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Modulation of apoptosis by chemopreventive agents
Abstract
A review of almost 2000 studies showed that the large majority of 39 putative cancer chemopreventive agents induced "spontaneous" apoptosis. Inhibition of the programmed cell death triggered by a variety of stimuli was consistently reported only with ascorbic acid, alpha-tocopherol, and N-acetylcysteine (NAC). We performed experimental studies in rodents exposed to cigarette smoke, either mainstream (MCS) or environmental (ECS), and UV-A/B-containing light. The nonsteroidal anti-inflammatory drug sulindac did not affect the apoptotic process in the skin of light-exposed mice and in the lungs of ECS-exposed mice. Likewise, 5,6-benzoflavone, indole-3-carbinol, 1,2-dithiole-3-thione and oltipraz failed to modulate apoptosis in the respiratory tract of ECS-exposed rats. Phenethyl isothiocyanate further enhanced the frequency of apoptosis in pulmonary alveolar macrophages and bronchial epithelial cells, and upregulated several genes in the lung of ECS-exposed rats. Both individually and in combination with oltipraz, NAC inhibited apoptosis in the respiratory tract of rats exposed either to MCS or ECS. Moreover, NAC attenuated the ECS-related overexpression of proapoptotic genes and normalized the levels of proapoptotic proteins in rat lung. The transplacental administration of NAC to mice considerably attenuated gene overexpression in the liver of fetuses exposed to ECS throughout pregnancy. Inhibition of apoptosis by chemopreventive agents reflects their ability to counteract certain upstream signals, such as genotoxic damage, redox imbalances, and other forms of cellular stress that trigger apoptosis. On the other hand, enhancement of apoptosis is a double-edged sword, since it represents a protective mechanism in carcinogenesis but may contribute to the pathogenesis of other degenerative diseases. We suggest that stimulation of apoptosis by so many chemopreventive agents, as reported in the literature, may often reflect the occurrence of toxic effects at high doses.
... In these studies, we used Sprague-Dawley rats and mice belonging to various strains and genotypes, including B6-129(F 1 ) mice, either wild type or Fhit +/À ; SKH-1 hairless mice, A/J mice, and (UL53-3 Â A/J)F 1 mice, either wild type or P53 +/À . Part of these animals have been used in parallel studies evaluating the occurrence of ECS-related induction of lung tumors and modulation of intermediate biomarkers in cells of the respiratory tract (18)(19)(20)(21)(22)(23). In addition, in one of the herein reported studies, we exposed hairless mice not only to ECS but also to ECS plus light or light alone, which induced skin tumors in mice (24,25) as well as molecular and biochemical alterations not only in skin but, surprisingly, even in the respiratory tract and bone marrow cells (19,21). ...
... Interestingly, in the same rats used in the present study, N-acetylcysteine and its combination with oltipraz were the only treatments capable of significantly decreasing the frequency of apoptotic cells in the bronchial/bronchiolar epithelium. In contrast, phenethyl isothiocyanate showed an opposite trend, and all other treatments had no significant effect (23). These data are in line with the results of other studies showing a decrease by N-acetylcysteine and an increase by phenethyl isothiocyanate of apoptotic PAM in rats exposed to ECS, and a decrease by N-acetylcysteine of apoptotic cells in the bronchial/bronchiolar epithelium of rats exposed to mainstream cigarette smoke (23). ...
... In contrast, phenethyl isothiocyanate showed an opposite trend, and all other treatments had no significant effect (23). These data are in line with the results of other studies showing a decrease by N-acetylcysteine and an increase by phenethyl isothiocyanate of apoptotic PAM in rats exposed to ECS, and a decrease by N-acetylcysteine of apoptotic cells in the bronchial/bronchiolar epithelium of rats exposed to mainstream cigarette smoke (23). Enhancement of apoptosis is conceptually a double-edged sword, because it provides a protective mechanism in carcinogenesis but may contribute to the pathogenesis of other degenerative diseases. ...
The Fhit gene, encompassing the most active common human chromosomal fragile region, FRA3B, has been shown to act as a tumor suppressor. Several studies have shown significant Fhit alterations or Fhit protein loss in lung cancers from smokers compared with lung cancers from nonsmokers. To evaluate the role of Fhit under controlled experimental conditions, we exposed rodents to environmental cigarette smoke (ECS) and evaluated Fhit expression or Fhit protein in the respiratory tract. After 14 days of exposure to ECS, loss of Fhit protein in the bronchial/bronchiolar epithelium affected half of the tested B6-129(F(1)) mice, either wild type or Fhit(+/-). After 28 days, it affected the vast majority of the tested SKH-1 hairless mice and of A/J mice and all (UL53-3 x A/J)F(1) mice, either wild type or P53(+/-). In Sprague-Dawley rats, exposure to ECS for up to 30 days caused a time-dependent loss of Fhit in pulmonary alveolar macrophages. Moreover, ECS down-regulated Fhit expression and significantly decreased Fhit protein in the rat bronchial epithelium. The oral administration of N-acetylcysteine attenuated the ECS-related loss of Fhit, whereas oltipraz, 5,6-benzoflavone, phenethyl isothiocyanate, and indole 3-carbinol, and their combinations had no significant effect. Parallel studies evaluated a variety of molecular, biochemical, and cytogenetic alterations in the respiratory tract of the same animals. In conclusion, there is unequivocal evidence that Fhit is an early, critical target in smoke-related lung carcinogenesis in rodents, and that certain chemopreventive agents can attenuate the occurrence of this gene alteration.
... Finally, all studies evaluating apoptosis (D'Agostini et al. 2001(D'Agostini et al. , 2005Johnston et al. 2000;Kirichenko et al. 1996;Lam et al. 2002;Leigh et al. 1997;Ortiz et al. 1998;Thakur and Sanyal 2010;Wang et al. 1997bWang et al. , 2005Zhao et al. 2004a) have shown a significant increase following the different genotoxic treatments. ...
