl-2 and bcl-XL protects against chemotherapy-induced apoptosis, at both the level of the mitochondrial dysfunction preceding nuclear apoptosis and the level of late nuclear apoptotic events. Bcl-2-mediated inhibition of ceramide-induced delta psi m disruption is observed in normal as well as anucleate cells, indicating that bcl-2 acts on an extranuclear pathway of apoptosis. In contrast to Bcl-2 and Bcl-XL, hyperexpression of the protease inhibitor cytokine response modifier A fails to protect tumor cells against chemotherapy-induced delta psi m disruption and apoptosis, although cytokine response modifier A does prevent the delta psi m collapse and posterior nuclear apoptosis triggered by cross-linking of Fas/Apo-1/CD95. In conclusion, delta psi m disruption seems to be an obligatory step of early (pre-nuclear) apoptosis, and delta psi m is stabilized by two members of the bcl-2 gene family conferring resistance to chemotherapy.
In this study we sought to clarify the role of the proapoptotic potential of mitochondria in the death pathway emanating from the TRAIL (APO-2L) and CD95 receptors in pancreatic carcinoma cells. We focused on the role of the Bcl-2 family member Bcl-XL, using three pancreatic carcinoma cell lines as a model system, two of which have high (Panc-1, PancTuI) and one has low (Colo357) Bcl-XL expression. In these cell lines, the expression of Bcl-XL correlated with sensitivity to apoptosis induced by TRAIL or anti-CD95. Flow cytometric analysis revealed cell surface expression of TRAIL-R1 and TRAIL-R2 on PancTuI and Colo357, and TRAIL-R2 on Panc-1 cells. In Colo357 cells retrovirally transduced with Bcl-XL, caspase-8 activation in response to treatment with TRAIL or anti-CD95 antibody was not different from parental cells and EGFP-transfected controls, however, apoptosis was completely suppressed as measured by the mitochondrial transmembrane potential deltapsim, caspase-3 activity (PARP cleavage) and DNA-fragmentation. Inhibition of Bcl-XL function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-XL mRNA resulted in sensitization of Panc-1 cells to TRAIL and PancTuI cells to anti-CD95 antibody-induced cell death. The results show that Bcl-XL can protect pancreatic cancer cells from CD95- and TRAIL-mediated apoptosis. Thus, in these epithelial tumour cells the mitochondrially mediated 'type II' pathway of apoptosis induction is not only operative regarding the CD95 system but also regarding the TRAIL system.
Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.
Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 'apoptosome', which seems to amplify rather than initiate the caspase cascade.
Caspase-3 is a member of the cysteine protease family, which plays a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins. Caspase-3 can be activated by diverse death-inducing signals, including the chemotherapeutic agents. The purpose of this study was to determine the levels of caspase-3 expression in breast tumor samples and to determine whether alterations in its expression can affect their ability to undergo apoptosis. Primary breast tumor and normal breast parenchyma samples were obtained from patients undergoing breast surgery and the expression of caspases-3 was studied. Similarly, normal mammary epithelial cells and several established mammary cancer cell lines were studied for caspases-3 expression by reverse transcriptase-polymerase chain reaction, Northern blot analysis, and Western blot analysis. Approximately 75% of the tumor as well as morphologically normal peritumoral tissue samples lacked the caspase-3 transcript and caspase-3 protein expression. In addition, the caspases-3 mRNA levels in commercially available total RNA samples from breast, ovarian, and cervical tumors were either undetectable (breast and cervical) or substantially decreased (ovarian). Despite the complete loss of caspase-3, the expression levels of other caspases, such as caspase-8 and caspase-9, were normal in all of the tumor samples studied. The sensitivity of caspase-3-deficient breast cancer (MCF-7) cells to undergo apoptosis in response to doxorubicin and other apoptotic stimuli could be augmented by reconstituting caspase-3 expression. These results suggest that the loss of caspases-3 expression may represent an important cell survival mechanism in breast cancer patients.
Silymarin, a defined mixture of natural flavonoid, has recently been shown to have potent cancer chemopreventive efficacy against colon carcinogenesis in rat model; however, the mechanism of such efficacy is not elucidated. Here, using pure active agent in silymarin, namely silibinin, we show its antiproliferative and apoptotic effects, and associated molecular alterations in human colon carcinoma HT-29 cells. Silibinin treatment of cells at 50-100 microg/ml doses resulted in a moderate to very strong growth inhibition in a dose- and a time-dependent manner, which was largely due to a G0/G1 arrest in cell cycle progression; higher dose and longer treatment time also caused a G2/M arrest. In mechanistic studies related its effect on cell cycle progression, silibinin treatment resulted in an upregulation of Kip1/p27 and Cip1/p21 protein as well as mRNA levels, and decreased CDK2, CDK4, cyclin E and cyclin D1 protein levels together with an inhibition in CDK2 and CDK4 kinase activities. In other studies, we observed that G2/M arrest by silibinin was associated with a decrease in cdc25C, cdc2/p34 and cyclin B1 protein levels, as well as cdc2/p34 kinase activity. In the studies assessing biological fate of silibinin-treated cells, silibinin-induced cell cycle arrest and growth inhibition were not associated with cellular differentiation, but caused apoptotic death. The quantitative apoptosis analysis showed up to 15% apoptotic cell death after 48 h of silibinin treatment. Interestingly, silibinin-induced apoptosis in HT-29 cells was independent of caspases activation, as all caspases inhibitor did not reverse silibinin-induced apoptosis. This observation was further confirmed by the findings showing a lack in caspases activity increase and caspases and PARP cleavage as well as a lack in cytochrome c release in cytosol following silibinin treatment of HT-29 cells. Additional studies conducted in mice showed that silibinin doses found effective in HT-29 cells are achievable in plasma, which increases the significance of the present findings and their possible translation in in vivo anticancer efficacy of silibinin against colon cancer. Together, these results identify molecular mechanisms of silibinin efficacy as a cell cycle regulator and apoptosis inducer in human colon carcinoma HT-29 cells, and justify further studies to investigate potential usefulness of this nontoxic agent in colon cancer prevention and intervention.
Proteasome inhibitors exhibit antitumor activity against malignancies of different histology. Yet, the mechanisms underlying this effect are poorly understood. Recent evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, possibly reducing their cytotoxicity. These include the Bcl-2 family member Mcl-1, whose down-regulation has been proposed to initiate apoptosis in response to genotoxic stimuli. In this study, we found that proteasome inhibitors release cyotochrome c and second mitochondria-derived activator of caspase (SMAC)/Diablo and trigger the subsequent apoptotic cascade in spite of concomitant Mcl-1 increase. However, our data indicate that subtraction of Mcl-1 during apoptosis, although not required for early release of proapoptotic factors, is probably relevant in speeding up cell demise, since RNA interference-mediated Mcl-1 silencing is lethal in lymphoma cells. Consistent with this, the cytotoxic effects of proteasome inhibitors are enhanced when Mcl-1 increase is impeded. Thus, this study identifies Mcl-1 accumulation as an unwanted molecular consequence of exposure to proteasome inhibitors, which slows down their proapoptotic effects. Pharmacologic or genetic approaches targeting Mcl-1, including therapeutic RNAi, may increase the effectiveness of these compounds.