... Finally, two studies showed a significant inhibition of apoptosis induced by cigarette smoke in rats after oral administration of various pharmacological agents (D'Agostini et al. 2001(D'Agostini et al. , 2005. In another study, administration of diclofenac caused an increase in apoptosis in the PAMs of rats treated with dimethylbenz(a)anthracene. In this case, however, the increase in apoptosis may represent an enhancement of the protective mechanism rather than an expression of genotoxic damage (Thakur and Sanyal 2010). ...
DNA damage is one of the primary mechanisms underlying cancer and other chronic degenerative diseases. Early evaluation of this damage in the affected cells and tissues is crucial for understanding pathogenesis and implementing effective prevention strategies. However, isolating target cells from affected organs, such as the lungs, can be challenging. Therefore, an alternative approach is to evaluate genotoxic damage in surrogate cells. Pulmonary alveolar macrophages are ideally suited for this purpose because they are in close contact with the target cells of the bronchial and alveolar epithelium, share the exact mechanisms and levels of exposure, and are easily recoverable in large numbers. This review comprehensively lists all studies using alveolar macrophages as surrogate cells to show genotoxic lung damage in humans or laboratory animals. These investigations provide fundamental information on the mechanisms of DNA damage in the lung and allow for better assessment and management of risk following exposure to inhalable genotoxic agents. Furthermore, they may be a valuable tool in cancer chemoprevention, helping the right choice of agents for clinical trials.
... ITCs have drawn the attention of the scientific community since the mid-1980s due to their anticancer potential (2)(3)(4)(5). ITCs inhibit carcinogenesis by several mechanisms including (I) inhibition of carcinogen metabolism by drug-metabolizing enzymes (DMEs) (6), (II) apoptosis of cancer cells (7), and (III) cell cycle inhibition (8). Recent evidence also suggests that ITCs are anti-angiogenic, and this may contribute to their cancer preventive effects (9). ...
... 47). Of all the ITCs, PEITC is one of the most comprehensively studied ITCs in various cancers (5,7,(11)(12)(13)(14) and is currently being evaluated in a National Cancer Institute phase II clinical trial to prevent lung cancer in smokers. The beneficial role of PEITC in lung cancer (6) and prostate cancer (13) has been documented. ...
Phenethyl isothiocyanate (PEITC)-a naturally occurring isothiocyanate in cruciferous vegetables-has been extensively studied as a chemopreventive agent in several preclinical species and in humans. Pharmacokinetic features of unchanged PEITC are (I) linear and first-order absorption, (II) high protein binding and capacity-limited tissue distribution, and (III) reversible metabolism and capacity-limited hepatic elimination. Membrane transport of PEITC is mediated by BCRP, multidrug resistance-associated protein (MRP) 1, and MRP2 transporters belonging to the ATP-binding-cassette (ABC) family. PEITC is metabolized by glutathione S-transferase (GST) in the liver, with the glutathione conjugate of PEITC undergoing further conversion to mercapturic acid by N-acetyl transferase in rats and humans. PEITC modulates the activity and expression of numerous phase I and phase II drug-metabolizing enzymes and can inhibit the metabolism of procarcinogens to form carcinogens and increase carcinogen elimination. In recent years, several in vitro and in vivo studies have elucidated molecular mechanisms underlying the pharmacodynamics of PEITC in breast cancer that include cancer cell apoptosis by upregulation of apoptotic genes, cell cycle arrest at G2/M phase by generation of reactive oxygen species and depletion of intracellular glutathione, downregulation of the estrogen receptor, decrease in sensitivity to estrogen, and inhibition of tumor metastasis. Inhibition of angiogenesis is one of the recently reported mechanisms of breast cancer prevention by PEITC. Complex pharmacokinetics and pharmacodynamics of PEITC necessitate a systems-biology approach in parallel with PK/PD modeling to develop PEITC as a therapeutic agent for treating cancers.
... Therefore, the cell proliferation rate is crucial in discriminating whether DNA alterations will evolve towards a proliferative disease or a genuinely degenerative disease [7]. (e) Cell apoptosis: This mechanism is another double-edged sword, since apoptosis has a protective meaning when it occurs in damaged proliferating cells but is detrimental in perennial cells [8]. ...
... The protective effect of budesonide is likely to be ascribed to the antiinflammatory properties of glucocorticoids [68]. NAC has antiinflammatory properties and inhibits triggering of apoptosis consequent to DNA damage and redox imbalances [8,22]. ...
Genomic and postgenomic changes are extensively investigated in cancer research. Similar alterations, affecting genome, transcriptome, mirnome and/or proteome end-points, have been detected in a variety of other chronic degenerative diseases, such as atherosclerosis, degenerative heart diseases, chronic obstructive pulmonary diseases, neurological disorders, eye diseases, diabetes, metabolic syndrome, skin ageing and alopecia. No generalization can be made due to the myriad of diverse clinical entities classified as chronic degenerative diseases. Moreover, the detection of molecular changes does not automatically imply their causal role. Nevertheless, common mechanisms, such as DNA damage, epigenetic alterations, oxidative stress, and chronic inflammation, in addition to genetic predisposition, are often involved in noncancer diseases. We debate here in more detail the subjects of cardiovascular diseases and of skin diseases. Moreover, we discuss our experimental studies suggesting that genomic and postgenomic changes do also occur during critical periods of life, including the prenatal life, the perinatal period, and ageing. In addition, we comment on the finding that stem-derived cells are more susceptible to molecular damage than more differentiated cells. All these data are viewed in the perspective of preventive medicine. In fact, there is evidence that the genomic and postgenomic alterations occurring not only in several pathological conditions but also in paraphysiological situations that affect critical periods of life can be modulated by means of dietary and pharmacological agents. The discovery that chemopreventive agents are also able to attenuate nucleotide damage in stem-derived cells warrants further studies in view of possible clinical applications.
... While a broad literature is available regarding modulation of apoptosis by NAC in various cell types, either in vitro or in vivo [28], the ability of L-cystine to inhibit apoptosis has been so far demonstrated only in vitro [29][30][31][32]. On the contrary, this activity had never been investigated earlier in vivo. ...
... Therefore, it could be hypothesized that the protective effects exerted by oral administration of L-cystine against ECSinduced alopecia, as demonstrated in the present study, should be mainly ascribed to a variety of mechanism which inhibit apoptosis. Presumably, Lcystine does not possess direct anti-apoptotic properties but, like NAC and other chemopreventive agents, it works upstream by counteracting apoptosis-triggering events [28]. ...
We previously demonstrated that high doses of environmental cigarette smoke (ECS) induce alopecia in mice. This effect was prevented by the oral administration of N-acetylcysteine (NAC), an analogue and precursor of L-cysteine and reduced glutathione.
The present study aimed at assessing whether L-cystine, the oxidized form of L-cysteine, which is a key hair component, may behave like NAC in inhibiting ECS-induced alopecia and modulating the mechanisms responsible for this condition.
C57BL/6 mice were exposed whole-body to ECS in a smoking machine. Groups of mice received in the diet, at three dose levels, a mixture of L-cystine with vitamin B6, which plays a role in L-cystine incorporation in hair cells. Occurrence of alopecia areas and apoptosis of hair bulb cells were evaluated for up to 6 months of exposure, and the time course induction of micronucleated erythrocytes in peripheral blood was investigated.
The frequency of micronucleated erythrocytes was increased by ECS, irrespective of treatment with L-cystine/vitamin B6. ECS-induced alopecia and apoptosis of hair bulb cells in all exposed mice. L-Cystine/vitamin B6 inhibited alopecia in a dose-dependent fashion.
High-dose ECS induces apoptosis-related alopecia in mice, and oral administration of L-cystine/vitamin B6 is an effective preventive treatment.
... It has been shown that oltipraz stimulates transcription of the mitochondrial superoxide dismutase (manganese SOD; Mn-SOD) gene through the increase of ROS [32] and it has also been shown to inhibit apoptosis [33]. In mammalian cells, the mitochondria are a major source of reactive oxygen species [34]. ...
... On a further point of discussion, it has been demonstrated that oltipraz inhibited apoptosis in the respiratory tract of rats exposed to cigarette smoke [33]. It is interesting to note that conformational change in cyt c is an early event in apoptosis [68]. ...
The major metabolite of the cancer chemopreventive agent oltipraz, a pyrrolopyrazine thione (PPD), has been shown to be a phase 2 enzyme inducer, an activity thought to be key to the cancer chemopreventive action of the parent compound. In cells, mitochondria are the major source of reactive oxygen species (ROS) and cytochrome c (cyt c) is known to participate in mitochondrial electron transport and confer antioxidant and peroxidase activities. To understand possible mechanisms by which PPD acts as a phase 2 enzyme inducer, a study of its interaction with cyt c was undertaken. UV-visible spectroscopic results demonstrate that PPD is capable of reducing oxidized cyt c. The reduced cyt c is stable for a long period of time in the absence of an oxidizing agent. In the presence of ferricyanide, the reduced cyt c is rapidly oxidized back to its oxidized form. Further, UV-visible spectroscopic studies show that during the reduction process the coordination environment and redox state of iron in cyt c are changed. Low-temperature EPR studies show that during the reduction process, the heme iron changes from a low-spin state of s = 1/2 to a low-spin state of s = 0. Room-temperature EPR studies demonstrate that PPD inhibits the peroxidase activity of cyt c. EPR spin trapping experiments using DMPO show that PPD inhibits the superoxide radical scavenging activity of oxidized cyt c. From these results, we propose that PPD interacts with cyt c, binding to and then reducing the heme, and this may enhance ROS levels in mitochondria. This in turn could contribute to the mechanism by which the parent compound, oltipraz, might trigger the cancer chemopreventive increase in transcription of phase 2 enzymes. The modifications of cyt c function by the oltipraz metabolite may have implications for the regulation of apoptotic cell death.
... These agents should also meet specific requirements, including 1) low price, as defined by standard costing and the target population, 2) pragmatism of use, as determined by accessibility, storage environments, Frontiers in Pharmacology frontiersin.org and route of administration, 3) efficacy, and 4) safety (Kelloff et al., 2004;Ferguson et al., 2004;D'Agostini et al., 2005). The Latin word primum non-nocere serves as a reminder that the most important condition for a medical treatment is that it not cause harm to healthy people. ...
Cancer is commonly thought to be the product of irregular cell division. According to the World Health Organization (WHO), cancer is the major cause of death globally. Nature offers an abundant supply of bioactive compounds with high therapeutic efficacy. Anticancer effects have been studied in a variety of phytochemicals found in nature. When Food and Drug Administration (FDA)-approved anticancer drugs are combined with natural compounds, the effectiveness improves. Several agents have already progressed to clinical trials based on these promising results of natural compounds against various cancer forms. Natural compounds prevent cancer cell proliferation, development, and metastasis by inducing cell cycle arrest, activating intrinsic and extrinsic apoptosis pathways, generating reactive oxygen species (ROS), and down-regulating activated signaling pathways. These natural chemicals are known to affect numerous important cellular signaling pathways, such as NF-B, MAPK, Wnt, Notch, Akt, p53, AR, ER, and many others, to cause cell death signals and induce apoptosis in pre-cancerous or cancer cells without harming normal cells. As a result, non-toxic “natural drugs” taken from nature’s bounty could be effective for the prevention of tumor progression and/or therapy of human malignancies, either alone or in combination with conventional treatments. Natural compounds have also been shown in preclinical studies to improve the sensitivity of resistant cancers to currently available chemotherapy agents. To summarize, preclinical and clinical findings against cancer indicate that natural-sourced compounds have promising anticancer efficacy. The vital purpose of these studies is to target cellular signaling pathways in cancer by natural compounds.