Recent studies have indicated that lipid rafts (LRs) in the cell membrane are clustered in response to different stimuli to form signaling platforms for transmembrane transduction. It remains unknown whether this LR clustering participates in redox signaling in endothelial cells. The present study tested a hypothesis that clustering of LRs on the membrane of coronary endothelial cells produces aggregation and activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, thereby forming a redox signaling platform. By confocal microscopic analysis of agonist-stimulated rafts patch formation, we found that several death receptor ligands or apoptotic factors, including tumor necrosis factor alpha, Fas ligand, or endostatin, stimulated the clustering and trafficking of individual LRs on the plasma membrane of coronary endothelial cells. Interestingly, double labeling of a membrane-bound NADPH oxidase subunit, gp91phox, and LRs showed that gp91phox colocalized within the LR patches when endothelial cells were stimulated by Fas ligand. In isolated LR fractions from Fas-stimulated endothelial cells, gp91phox, p47phox (a crucial cytosolic regulatory subunit of NADPH oxidase), and Rac GTPase were markedly increased and blocked by nystatin, a compound that disrupts LRs. These clustered LRs contained high NADPH oxidase activity, which increased in response to Fas stimulation. Functionally, Fas ligand-induced inhibition of endothelium-dependent vasorelaxation was reduced if LRs were disrupted or NADPH oxidase was inhibited. These results suggest that LR clustering occurs in coronary endothelial cells. The formation of redox signaling platforms on the cell membrane mediates transmembrane signaling of death receptors, resulting in endothelial dysfunction.
Quantifiable, well‐characterized cancer risk factors demonstrate the need for chemoprevention and define cohorts for chemopreventive intervention. For chemoprevention, the important cancer risk factors are those that can be measured quantitatively in the subject at risk. These factors, called risk biomarkers, can be used to identify cohorts for chemoprevention. Those modulated by chemopreventive agents may also be used as endpoints in chemoprevention studies. Generally, the risk biomarkers fit into categories based on those previously defined by Hulka: 1) carcinogen exposure, 2) carcinogen exposure/effect, 3) genetic predisposition, 4) intermediate biomarkers of cancer, and 5) previous cancers.
Besides their use in characterizing cohorts for chemoprevention trials, some risk biomarkers can be modulated by chemopreventive agents. These biomarkers may be suitable surrogate endpoints for cancer incidence in chemoprevention intervention trials. The criteria for risk biomarkers defining cohorts and serving as endpoints are the same, except that those defining cohorts are not necessarily modulated by chemopreventive agents. A primary criterion is that the biomarkers fit expected biological mechanisms of early carcinogenesis—i.e., differential expression in normal and high‐risk tissue, on or closely linked to the causal pathway for the cancer, and short latency compared with cancer. They must occur in sufficient number to allow their biological and statistical evaluation. Further, the biomarkers should be assayed reliably and quantitatively, measured easily, and correlated to cancer incidence. Particularly important for cancer risk screening in normal subjects is the ability to use noninvasive techniques that are highly specific, sensitive, and quantitative.
Since carcinogenesis is a multipath process, single biomarkers are difficult to correlate to cancer, as they may appear on only one or a few of the many possible causal pathways. As shown in colorectal carcinogenesis, the risks associated with the presence of biomarkers may be additive or synergistic. That is, the accumulation of genetic lesions is the more important determinant of colorectal cancer compared with the presence of any single lesion. Thus, batteries of biomarker abnormalities, particularly those representing the range of carcinogenesis pathways, may prove more useful than single biomarkers both in characterizing cohorts at risk and defining modulatable risks.
Risk biomarkers are already being integrated into many chemoprevention intervention trials. One example is the phase II trial of oltipraz inhibition of carcinogen‐DNA adducts in a Chinese population exposed to aflatoxin B1. Also, urine samples from subjects in this trial will be screened for the effect of oltipraz on urinary mutagens. A second example is a chemoprevention protocol developed for patients at high risk for breast cancer; the cohort is defined both by hereditary risk and the presence of biomarker abnormalities. Modulation of the biomarker abnormalities is a proposed endpoint. Also, dysplastic lesions, such as prostatic intraepithelial neoplasia, oral leukoplakia and colorectal adenomas, have been used to define high‐risk cohorts and as potential modulatable surrogate endpoints in chemoprevention trials. J. Cell. Biochem. 25S:1–14. © 1997 Wiley‐Liss, Inc.
• 1 This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
The Breast Cancer Association Consortium (BCAC) has been established to conduct combined case-control analyses with augmented statistical power to try to confirm putative genetic associations with breast cancer. We genotyped nine SNPs for which there was some prior evidence of an association with breast cancer: CASP8 D302H (rs1045485), IGFBP3 -202 C --> A (rs2854744), SOD2 V16A (rs1799725), TGFB1 L10P (rs1982073), ATM S49C (rs1800054), ADH1B 3' UTR A --> G (rs1042026), CDKN1A S31R (rs1801270), ICAM5 V301I (rs1056538) and NUMA1 A794G (rs3750913). We included data from 9-15 studies, comprising 11,391-18,290 cases and 14,753-22,670 controls. We found evidence of an association with breast cancer for CASP8 D302H (with odds ratios (OR) of 0.89 (95% confidence interval (c.i.): 0.85-0.94) and 0.74 (95% c.i.: 0.62-0.87) for heterozygotes and rare homozygotes, respectively, compared with common homozygotes; P(trend) = 1.1 x 10(-7)) and weaker evidence for TGFB1 L10P (OR = 1.07 (95% c.i.: 1.02-1.13) and 1.16 (95% c.i.: 1.08-1.25), respectively; P(trend) = 2.8 x 10(-5)). These results demonstrate that common breast cancer susceptibility alleles with small effects on risk can be identified, given sufficiently powerful studies.
Nature Genetics publishes the very highest quality research in genetics. It encompasses genetic and functional genomic studies on human traits and on other model organisms, including mouse, fly, nematode and yeast. Current emphasis is on the genetic basis for common and complex diseases and on the functional mechanism, architecture and evolution of gene networks, studied by experimental perturbation.