... Programmed cell death via apoptosis cascade is necessary for the proper development of multicellular organisms. The morphological and biochemical changes in the apoptosis pathway are chromatin condensation, inter nucleosomal DNA fragmentation, the origination of apoptotic bodies, and cell shrinkage [32]. Apoptosis can destroy genetically modified, pre-cancerous, or cancerous cells. ...
In developing and developed countries, cancer is a significant health problem in people. Cancer becomes the second greatest cause of death in human after cardiovascular disease. However, significant advancements in modern cancer therapies have a beneficial impact on survival, chemotherapy and radiation therapy. Plants fulfill our basic needs to continue life and provide natural products that help to cure disease. The medicinal plants are readily available and have no toxicity as compared to modern drugs. Phytochemicals act on metabolic pathways and inhibit tumor growth, the development of cancerous cells, and replication by different mechanisms. Apigenin's chemo-preventive and anticancer activities have been demonstrating in numerous studies. Curcumin is a polyphenolic compound isolated from the Curcuma longa plant. EGCG, a polyphenol in black, white, and green tea is a chemo-preventive effect against many cancers by targeting multiple pathways. Normal cell growth and cell proliferation are closely regulated processes. The JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway controls gene expression during different processes, including proliferation, initiation, and apoptosis. The transcription factors are associated with the growth of cancer cells and control a cellular function in the disease. Mitogen-activated protein kinase (MAPK) is a class of serine and threonine kinase that includes ERK (extracellular regulated kinase), JNK (c-Jun N-terminal kinases), and p38. This review paper describes natural phytochemical compounds, their molecular targets and mechanisms of action.
... In vitro studies have also shown antimicrobial and anthelmintic activities. Moreover, it has an important anticancer function, increasing the occurrence of apoptosis of cancer cells (Kermanshai et al., 2001;D`agostini et al., 2005;Morant et al., 2008;Volden et al., 2008;Sofrata et al., 2011). Koriem et al. (2010), dosed the fatty acids content in the leaves and flowers of T. majus's methyl alcohol extract through liquid chromatography/mass spectra (LC/MS). ...
Tropaeolum majus presents medicinal, nutritional and ornamental value. Plant extracts and fractions have been found to exhibit diuretic, antihypertensive, anti-inflammatory, antimicrobial and antioxidant activities. Moreover, protective effects on blood and liver, scurvy’s treatment, antithrombin activity and prevention against macular degeneration have also been observed. T. majus contains biologically active compounds such as flavonoids, glucosilonates, fatty acids, essential oil, chlorogenic acid, aminoacids, cucurbitacins, proteins and carotenoids. Acute and subchronic studies demonstrated a lack of toxic effects, but the extracts of this plant can have deleterious consequences during the pregnancy. The revised databases were SciELO, PubMed, ScienceDirect and Portal da Capes, considering studies between 1963 and 2014 and by searching for terms like Tropaeolum majus, Tropaeolaceae, Tropaeolum majus constituents, Tropaeolum majus use and Tropaeolum majus toxicity.
... The main mechanism of PEITC and other isothiocyanates is that these agents modify the metabolism of carcinogens both by inhibiting phase I CYP activities involved in the activation of procarcinogens and by inducing phase II detoxifying enzymes [107]. In addition, PEITC affects multigene expression in the lung of smoke-exposed rats [108] and is also an inducer of apoptosis [109]. PEITC significantly reduced both lung tumor multiplicity and incidence in mice treated with NNK but failed to affect the weak tumorigenicity of ECS in A/J mice [110]. ...
Many drugs in common use possess pleiotropic properties that make them capable of interfering with carcinogenesis mechanisms. We discuss here the ability of pharmacological agents to mitigate the pulmonary carcinogenicity of mainstream cigarette smoke. The evaluated agents include anti-inflammatory drugs (budesonide, celecoxib, aspirin, naproxen, licofelone), antidiabetic drugs (metformin, pioglitazone), antineoplastic agents (lapatinib, bexarotene, vorinostat), and other drugs and supplements (phenethyl isothiocyanate, myo-inositol, N-acetylcysteine, ascorbic acid, berry extracts). These drugs have been evaluated in mouse models mimicking interventions either in current smokers or in ex-smokers, or in prenatal chemoprevention. They display a broad spectrum of activities by attenuating either smoke-induced preneoplastic lesions or benign tumors and/or malignant tumors. Together with epidemiological data, these findings provide useful information to predict the potential effects of pharmacological agents in smokers.
... The major component described in the bark's extracts is the condurango glycoside A (7), which is capable of inducing DNA damage resulting in reduction of the cell viability of HeLa cervical cancer cells, reactive oxygen species (ROS) induction and subsequent p53 activation, and induction of cell death through apoptosis (Bishayee et al., 2013). A large amount of coumarins and flavonoids have been isolated from the bark of M. condurango (Table 2), many possesses antitumor activity (Gurib-Fakim, 2006;Agostini et al., 2005), as the case of quercetin un flavonol (Ji et al., 2009). Quercetin (8) is an efficient anticancer agent that induces apoptosis in HeLa cervical cancer cells, HT29 colon carcinoma (Xavier et al., 2009) and A431 epidermal carcinoma cells with the modulation of tyrosine kinase epidermal growth factor receptor (EGFR). ...
Ecuador is well known for its biodiversity and its ancient richness. This review provides an overview of Ecuadorian plant species in terms of the ethnobotany and chemistry of natural products in relation to anticancer activity. Plant species were classified into two groups: (a) Ethnomedical species with confirmed antitumor activity and (b) species indigenous to Ecuador with anticancer potential. This review shows that there is a great chemical diversity in Ecuadorian plants that can be used for potential antitumor therapeutics, and chemical and biological analysis confirms the biomedical use of the plant-derived compounds as cytotoxic agents for cancer cells.
Graphical Abstract
... Most of the chemopreventive agents exert their anticancer effects by inducing apoptosis [40]. Here we show that BA, NA and CAG alone or in combination also induce the apoptotic pathways against DMBA induced mouse skin tumorigenesis by modulating the anti and pro-apoptotic proteins of the Bcl-2 family even before the tumor developed, at 4 weeks and these changes progressed with the appearance of the tumor, at 16 weeks. ...
We explored the basis of the combinatorial chemopreventive effect of Butyric acid (BA), Nicotinamide (NA) and Calcium Glucarate (CAG) on mouse skin exposed to 7, 12-dimethylbenz (a) anthracene (DMBA). We studied the effects of topical application of DMBA in the presence or absence of BA, NA and CAG on the regulators of apoptosis. DMBA treatment suppressed Bax, Bax/Bcl-2 ratio, release of cyt c, Apaf1, caspase-9, -3 mediated apoptosis. Downregulation of p21 and upregulation of Bcl-2, mut p53 were also observed in only DMBA treated mice. Simultaneous application of BA, NA and CAG induced a mitochondria-mediated apoptosis, characterized by a rise in the Bax, Bax/Bcl-2 ratio, release of cyt c, upregulation of Apaf1 with down-stream activation of caspase-9, -3. Furthermore treatment with BA, NA and CAG demonstrated an upregulation of p21 and downregulation of Bcl-2, mut p53. But this effect was enhanced in the presence of all the three compounds together in combination. Chemoprevention by a combination of BA, NA and CAG by inducing the apoptosis, the natural cell death, suggest the importance of the potential combinational strategies capable of preventing skin tumor development.
Copyright © 2014. Published by Elsevier Ireland Ltd.
... The release of cytochrome c into the cytosol triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex, while Smac/DIABLO and Omi/HtrA2 promote caspase activation through neutralizing the inhibitory effects to inhibitor of apoptotic proteins (IAPs) ( Van Loo et al., 2002;Du et al., 2000). The selec-tive induction of apoptosis in malignant and premalignant cells indicate a protective mechanism of both chemoprevention and chemoptherapy in carcinogenesis while enhancement of apoptosis is considered as a double-edged sword because of its potential implication in neuronal cell death in degenerative diseases (D'Agostini et al., 2005). ...
... Necrosis involves the death of cells via external damage, mediated by destruction of the plasma membrane or the biochemical supports of its integrity. Apoptosis, a form of programmed cell death, is characterized by cell shrinkage, chromatin condensation; inter nucleosomal DNA fragmentation, and the formation of apoptotic bodies (D'Agostini et al., 2005). Apoptosis is arguably one of the most potent forms of defense against cancer (Ghavami et al., 2009). ...
... Induction of cell death by chemopreventive agents has been reported to be mediated by apoptosis [26,[40][41][42]. To confirm whether genistein induced cell death via apoptosis in HeLa cells, nuclear morphological changes were assessed before and after treatment with genistein at different time points. ...
... Necrosis involves the death of cells via external damage, mediated by destruction of the plasma membrane or the biochemical supports of its integrity. Apoptosis, a form of programmed cell death, is characterized by cell shrinkage, chromatin condensation; inter nucleosomal DNA fragmentation, and the formation of apoptotic bodies (D'Agostini et al., 2005). Apoptosis is arguably one of the most potent forms of defense against cancer (Ghavami et al., 2009). ...
Cervical cancer is the second most common female cancer worldwide, and it remains a challenge to manage preinvasive and invasive lesions. Fruit-based cancer prevention entities, such as flavonoid and their derivatives, have demonstrated a marked ability to inhibit preclinical models of epithelial cancer cell growth and tumor formation. Here, we extend the role of naringin-mediated chemoprevention to that of cervical carcinogenesis. The present study sought to investigate the therapeutic potential effect of naringin on apoptosis in human cervical SiHa cancer cells. Viability of SiHa cells was evaluated by the MTT assay, apoptosis and mitochondrial transmembrane potential by flow cytometry, and pro-apoptotic related genes by Real-time quantitative PCR. Naringin showed a 50% inhibition of SiHa human cervical cancer cells at a concentration of 750μM. SiHa cells exhibited apoptotic cell death, intranucleosomal DNA fragmentation, morphological changes and decline in the mitochondrial transmembrane potential. In addition, administration of naringin increased the expression of caspases, p53 and Bax, Fas death receptor and its adaptor protein FADD. These results suggest that the induction of apoptosis by naringin is through both death-receptor and mitochondrial pathways. Taken together, our results suggest that naringin might be an effective agent to treat human cervical cancer.
... Chemopreventive agents prevent the transformation of pre-cancerous cells from reaching a final stage of carcinogenesis by stopping uncontrolled molecular events which activated by carcinogens. Thus, the net effect of chemopreventive agents on pre-cancerous or cancerous cells includes suppressing transformation, halting proliferation and finally inducing apoptotic cell death (Crowell, 2005; D'Agostini et al., 2005). The benefits and molecular targets of compounds from dietary and medicinal plants for use in cancer chemoprevention have been reviewed by several authors (Neergheen et al., 2009; Pan et al., 2008). ...
Orthosiphon stamineus (Lamiaceae) is a medicinal plant containing several biologically active components that have chemopreventive activity. To investigate the chemopreventive properties of O. stamineus, we studied the apoptotic activity of the ethyl acetate fraction (EAF) derived from the hot water extract of O. stamineus leaves on the human hepatocellular carcinoma cell line, HepG2. The sulforhodomine B assay indicated that the EAF inhibited the viability of HepG2 cells in a concentration dependent manner. Hoechst 33342 staining showed that EAF-treated cells exhibited typical apoptotic morphologic changes such as nuclear condensation and fragmentation. JC-1 assays indicated that the EAF disrupted the mitochondrial transmembrane potential of HepG2 cells in a dose-dependent manner. Western blot analysis revealed that the EAF activated caspase-3, caspase-8 and caspase-9, increased Bax expression, downregulated Bcl-2, decreased Cox-2 expression and decreased level of the NF-κ κ κ κB p65 in nucleus. HPLC-DAD analysis identified the major components in the EAF as rosmarinic acid (31.8%) and caffeic acid (20.2%). Taken together, our study suggests that the EAF has the potential to be developed as an agent for human liver cancer prevention.