Quantifiable, well-characterized cancer risk factors demonstrate the need for chemoprevention and define cohorts for chemopreventive intervention. For chemoprevention, the important cancer risk factors are those that can be measured quantitatively in the subject at risk. These factors, called risk biomarkers, can be used to identify cohorts for chemoprevention. Those modulated by chemopreventive agents may also be used as endpoints in chemoprevention studies. Generally, the risk biomarkers fit into categories based on those previously defined by Hulka: 1) carcinogen exposure, 2) carcinogen exposure/effect, 3) genetic predisposition, 4) intermediate biomarkers of cancer, and 5) previous cancers.
Grape seeds accumulate in huge quantities as byproduct during wine production and are therefore a cheap source for pharmacologically active agents. However, studies prove poor antibacterial activity, and results of analyses are sometimes contradictory. The aim of this study was, thus, to determine the antibacterial activity of grape seed extracts with special focus on the chromatographic characterization of active fractions. In the course of these investigations, extraction protocols were optimized so that microwave-assisted extraction (MAE) guaranteed highest preconcentration efficiency. Proanthocyanidins, monomeric flavonoid aglycones, as well as some of their glycosides could be identified within yielded extracts via high-performance liquid chromatography-mass spectrometry (HPLC-MS). By that means the coherence number of possible isomers of procyanidins was approximated by a newly developed equation. As far as antibacterial activity determined via screening tests is concerned, the extracts generally have been found to be positively responsive toward 10 different gram-positive and gram-negative bacteria strains. After fractionation of the raw extracts, proanthocyanidins P2, P3, P4 and gallate esters P2G and P3G (P = proanthocyanidin consisting of catechin and epicatechin units, n = oligomerization degree, G = gallate ester) were determined as active antibacterial agents toward 10 different pathogens. Only moderate activity was found for monomeric flavonoid fractions.
The success of current treatment strategies is limited by the development of therapy resistance as evidenced by recurrence of the primary tumor or distant metastasis. Eradication of primary and metastatic disease requires interventions at both the cancer cell and tumor microenvironment levels. In this review, we will discuss mechanisms that are intrinsic to cancer cells, and those that are mediated by the tumor microenvironment as contributors to drug resistance. Mechanisms contributing to multidrug resistance phenotype and the challenges facing molecular targeted therapy are discussed. The DNA damage tolerance pathway confers tolerance to a variety of structurally and functionally unrelated drugs. A rationale for targeting the DNA damage tolerance pathway as a novel tool for overcoming drug resistance is discussed. We have also addressed the need for employing clinically relevant model systems for performing drug sensitivity evaluations. These model systems must take into account the three-dimensional organization and in vivo relationship of tumor with its microenvironment. Such integrative efforts would not only yield a more global understanding of the tumor- and microenvironment-derived mechanisms involved in emergence of drug resistance but would also provide novel therapeutic targets that will disrupt the interactions between the tumor cells and its microenvironment.
Dietary antioxidants may provide a cost-effective strategy to promote health in obesity by targeting oxidative stress and inflammation. We recently found that the antioxidant-rich grape skin extract (GSE) also exerts a novel anti-hyperglycemic activity. This study investigated whether 3-month GSE supplementation can improve oxidative stress, inflammation, and hyperglycemia associated with a Western diet-induced obesity. Young diet-induced obese (DIO) mice were randomly divided to three treatment groups (n = 12): a standard diet (S group), a Western high fat diet (W group), and the Western diet plus GSE (2.4 g GSE/kg diet, WGSE group). By week 12, DIO mice in the WGSE group gained significantly more weight (24.6 g) than the W (20.2 g) and S groups (11.2 g); the high fat diet groups gained 80% more weight than the standard diet group. Eight of 12 mice in the W group, compared to only 1 of 12 mice in the WGSE group, had fasting blood glucose levels above 140 mg/dL. Mice in the WGSE group also had 21% lower fasting blood glucose and 17.1% lower C-reactive protein levels than mice in the W group (P < 0.05). However, the GSE supplementation did not affect oxidative stress in diet-induced obesity as determined by plasma oxygen radical absorbance capacity, glutathione peroxidase, and liver lipid peroxidation. Collectively, the results indicated a beneficial role of GSE supplementation for improving glycemic control and inflammation in diet-induced obesity.
Abnormalities in cell cycle progression provide unlimited replicative potential to cancer cells, and therefore targeting of key cell cycle regulators could be a sound cancer chemopreventive strategy. Earlier, we found that grape seed extract (GSE) increases Cip/p21 protein level and inhibits growth and induces apoptosis in human colon carcinoma HT29 cells both in vitro and in vivo. However, the mechanism of GSE-induced p21 upregulation and its role in biological efficacy of GSE are not known, which were investigated here. GSE treatment of HT29 cells resulted in a strong dose- and time-dependent phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), consistent with p21 induction. The inhibition of sustained ERK1/2 activation by GSE using pharmacological inhibitors abrogated GSE-induced p21 upregulation. Furthermore, pretreatment of cells with N-acetylcysteine inhibited GSE-induced ERK1/2 phosphorylation as well as p21 upregulation, suggesting the involvement of GSE-induced oxidative stress as an upstream event. Consistent with this, GSE also decreased intracellular level of reduced glutathione. Next, we determined whether GSE-induced signaling regulates p21 expression at transcriptional and/or translational levels. GSE was found to increase the stability of p21 message with resultant increase in p21 protein level, but it did not alter the protein stability to a great extent. Importantly, knock-down of p21 abrogated GSE-induced G(1) arrest suggesting that p21 induction by GSE is essential for its G(1) arrest effect. Together, our results for the first time identify a central role of p21 induction and associated mechanism in GSE-induced cell cycle arrest in HT29 cells.
The incidence of colorectal cancer (CRC) has been increasing during the past decades, and the lifetime risk for CRC in industrialised countries is about 5%. CRC is a good candidate for screening, because it is a disease with high prevalence, has recognised precursors, and early treatment is beneficial. This paper outlines the evidence for efficacy from randomised trials for the most commonly used CRC screening tests to reduce CRC incidence and mortality in the average-risk population. Four randomised trials have investigated the effect of guaiac-based fecal occult blood screening on CRC mortality, with a combined CRC mortality risk reduction of 15-17% in an intention-to-screen analysis, and 25% for those people who attended screening. Flexible sigmoidoscopy screening has been evaluated in three randomised trials. The observed reduction in CRC incidence varied between 23 and 80%, and between 27 and 67% for CRC mortality, respectively (intention-to-screen analyses) in the trials with long follow-up time. No randomised trials exist in other CRC screening tools, included colonoscopy screening. FOBT and flexible sigmoidoscopy are the two CRC screening methods which have been tested in randomised trials and shown to reduce CRC mortality. These tests can be recommended for CRC screening.
Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six-week-old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in pre- and post-AOM or only post-AOM experimental protocols. At 16 wk of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis, and molecular analyses by immunohistochemistry. GSE-feeding caused strong chemopreventive efficacy against AOM-induced ACF formation in terms of up to 60% (P < 0.001) reduction in number of ACF and 66% (P < 0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE-feeding inhibited AOM-induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX-2, iNOS, and survivin levels. Additional studies showed that GSE-feeding also decreased AOM-caused increase in beta-catenin and NF-kappaB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was nontoxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of beta-catenin and NF-kappaB signaling, and suggest its potential usefulness for the prevention of human CRC.
Consumption of grape seed extract (GSE) is widely marketed as a dietary supplement and is considered safe for human health. Nevertheless, the analytical composition of GSE from different grape cultivars, growing in special agronomic constraints, differs greatly in flavan-3-ols content. The major concern with GSE studies is a lack of availability of uniformly standardised preparations, which raises an important question whether different GSE samples have comparable activity and trigger the same mechanisms of action on a given biological system. Therefore, it is tempting to speculate that GSE, obtained from different cultivars, could exert differentiated anticancer effects. The focus of the present study is to determine the selective biological efficacy of GSE obtained from three different sources on the human colon cancer cell line Caco-2. Irrespective of its source, high doses of GSE induced a significant inhibition on Caco-2 cell growth. Moreover, apoptosis was enhanced through both caspase-dependent and caspase-independent mechanisms, leading to an early apoptosis-inducing factor release and, further, to a dramatic increase in caspase 7 and 3 activity. However, a significant difference in apoptotic rates induced by the three grape sources clearly emerged when treating cancer cells with low and intermediate GSE concentrations (25 and 50 microg/ml).
With emerging trends in the incidence of cancer of various organ sites, additional approaches are needed to control human malignancies. Intervention or prevention of cancer by dietary constituents, a strategy defined as chemoprevention, holds great promise in our conquest to control cancer, because it can be implemented on a broader population base with less economic burden. Consistent with this, several epidemiological studies have shown that populations that consume diets rich in fruits and vegetables have an overall lower cancer incidence. Based on these encouraging observations, research efforts from across the globe have focused on identifying, characterizing, and providing scientific basis to the efficacy of various phytonutrients in an effort to develop effective strategy to control various human malignancies. Cancer induction, growth, and progression are multi-step events and numerous studies have demonstrated that various dietary agents interfere with these stages of cancer, thus blocking malignancy. Fruits and vegetables represent untapped reservoir of various nutritive and nonnutritive phytochemicals with potential cancer chemopreventive activity. Grapes and grape-based products are one such class of dietary products that have shown cancer chemopreventive potential and are also known to improve overall human health. This review focuses on recent advancements in cancer chemopreventive and anticancer efficacy of grape seed extract and other grape-based products. Overall, completed studies from various scientific groups conclude that both grapes and grape-based products are excellent sources of various anticancer agents and their regular consumption should thus be beneficial to the general population.
Grape seed procyanidins (GSP) can inhibit cell proliferation and tumorigenesis, and induce apoptosis in human breast, prostate, skin and colorectal carcinoma cell lines.
In order to study the mechanism of apoptosis, four colorectal cell lines, HT-29, SW-480, LoVo and Colo 320DM, were used. GSP-treated cells were assessed for viability by trypan blue exclusion, for loss of mitochondrial membrane potential by rhodamine 123 staining, for increased apoptosis by annexin V labeling, and for changes in the levels of proteins involved in apoptosis by immunoblotting.
GSP had no significant pro-apoptotic effect on the Colo 320DM cell line. In HT-29, SW-480 and LoVo cells, GSP (12.5-50 mg/l) inhibited proliferation in a dose-dependent manner. In these three lines, GSP treatment increased the proportion of rhodamine 123-negative cells and annexin V-positive cells, while immunoblotting revealed increased levels of apoptosis activation protein, caspase-3 and the cleavage fragment of PARP (a caspase-3 substrate), but the level of Bcl-2 did not change.
GSP inhibited the proliferation of some colorectal carcinoma cell lines and was associated with an apoptotic mechanism involving a loss of mitochondrial membrane potential and caspase-3 activation in these cells.
Proanthocyanidin rich plant extracts derived from grape seed extract (GSE), hawthorn and cranberry are on markets for their preventive effects against cardiovascular diseases and uroinfections in woman. However, the importance of these health beneficial effects of these botanicals remains elusive due to incomplete understanding of uptake, metabolism and bioavailability of proanthocyanidins in vivo. In the present study rats were given GSE orally (300 mg/kg, twice a day) and blood and urine were collected over a 24 h period. Monomeric catechins and their methylated metabolites, and proanthocyanidins up to trimers were detected in blood samples treated with GSE using LC-MS/MS operating in the multiple reaction monitoring (MRM) mode. A new tetramethylated metabolite of dimeric proanthocyanidin (m/z 633) in GSE-treated urine was tentatively identified. Using LC-MS/MS, (+)-catechin and (-)-epicatechin were identified in the brain conclusively. These data suggested that GSE catechins cross the blood brain barrier and may be responsible for the neuroprotective effects of GSE.
One approach to control colorectal cancer (CRC) is its preventive intervention by dietary agents or those consumed as supplements. However, because most of these products are often consumed by patients as an complementary and alternative medicine practice, a scientific base such as efficacy, mechanism, and standardized preparation needs to be developed. Grape seed extract (GSE) is one such supplement widely consumed by humans for its several health benefits. We reported recently that GSE inhibits CRC cell HT29 growth in culture and nude mice xenograft. Because GSE is available commercially through different vendors, here we assessed whether GSE from 2 different manufacturers produces comparable biological effects in a panel of human CRC cell lines. Our results show that irrespective of source, GSE strongly inhibits LoVo, HT29, and SW480 cell growth, with a G1 arrest in LoVo and HT29 cells but an S and/or G2/M arrest in SW480 cell cycle progression. GSE also induced Cip/p21 levels in all 3 cell lines. Furthermore, an induction of apoptosis was observed in all 3 cell lines by GSE. Taken together, our findings suggest that GSE could be an effective CAM agent against CRC possibly due to its strong growth inhibitory and apoptosis-inducing effects.