... The mouse skin carcinogenesis model is a well-established model to study the genetic and biological changes leading to tumor promotion (Samaha et al., 1997;Holden et al., 1997). Some of the genetic changes like lesions and transition to squamous cell carcinoma are associated with the chemical initiation of benign papillomas and are characterized in this model (D'Agostini et al., 2005). Dimethylbenz[a]anthracene (DMBA), a potent polycyclic aromatic hydrocarbon, is repeatedly applied on mouse skin to induce skin lesions (Ishikawa et al., 2010). ...
Tamoxifen (TAM) is a non-steroidal estrogen receptor modulator known for its anticancer activity. Apart from marked breast cancer activity, this drug has also shown potential in treating other types of cancers including skin cancers. TAM is reported to be associated with serious side effects primarily due to its systemic distribution. The localized delivery of this drug in this regard would be highly beneficial with respect to safety as well as efficacy.
In the current studies, an endeavor has been made to investigate the efficacy of topically applied liposome-encapsulated TAM on skin cancer model. The drug was encapsulated in phospholipid-based vesicular systems viz. conventional liposomes and elastic liposomes. Incidence of papillomas and histopathological examination were employed to determine the efficacy of the tested formulations.
The results demonstrated carrier-dependent strong inhibition of skin carcinogenesis with encapsulated drug vis-à-vis drug in the solution form. The encouraging findings from the current work construe immense potential of the TAM-loaded liposomal systems in the management of skin cancer.
... The ability of a high CS dose to induce apoptosis in mouse cortical neurons, as detectable by TUNEL, is certainly consistent with the results of the neutral comet assay. Apoptosis leads to the death of damaged cells, including postmitotic neurons in the developing brain (D'Agostini et al., 2005). Consistent with this conclusion , smoking was associated with reduced cortical gray matter in the Alzheimer-diseased brain (Almeida et al., 2008). ...
The prenatal and perinatal periods of brain development are especially vulnerable to insults by environmental agents. Early life exposure to cigarette smoke (CS), which contains both genotoxicants and oxidants, is considered an important risk factor for both neurodevelopmental and neurodegenerative disorders. Yet, little is known regarding the underlying pathogenetic mechanisms. In the present study, neonatal Swiss ICR (CD-1) albino mice were exposed to various concentrations of CS for 4 weeks and the brain examined for lipid peroxides, DNA damage, base-excision repair (BER) enzymes, apoptosis, and levels of the microtubule protein tau. CS induced a dose-dependent increase in both malondialdehyde and various types of DNA damage, including single-strand breaks, double-strand breaks, and DNA-protein cross-links. However, the CS-induced DNA damage in the brain returned to basal levels 1 week after smoking cessation. CS also modulated the activity and distribution of the BER enzymes 8-oxoguanine-DNA-glycosylase (OGG1) and apyrimidinic/apurinic endonuclease (APE1) in several brain regions. Normal tau (i.e., three-repeat tau, 3R tau) and various pathological forms of tau were also measured in the brain of CS-exposed neonatal mice, but only 3R tau and tau phosphorylated at serine 199 were significantly elevated. The oxidative stress, genomic dysregulation, and alterations in tau metabolism caused by CS during a critical period of brain development could explain why CS is an important risk factor for both neurodevelopmental and neurodegenerative disorders appearing in later life.
... Modulation of apoptosis is also a mechanism included in the first level of prevention. However, in light of the complex network of events involved in chemoprevention, it has been argued if it is actually a mechanism rather than the consequence of another mechanism working upstream in the sequence of events leading to mutation and cancer (De Flora and Ferguson, 2005;D'Agostini et al., 2005). No previous studies with GJ regarding its effect on apoptosis/necrosis are known, although there are some studies with its flavonoid components. ...
By means of the comet assay we demonstrated a strong effect by hydrogen peroxide (HP) and no damage by grapefruit juice (GJ) in human lymphocytes. Cells exposed to HP and treated with three concentrations of GJ (10-90 min) showed an increase of DNA damage by HP over the control level, and a decrease of such damage by GJ. With the comet assay plus formamidopyrimidine-DNA-glycosylase we found the strongest increase of DNA damage by HP over the control level, and the strongest reduction of such damage by GJ. By applying the comet/FISH method we determined 98% of the p53 gene signals in the comet head of control cells along the experiment (10-90 min), in contrast with about 90% signals in the comet tail of cells exposed to HP. Cells treated with both agents showed a significant, concentration/time dependent return of p53 signals to the head, suggesting enhancement of the gene repair. Finally, with the annexin V assay we found an increase in apoptosis and necrosis by HP, and no effect by GJ; when GJ was added to HP treated cells no modification was observed in regard to apoptosis, although a decrease of necrosis was observed.
... The release of cytochrome c into the cytosol triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex, while Smac/DIABLO and Omi/HtrA2 promote caspase activation through neutralizing the inhibitory effects to inhibitor of apoptotic proteins (IAPs) (Van Loo et al., 2002;Du et al., 2000). The selec-tive induction of apoptosis in malignant and premalignant cells indicate a protective mechanism of both chemoprevention and chemoptherapy in carcinogenesis while enhancement of apoptosis is considered as a double-edged sword because of its potential implication in neuronal cell death in degenerative diseases (D'Agostini et al., 2005). ...
Natural phytochemicals derived from dietary sources or medicinal plants have gained significant recognition in the potential management of several human clinical conditions. Much research has also been geared towards the evaluation of plant extracts as effective prophylactic agents since they can act on specific and/or multiple molecular and cellular targets. Plants have been an abundant source of highly effective phytochemicals which offer great potential in the fight against cancer by inhibiting the process of carcinogenesis through the upregulation of cytoprotective genes that encode for carcinogen detoxifying enzymes and antioxidant enzymes. The mechanistic insight into chemoprevention further includes induction of cell cycle arrest and apoptosis or inhibition of signal transduction pathways mainly the mitogen-activated protein kinases (MAPK), protein kinases C (PKC), phosphoinositide 3-kinase (PI3K), glycogen synthase kinase (GSK) which lead to abnormal cyclooxygenase-2 (COX-2), activator protein-1 (AP-1), nuclear factor-kappaB (NF-κB) and c-myc expression. Effectiveness of chemopreventive agents reflects their ability to counteract certain upstream signals that leads to genotoxic damage, redox imbalances and other forms of cellular stress. Targeting malfunctioning molecules along the disrupted signal transduction pathway in cancer represent a rational strategy in chemoprevention. NF-κB and AP-1 provide mechanistic links between inflammation and cancer, and moreover regulate tumor angiogenesis and invasiveness, indicating that signaling pathways that mediate their activation provide attractive targets for new chemotherapeutic approaches. Thus cell signaling cascades and their interacting factors have become important targets of chemoprevention and phenolic phytochemicals and plant extracts seem to be promising in this endeavor.
... 30 PEITC is also an inducer of apoptosis. 31 Budesonide is a potent anti-inflammatory agent and is therefore expected to work also in advanced carcinogenesis stages, as it was previously demonstrated in A/J mice treated with B(a)P, in which this glucocorticoid inhibited all stages of tumor progression, from hyperplasia to cancer. 32 Budesonide was also found to decrease the size of lung tumors, and reversed DNA hypomethylation and gene expression in lung tumors induced by vinyl carbamate in A/J mice. ...
Lung cancer is the most important cause of death among neoplastic diseases worldwide, and cigarette smoke (CS) is the major risk factor for cancer. Complementarily to avoidance of exposure to CS, chemoprevention will lower the risk of cancer in passive smokers, ex-smokers, and addicted current smokers who fail to quit smoking. Unfortunately, chemoprevention clinical trials have produced disappointing results to date and, until recently, a suitable animal model evaluating CS carcinogenicity was not available. We previously demonstrated that mainstream CS induces a potent carcinogenic response when exposure of mice starts at birth. In the present study, neonatal mice (strain H) were exposed to CS for 120 consecutive days, starting at birth. The chemopreventive agents budesonide (2.4 mg/kg diet), phenethyl isothiocyanate (PEITC, 1,000 mg/kg diet), and N-acetyl-L-cysteine (NAC, 1,000 mg/kg body weight) were administered orally according to various protocols. The experiment was stopped after 210 days. Exposure to CS resulted in a high incidence and multiplicity of benign lung tumors and in significant increases of malignant lung tumors and other histopathological alterations. All three chemopreventive agents, administered to current smokers after weaning, were quite effective in protecting both male and female mice from CS pulmonary carcinogenicity. When given to ex-smokers after withdrawal of exposure to CS, the protective capacity of budesonide was unchanged, while PEITC lost part of its cancer chemopreventive activity. In conclusion, the proposed experimental model provides convincing evidence that it is possible to prevent CS-induced lung cancer by means of dietary and pharmacological agents.
... Conversely, exposure of mice either to ECS or ECS -122a, miR-124a, and miR-125b), with special reference to NF-B (miR-30b), protein repair (miR-30b and miR-431), and apoptosis (miR-99b). Previous studies in rodents have demonstrated that ECS is a potent inducer of apoptosis in pulmonary alveolar macrophages and in the bronchial/bronchiolar epithelium (27,28). Many other ECS-down-regulated miRNAs are involved in cell proliferation (let-7a, miR-30b, miR-30c, miR-124a, miR-219, and miR-376), which is known to be stimulated in the respiratory tract of ECS-exposed rodents (18,27). ...
MicroRNAs provide a formidable tool not only in cancer research but also to investigate physiological mechanisms and to assess the effect of environmental exposures in healthy tissues. Collectively, cigarette smoke and sunlight have been estimated to account for 40% of all human cancers, and not only smoke but also, surprisingly, UV light induced genomic and postgenomic alterations in mouse lung. Here we evaluated by microarray the expression of 484 microRNAs in the lungs of CD-1 mice, including newborns, postweanling males and females, and their dams, either untreated or exposed to environmental cigarette smoke and/or UV-containing light. The results obtained highlighted age-related variations in microRNA profiles, especially during the weanling period, due to perinatal stress and postnatal maturation of the lung. UV light alone did not affect pulmonary microRNAs, whereas smoke produced dramatic changes, mostly in the sense of down-regulation, reflecting both adaptive mechanisms and activation of pathways involved in the pathogenesis of pulmonary diseases. Both gender and age affected smoke-related microRNA dysregulation in mice. The data presented provide supporting evidence that microRNAs play a fundamental role in both physiological and pathological changes occurring in mouse lung.
... These compounds directly attack random or specified site of cancer cell DNA and also trigger a wide array of intracellular signaling pathways. Both direct and delicate, a complex network of genotoxic signals synergistically induce efficient apoptosis of targeted cancer cells (Janssens and Tschopp, 2006;Shin et al., 2006;D'Agostini et al., 2005). While the apoptotic signal path of DSB inducer has been well documented, how DNA intercalating agents initiate or elicit apoptotic signaling is unknown. ...