Quantifiable, well-characterized cancer risk factors demonstrate the need for chemoprevention and define cohorts for chemopreventive intervention. For chemoprevention, the important cancer risk factors are those that can be measured quantitatively in the subject at risk. These factors, called risk biomarkers, can be used to identify cohorts for chemoprevention. Those modulated by chemopreventive agents may also be used as endpoints in chemoprevention studies. Generally, the risk biomarkers fit into categories based on those previously defined by Hulka: 1) carcinogen exposure, 2) carcinogen exposure/effect, 3) genetic predisposition, 4) intermediate biomarkers of cancer, and 5) previous cancers. Besides their use in characterizing cohorts for chemoprevention trials, some risk biomarkers can be modulated by chemopreventive agents. These biomarkers may be suitable surrogate endpoints for cancer incidence in chemoprevention intervention trials. The criteria for risk biomarkers defining cohorts and serving as endpoints are the same, except that those defining cohorts are not necessarily modulated by chemopreventive agents. A primary criterion is that the biomarkers fit expected biological mechanisms of early carcinogenesis-i.e., differential expression in normal and high-risk tissue, on or closely linked to the causal pathway for the cancer, and short latency compared with cancer. They must occur in sufficient number to allow their biological and statistical evaluation. Further, the biomarkers should be assayed reliably and quantitatively, measured easily, and correlated to cancer incidence. Particularly important for cancer risk screening in normal subjects is the ability to use noninvasive techniques that are highly specific, sensitive, and quantitative. Since carcinogenesis is a multipath process, single biomarkers are difficult to correlate to cancer, as they may appear on only one or a few of the many possible causal pathways. As shown in colorectal carcinogenesis, the risks associated with the presence of biomarkers may be additive or synergistic. That is, the accumulation of genetic lesions is the more important determinant of colorectal cancer compared with the presence of any single lesion. Thus, batteries of biomarker abnormalities, particularly those representing the range of carcinogenesis pathways, may prove more useful than single biomarkers both in characterizing cohorts at risk and defining modulatable risks. Risk biomarkers are already being integrated into many chemoprevention intervention trials. One example is the phase II trial of oltipraz inhibition of carcinogen-DNA adducts in a Chinese population exposed to aflatoxin B1. Also, urine samples from subjects in this trial will be screened for the effect of oltipraz on urinary mutagens. A second example is a chemoprevention protocol developed for patients at high risk for breast cancer; the cohort is defined both by hereditary risk and the presence of biomarker abnormalities. Modulation of the biomarker abnormalities is a proposed endpoint. Also, dysplastic lesions, such as prostatic intraepithelial neoplasia, oral leukoplakia and colorectal adenomas, have been used to define high-risk cohorts and as potential modulatable surrogate endpoints in chemoprevention trials.
Proanthocyanidins, a group of polyphenolic bioflavonoids, have been reported to exhibit a wide range of biological, pharmacological and chemoprotective properties against oxygen free radicals. We have assessed the concentration-dependent oxygen free radical scavenging abilities of a grape seed proanthocyanidin extract (GSPE), vitamin C and vitamin E succinate (VES) as well as superoxide dismutase, catalase and mannitol against biochemically generated superoxide anion and hydroxyl radical using a chemiluminescence assay and cytochrome c reduction. A concentration-dependent inhibition was demonstrated by GSPE. At a 100 mg/l concentration, GSPE exhibited 78-81% inhibition of superoxide anion and hydroxyl radical. Under similar conditions, vitamin C inhibited these two oxygen free radicals by approximately 12-19%, while VES inhibited the two radicals by 36-44%. The combination of superoxide dismutase and catalase inhibited superoxide anion by approximately 83%, while mannitol resulted in an 87% inhibition of hydroxyl radical. The results demonstrate that GSPE is a more potent scavenger of oxygen free radicals as compared to vitamin C and VES.
The observation that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family, induces apoptosis in a number of different tumor cell types led us to compare the tumoricidal effects of TRAIL to those of other TNF family molecules on human melanoma cells. We found that a high proportion of the melanoma cell lines tested were killed by TRAIL, whereas all the melanoma lines were resistant to the other TNF family cytokines tested. TRAIL-induced death was characterized by caspase activation and cellular protein cleavage within minutes of TRAIL addition, and death could be completely inhibited by the caspase inhibitors Ile-Glu-Thr-Asp (IETD) and Val-Ala-Asp (VAD), indicating the presence of a TRAIL receptor signaling pathway similar to that identified for Fas and TNF receptors. Specific TRAIL receptor expression was determined by RT-PCR, and the presence of mRNA encoding the "protective" TRAIL receptors did not correspond to resistance or sensitivity to TRAIL-induced apoptosis. Addition of protein synthesis inhibitors to TRAIL-resistant melanomas rendered them sensitive to TRAIL, indicating that the presence or the absence of intracellular apoptosis inhibitors may mediate resistance or sensitivity to TRAIL-mediated apoptosis. Expression of one such inhibitor, FLICE-inhibitory protein (FLIP), was highest in the TRAIL-resistant melanomas, while being low or undetectable in the TRAIL-sensitive melanomas. Furthermore, addition of actinomycin D to TRAIL-resistant melanomas resulted in decreased intracellular concentrations of FLIP, which correlated with their acquisition of TRAIL sensitivity. Collectively, our results indicate that TRAIL-induced apoptosis occurs through a caspase signaling cascade and that resistance is controlled by intracellular regulators of apoptosis.
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a potent inducer of death of cancer but not normal cells, which suggests its potential use as a tumor-specific antineoplastic agent. TRAIL binds to the proapoptotic death receptors DR4 and the p53-regulated proapoptotic KILLER/DR5 as well as to the decoy receptors TRID and TRUNDD. In the present studies, we identified a subgroup of TRAIL-resistant cancer cell lines characterized by low or absent basal DR4 or high expression of the caspase activation inhibitor FLIP. Four of five TRAIL-sensitive cell lines expressed high levels of DR4 mRNA and protein, whereas six of six TRAIL-resistant cell lines expressed low or undetectable levels of DR4 (chi 2; P < 0.01). FLIP expression appeared elevated in five of six (83%) TRAIL-resistant cell lines and only one of five (20%) TRAIL-sensitive cells (chi 2; P < 0.05). Two TRAIL-resistant lines that expressed DR4 contained an A-to-G alteration in the death domain encoding arginine instead of lysine at codon 441. The K441R polymorphism is present in 20% of the normal population and can inhibit DR4-mediated cell killing in a dominant-negative fashion. The expression level of KILLER/DR5, TRID, TRUNDD or TRID, and TRUNDD did not correlate with TRAIL sensitivity (P > 0.05). These results suggest that the major determinants for TRAIL sensitivity may be the expression level of DR4 and FLIP. TRAIL-resistant cells became susceptible to TRAIL-mediated apoptosis in the presence of doxorubicin. In TRAIL-sensitive cells, caspases 8, 9, and 3 were activated after TRAIL treatment, but in TRAIL-resistant cells, they were activated only by the combination of TRAIL and doxorubicin. Our results suggest: (a) evaluation of tumor DR4 and FLIP expression and host DR4 codon 441 status could be potentially useful predictors of TRAIL sensitivity, and (b) doxorubicin, in combination with TRAIL, may effectively promote caspase activation in TRAIL-resistant tumors.
Resistance of normal cells to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis is believed to be mediated by expression of two decoy receptors. Here we show that the expression and localisation of TRAIL receptors (TRAIL-Rs) vary between different cells and that resistance to TRAIL is mediated by different mechanisms. The decoy receptor, TRAIL-R3, appeared important in protection of endothelial cells, whereas lack of surface death receptor expression and as yet unknown intracellular inhibitor(s) of apoptosis downstream of caspase-3 may play a major role in protection of melanocytes and fibroblasts from TRAIL induced apoptosis, respectively. Differential subcellular location of decoy receptors may be an important determinant of their effectiveness in different types of normal cells.
Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines.
In this study we analyzed the expression and function of TRAIL, its agonistic and antagonistic receptors, and important intracellular signaling elements in 18 NB cell lines.
Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cFLIP. Surprisingly, caspase-8 and caspase-10 mRNA was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner.
Treatment with 5-aza-2'-deoxycytidine restored mRNA expression of caspase-8 and -10 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. Since many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.
The p53 tumor suppressor limits cellular proliferation by inducing cell cycle arrest and apoptosis in response to cellular stresses such as DNA damage, hypoxia, and oncogene activation. Many apoptosis-related genes that are transcriptionally regulated by p53 have been identified. These are candidates for implementing p53 effector functions. In response to oncogene activation, p53 mediates apoptosis through a linear pathway involving bax transactivation, Bax translocation from the cytosol to membranes, cytochrome c release from mitochondria, and caspase-9 activation, followed by the activation of caspase-3, -6, and -7. p53-mediated apoptosis can be blocked at multiple death checkpoints, by inhibiting p53 activity directly, by Bcl-2 family members regulating mitochondrial function, by E1B 19K blocking caspase-9 activation, and by caspase inhibitors. Understanding the mechanisms by which p53 induces apoptosis, and the reasons why cell death is bypassed in transformed cells, is of fundamental importance in cancer research, and has great implications in the design of anticancer therapeutics.
Cellular stresses, such as growth factor deprivation, DNA damage or oncogene expression, lead to stabilization and activation of the p53 tumour suppressor protein. Depending on the cellular context, this results in one of two different outcomes: cell cycle arrest or apoptotic cell death. Cell death induced through the p53 pathway is executed by the caspase proteinases, which, by cleaving their substrates, lead to the characteristic apoptotic phenotype. Caspase activation by p53 occurs through the release of apoptogenic factors from the mitochondria, including cytochrome c and Smac/DIABLO. Released cytochrome c allows the formation of a high-molecular weight complex, the apoptosome, which consists of the adapter protein Apaf-1 and caspase 9, which is activated following recruitment into the apoptosome. Active caspase 9 then cleaves and activates the effector caspases, such as caspases-3 and -7, which execute the death program. Released Smac/DIABLO facilitates caspase activation through repression of the IAP caspase inhibitor proteins. The release of mitochondrial apoptogenic factors is regulated by the pro- and anti-apoptotic Bcl-2 family proteins, which either induce or prevent the permeabilization of the outer mitochondrial membrane. The mechanism by which p53 signals to the Bcl-2 family proteins is unclear. It was shown that some of the pro-apoptotic family members, such as Bax, Noxa or PUMA, are transcriptional targets of p53. In addition, transcription-independent, pro-apoptotic activities of p53 have been described. The elucidation of the p53-dependent pathway, resulting in mitochondrial outer membrane permeabilization through the pro-apoptotic Bcl-2 family proteins, is a key to unveiling the mechanism of stress-induced apoptosis.
Every cell in a multicellular organism has the potential to die by apoptosis, but tumour cells often have faulty apoptotic pathways. These defects not only increase tumour mass, but also render the tumour resistant to therapy. So, what are the molecular mechanisms of tumour resistance to apoptosis and how can we use this knowledge to resensitize tumour cells to cancer therapy?
Grape seed extract (GSE), rich in the bioflavonoids commonly known as procyanidins, is one of the most commonly consumed dietary supplements in the United States because of its several health benefits. Epidemiological studies show that many prostate cancer (PCA) patients use herbal extracts as dietary supplements in addition to their prescription drugs. Accordingly, in recent years, we have focused our attention on assessing the efficacy of GSE against PCA. Our studies showed that GSE inhibits growth and induces apoptotic death of human PCA cells in culture and in nude mice. Here, we performed detailed studies to define the molecular mechanism of GSE-induced apoptosis in advanced human PCA DU145 cells. GSE treatment of cells at various doses (50-200 micro g/ml) for 12-72 h resulted in a moderate to strong apoptotic death in a dose- and time-dependent manner. In the studies assessing the apoptotic-signaling pathway induced by GSE, we observed an increase in cleaved fragments of caspases 3, 7 and 9 as well as PARP in GSE-treated cells after 48 and 72 h of treatment. Pre-treatment of cells with general caspases inhibitor, z-Val-Ala-Asp(OMe)-FMK or caspase 3-like proteases inhibitor [z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK], almost completely (approximately 90%) inhibited the GSE-induced apoptotic cell death. In a later case, GSE-induced caspase-3 activity was completely inhibited. Selective caspase 9 inhibitor [z-Leu-Glu(OMe)-His-Asp(OMe)-FMK] showed only partial inhibition of GSE-induced apoptosis whereas GSE-induced protease activity of caspase 9 was completely inhibited. Upstream of caspase cascade, GSE showed disappearance of mitochondrial membrane potential and an increase in cytochrome c release in cytosol. Together, these results suggest that GSE possibly causes mitochondrial damage leading to cytochrome c release in cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic death of human PCA DU145 cells. Furthermore, GSE-caused caspase 3-mediated apoptosis also involves other pathway(s) including caspase 9 activation.
A loss of functional androgen receptor and an enhanced expression of growth factor receptors and associated ligands are causal genetic events in prostate cancer (PCA) progression. These genetic alterations lead to an epigenetic mechanism where a feedback autocrine loop between membrane receptor and ligand (e.g. EGFR-TGFalpha) results in a constitutive activation of MAPK-Elk1-AP1-mediated mitogenic signaling in human PCA at an advanced and androgen-independent stage. We rationalized that inhibiting these epigenetic events could be useful in controlling advanced PCA growth. Recently, we found that grape seed extract (GSE), a dietary supplement rich in flavonoid procyanidins, inhibits advanced and androgen-independent human PCA DU145 cell growth in culture and nude mice. Here, we performed detailed mechanistic studies to define the effect of GSE on EGFR-Shc-MAPK-Elk1-AP1-mediated mitogenic signaling in DU145 cells. Pretreatment of serum-starved cells with GSE resulted in 70% to almost complete inhibition of EGF-induced EGFR activation and 50% to complete inhibition of Shc activation, which corroborated with a comparable decrease in EGF-induced Shc binding to EGFR. Conversely, EGF-induced ERK1/2 phosphorylation was inhibited only by lower doses of GSE; in fact, higher doses showed an increase. Additional studies showed that GSE alone causes a dose- and time-dependent increase in ERK1/2 phosphorylation in starved DU145 cells that is inhibited by an MEK1 inhibitor PD98059. Independent of this increase in ERK1/2 phosphorylation, GSE showed a strong inhibition of ERK1/2 kinase activity to Elk1 in both cellular and cell-free systems. GSE treatment of cells also inhibited both EGF-induced and constitutively active Elk1 phosphorylation and AP1 activation. GSE treatment also showed DNA synthesis inhibition in starved and EGF-stimulated cells as well as loss of cell viability and apoptotic death that was further increased by adding MEK1 inhibitor. Since GSE strongly induced apoptosis independent of its affect on an increase in phospho-ERK1/2, we hypothesized that apoptotic effect of GSE could be by other mechanism(s) including its effect on stress-associated MAPK, the JNK. Indeed, GSE-treated cells showed a strong and sustained increase in phospho-JNK1/JNK2 levels, JNK activity and phospho-cJun levels. An inhibition of GSE-induced JNK activation by a novel JNK inhibitor SP600125 resulted in a significant reversal of GSE-induced apoptotic death suggesting the involvement of JNK activation by GSE in its apoptosis response. Together, these results suggest that anticancer effects of GSE in PCA be mediated via impairment of EGFR-ERK1/2-Elk1-AP1-mediated mitogenic signaling and activation of JNK causing growth inhibition and apoptosis, respectively.
There has been evidence that dysregulation of apoptosis is involved in the pathogenesis of cancer development. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis. The aim of this study was to explore the possibility that mutation of the caspase-8 gene might be involved in the development of colorectal cancer.
We analyzed the entire coding region of the caspase-8 gene for the detection of somatic mutations in 180 colorectal tumors (98 invasive carcinomas and 82 adenomas) by polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing.
Overall, we detected a total of 5 somatic mutations in 98 invasive carcinomas (5.1%), but no mutations were detected in 82 adenomas (0%). The frequency of caspase-8 mutation in the carcinomas was significantly higher than that in adenomas (P < 0.05). The 5 mutations consisted of 1 frameshift, 1 nonsense mutation, and 3 missense mutations. We expressed the 5 tumor-derived caspase-8 mutants and found that 3 of the 5 mutations markedly decreased apoptosis activity of caspase-8. Furthermore, expression of the inactivating caspase-8 mutants interfered with apoptosis by death receptor overexpression, indicating that these mutants have dominant-negative inhibition of the death receptor-induced apoptosis.
The presence of caspase-8 mutation in colon carcinomas suggests that caspase-8 gene mutation might lead to the loss of its apoptotic function and contribute to the pathogenesis of colorectal carcinomas, especially at the late stage of colorectal carcinogenesis.
Tumor samples were fixed in 10% buffered formalin for 12 hr and processed conventionally. The paraffin-embedded tumor sections (5 μm thick) were heat immobilized, and deparaffinized using xylene and rehydrated in a graded series of ethanol with a final wash in distilled water. Antigen retrieval was done with 10 mM citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by immersing the sections in 3.0 % H2O2 in methanol (v/v). The sections were then incubated with mouse monoclonal anti-PCNA antibody IgG2a (Dako, Carpinteria, CA), 1:400 in PBS for 1 hr at 37°C in humidity chamber. Negative controls were treated only with PBS under identical conditions. The sections were then incubated with biotinylated rabbit anti-mouse antibody IgG (1:200 in 10% normal rabbit serum) followed by conjugated horseradish peroxidase streptavidin (Dako). The sections were then incubated with 3,3′-diaminobenzidine (Sigma, St. Louis, MO) working solution for 10 min at room temperature and counterstained with diluted Harris hematoxylin (Sigma). Finally, sections were viewed and photographed under inverted Nikon TE-300 microscope equipped with a Princeton Instrument Micromax camera. Images are acquired with Image Pro-plus software (Media Cybernetics, Silver Spring, MD). Proliferating cells were quantified by counting the PCNA-positive cells and the total number of cells at 10 arbitrarily selected fields at 400× magnification in a blinded manner. The proliferation index (per 400× microscope field) was determined as number of PCNA-positive cells × 100/total number of cells.
The present study is focused on the investigation of in vitro angiogenic potential of grape seed extract (GSE). Human umbilical vein endothelial cells (HUVEC) in culture were used to assess the effect of GSE on proliferation, survival, matrix metalloproteinases (MMPs) secretion and capillary tube formation. Our data show that GSE significantly inhibited cell growth (< or =91%, P<0.001) and cell viability (< or =64%, P<0.005) of HUVEC. Further studies by BrdU incorporation and annexin V staining showed that GSE strongly inhibits DNA synthesis (< or =76%, P<0.001) and induces apoptotic cell death (< or =42.8% versus control 2.6%, P<0.05) in HUVEC, respectively. Similar GSE treatment decreased secreted levels of MMP-2 from HUVEC. GSE also inhibited capillary tube formation on Matrigel by endothelial cells in a dose-dependent manner. These findings suggest that GSE possesses an anti-angiogenic potential, which is associated with its antiproliferative, proapoptotic and inhibition of MMP-2 secretion in endothelial cells. Further studies are warranted to evaluate the in vivo anti-angiogenic efficacy of GSE for its possible usefulness in the inhibition of tumor angiogenesis.
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, considerable numbers of cancer cells, especially some highly malignant tumors, are resistant to apoptosis induction by TRAIL, and some cancer cells that were originally sensitive to TRAIL-induced apoptosis can become resistant after repeated exposure (acquired resistance). Understanding the mechanisms underlying such resistance and developing strategies to overcome it are important for the successful use of TRAIL for cancer therapy. Resistance to TRAIL can occur at different points in the signaling pathways of TRAIL-induced apoptosis. Dysfunctions of the death receptors DR4 and DR5 due to mutations can lead to resistance. The adaptor protein Fas-associated death domain (FADD) and caspase-8 are essential for assembly of the death-inducing signaling complex, and defects in either of these molecules can lead to TRAIL resistance. Overexpression of cellular FADD-like interleukin-1beta-converting enzyme-inhibitory protein (cFLIP) correlates with TRAIL resistance in several types of cancer. Overexpression of Bcl-2 or Bcl-X(L), loss of Bax or Bak function, high expression of inhibitor of apoptosis proteins, and reduced release of second mitochondria-derived activator of caspases (Smac/Diablo) from the mitochondria to the cytosol have all been reported to result in TRAIL resistance in mitochondria-dependent type II cancer cells. Finally, activation of different subunits of mitogen-activated protein kinases or nuclear factor-kappa B can lead to development of either TRAIL resistance or apoptosis in certain types of cancer cells.
Several lines of evidence indicate that deregulation of apoptosis is involved in the mechanisms of cancer development. Caspase-8 activation plays a central role in the initiation phase of apoptosis. The aim of this study was to explore the possibility that genetic alteration of CASPASE-8 gene is involved in the development of human cancers, including gastric cancers. We have analyzed the entire coding region of human CASPASE-8 gene for the detection of somatic mutations in 162 gastric carcinomas (40 early and 122 advanced cancers), 185 non-small cell lung cancers, 93 breast carcinomas, and 88 acute leukemias by PCR-single-strand conformation polymorphism. Of the cancers analyzed, 13 cancers harbored CASPASE-8 somatic mutations. Interestingly, all of the mutations were detected in the advanced gastric cancers (10.7% of the 122 samples). We expressed the tumor-derived caspase-8 mutants in 293T, 293, and HT1080 cells and found that most of the mutants (9 of the 10 mutations tested) markedly decreased the cell death activity of caspase-8. In addition, in the cells with the inactivating caspase-8 mutants, cleavage of poly(ADP-ribose)polymerase was markedly reduced compared with that of wild-type caspase-8. The occurrence of CASPASE-8 mutation and the inactivation of cell death activity by the mutants suggest that CASPASE-8 gene mutation may affect the pathogenesis of gastric cancers, especially at the late stage of gastric carcinogenesis.
Previously, we showed that the proteasome inhibitor bortezomib/Velcade (formerly PS-341) synergizes with the protein tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL), a ligand for certain death receptors, to induce apoptosis in cell lines derived from prostate and colon cancers. Because apoptosis is often triggered by BH3-only proteins of the Bcl-2 family, we have explored the hypothesis that bortezomib contributes to the apoptosis by up-regulating their levels. Indeed, bortezomib induced increases of Bik and/or Bim in multiple cell lines but not notably of two other BH3-only proteins (Puma and Bid) nor other family members (Bax, Bak, Bcl-2, and Bcl-xL). The increase in Bik levels seems to reflect inhibition by bortezomib of its proteasome-mediated degradation. Importantly, both Bik and Bim seem central to the proapoptotic function of bortezomib, because mouse embryo fibroblasts in which the genes for both Bik and Bim had been disrupted were refractory to its cytotoxic action. Similarly, the synergy between bortezomib and TRAIL in killing human prostate cancer cells was impaired in cells in which both Bik and Bim were down-regulated by RNA interference. Further evidence that bortezomib acts through the mitochondrial pathway regulated by the Bcl-2 family is that deficiency for APAF-1, which acts downstream of Bcl-2, also blocked its apoptotic effect. These results implicate BH3-only proteins, in particular both Bik and Bim, as important mediators of the antitumor action of bortezomib and establish their role in its enhancement of TRAIL-induced apoptosis.
Mounting evidence indicates that deregulation of apoptosis is involved in the mechanisms of cancer development. Caspase-9 plays a crucial role in the initiation phase of the intrinsic apoptosis pathway. To explore the possibility that the genetic alterations of caspase-9 might be involved in the development of human cancers, we analyzed the entire coding region and all splice sites of the human caspase-9 gene for the detection of somatic mutations in a series of 353 cancers, including 180 gastric, 104 colorectal and 69 lung adenocarcinomas. Overall, we detected three somatic mutations of caspase-9, but all of the mutations were silent mutations. The mutations were observed in 2 of 104 colorectal carcinomas and 1 of 180 gastric carcinomas. These data indicate that the caspase-9 gene is rarely mutated in gastric, colorectal and lung adenocarcinomas, and suggest that caspase-9 gene mutation may not contribute to the pathogenesis of these cancers.
Prostate cancer is the second leading cancer diagnosed in elderly males in the Western world. Epidemiologic studies suggest that dietary modifications could be an effective approach in reducing various cancers, including prostate cancer, and accordingly cancer-preventive efficacy of dietary nutrients has gained increased attention in recent years. We have recently shown that grape seed extract (GSE) inhibits growth and induces apoptotic death of advanced human prostate cancer DU145 cells in culture and xenograft. Because prostate cancer is initially an androgen-dependent malignancy, here we used LNCaP human prostate cancer cells as a model to assess GSE efficacy and associated mechanisms. GSE treatment of cells led to their detachment within 12 hours, as occurs in anoikis, and caused a significant decrease in live cells mostly due to their apoptotic death. GSE-induced anoikis and apoptosis were accompanied by a strong decrease in focal adhesion kinase levels, but an increase in caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage; however, GSE caused both caspase-dependent and caspase-independent apoptosis as evidenced by cytochrome c and apoptosis-inducing factor release into cytosol. Additional studies revealed that GSE causes DNA damage-induced activation of ataxia telangiectasia mutated kinase and Chk2, as well as p53 Ser(15) phosphorylation and its translocation to mitochondria, suggesting this to be an additional mechanism for apoptosis induction. GSE-induced apoptosis, cell growth inhibition, and cell death were attenuated by pretreatment with N-acetylcysteine and involved reactive oxygen species generation. Together, these results show GSE effects in LNCaP cells and suggest additional in vivo efficacy studies in prostate cancer animal models.
Little has been published regarding clinical predictors of severe toxicity in patients with metastatic colorectal cancer (CRC) treated with combination chemotherapy (CT) with oxaliplatin and/or irinotecan.
We analyzed retrospectively 142 patients treated between 1996 and 2004 in our center with these regimes with regards to grade 3-4 toxicity and overall survival (OS) rates. Köhne's prognostic classification could be applied in all patients.
Köhne classification: good (54.2%), intermediate (26.8%), and poor prognosis (19%). 50.4% received irinotecan-based CT. Median number of cycles 6 with a total response rate of 38.9%. 23.2% stopped first-line CT due to toxicity. 50.7% suffered grade 3-4 toxicity: digestive (28.2%), hematologic (19.7%), and fatigue (25.4%). 7.7% episodes of neutropenic fever with 4.9% toxic deaths. 70.9% of grade 3-4 episodes occurred in the first four cycles. Median follow-up of 33.9 mo; median OS of 15.9 mo. For Köhne classification: good (20 mo), intermediate (15.8 mo), and poor (6.8 mo). Toxicity analysis: female sex and age > 70 yr predicted higher overall grade 3-4 toxicity, with no differences in CT efficacy; age > 70 yr and PS > 1 predicted higher grade 3-4 fatigue. No relationship could be found between baseline laboratory characteristics and higher toxicity, except baseline hemoglobin and grade 3-4 hematologic toxicity.
Female and elderly patients have a higher grade 3-4 toxicity rate when treated with combination CT with oxaliplatin or irinotecan. Prognostic classifications such as Köhne's can help differentiate subgroups of patients who benefit little with the use of combination CT.