The present study was to see whether echinomycin-induced apoptosis would be NF-kappaB-dependent and if so, whether echinomycin would activate or inhibit NF-kappaB as well as resultant chemokine IL-8 expression. In HT-29 cells echinomycin activated NF-kappaB in time-dependent manner. EMSA in the presence of antibodies specific for p50 and p65 subunits indicated that echinomycin-induces the translocation of p50-p65 heterodimeric subunits of NF-kappaB. Levels of IkappaB were detected at initial echinomycin treatment and thereafter decreased, faintly seen after a 6h treatment. In contrast p-IkappaB levels were clearly detected throughout 6-24h of echinomycin treatment, albeit initially fainted. To clarify the role of NF-kappaB on IL-8 expression in echinomycin-mediated apoptosis of HT-29 cells, ELISA plus RT-PCR clearly showed that IL-8 production is inducible by echinomycin treatment. Using a specific inhibitor, IL-8 regulation at echinomycin treatment in HT-29 cells occurred via both caspase-3 and NF-kappaB-dependent signal pathway. To confirm whether two different pathways (NF-kappaB and caspase) would be coupled, only NF-kappaB inhibitor (PDTC) and caspase-3 specific inhibitor (Z-DEVD-FMK) together significantly attenuated echinomycin-initiated apoptosis of HT-29 cells, pretreatment of HT-29 cells with PDTC rarely affected echinomycin-induced caspase-3 activation. So echinomycin-induced apoptosis in HT-29 cells occurs via NF-kappaB activation independent of caspase-3 activation modulating the resultant-linked key chemokine IL-8 expression and echinomycin-induced apoptosis is NF-kappaB-dependant and directly related to NF-kappaB activation, consequently regulating IL-8 expression.
... Modulation of programmed cell death (apoptosis) has been proposed as an important mechanism of action of cancer chemopreventive agents. However, a critical review by D'Agostini et al. [27] reveals that many such reports are based on in vitro experiments or high dose experiments that are simply revealing toxicity. When they retested several agents in vivo at physiologically relevant concentrations, they revealed that cancer prevention was not occurring through the apoptotic process. ...
This is the eighth special issue of 'Mutation Research' to focus on antimutagenesis and anticarcinogenesis. It covers a wide range of mechanisms from prevention of cancer initiation by antimutagens through to inhibition of tumour angiogenesis and selective estrogen receptor modulators. New screening methods and new biomarkers are also elucidated. There is increasing reason to believe that the long-term use of a combination of anticarcinogens, over an extended time span, may provide a realistic prospect of reducing the current burden of human cancers.
... • Since mutagenesis and carcinogenesis evolve through a cascade and a network of events, it is difficult to discriminate whether modulation of a given end-point is actually a specific mechanism or rather the consequence of other mechanisms working upstream in the sequence of events leading to mutation and cancer [11]. A typical example is provided by modulation of apoptosis [28]. ...
Epidemiological data provide evidence that it is possible to prevent cancer and other chronic diseases, some of which share common pathogenetic mechanisms, such as DNA damage, oxidative stress, and chronic inflammation. An obvious approach is avoidance of exposure to recognized risk factors. As complementary strategies, it is possible to render the organism more resistant to mutagens/carcinogens and/or to inhibit progression of the disease by administering chemopreventive agents. In a primary prevention setting, addressed to apparently healthy individuals, it is possible to inhibit mutation and cancer initiation by triggering protective mechanisms either in the extracellular environment or inside cells, e.g., by modifying transmembrane transport, modulating metabolism, blocking reactive species, inhibiting cell replication, maintaining DNA structure, modulating DNA metabolism and repair, and controlling gene expression. Tumor promotion can be counteracted by inhibiting genotoxic effects, favoring antioxidant and anti-inflammatory activity, inhibiting proteases and cell proliferation, inducing cell differentiation, modulating apoptosis and signal transduction pathways, and protecting intercellular communications. In a secondary prevention setting, when a premalignant lesion has been detected, it is possible to inhibit tumor progression via the same mechanisms, and in addition by affecting the hormonal status and the immune system in various ways, and by inhibiting tumor angiogenesis. Although tertiary prevention, addressed to cancer patients after therapy, is outside the classical definition of chemoprevention, it exploits similar mechanisms. It is also possible to affect cell-adhesion molecules, to activate antimetastasis genes, and to inhibit proteases involved in basement membrane degradation.
... Extensive data exist documenting the ability of chemotherapeutic agents to cause chromosomal damage and apoptosis (Rao et al., 2005;Agostini et al., 2005). In the present study, the animals treated with ADR intravenously at a dose of 20 mg/kg body weight exhibited Table 3 Effect of LA on the frequency of MNPCEs and PCEs in the peripheral blood of rats treated with ADR Values are expressed as mean ± S.D. for six rats in each group. ...
Adriamycin (ADR), an anthracycline antibiotic, which is widely used as an antineoplastic drug in the treatment of various solid tumors, has been shown to induce genotoxicity in erythropoietic system. The aim of the present study was to investigate the protective efficacy of DL-alpha-lipoic acid (LA) on ADR-induced clastogenicity and apoptosis in the bone marrow of rats. The animals were randomly divided into eight groups consisting of six rats each. Five groups were administered ADR (20 mg/kg body weight, i.v.) to induce genotoxicity; four of these groups received a single intraperitoneal injection of LA at a dose of either 100 or 200 mg/kg body weight, and either 30 or 60 min prior to ADR administration. A vehicle treated control group and LA control groups were also included. The beneficial effects of LA were monitored by DNA strand breaks, chromosomal aberrations, micronucleus assay and apoptotic studies in the bone marrow cells of rats after 24 h following single dose of ADR treatment. ADR treatment caused significant clastogenicity and apoptosis in rat bone marrow cells. The treatment with LA showed significant reduction in the frequency of chromosomal aberrations, DNA strand breaks and apoptosis in bone marrow cells as well as decreased the micronuclei formation in bone marrow and peripheral blood of rats treated with ADR. The protective effect of LA was found to be stronger at a dose of 200 mg/kg body weight than 100 mg/kg body weight dosage with respect to the above results, indicating the dose dependent effect of LA. However, the protection by LA was not dependent on the time intervals between LA and ADR administration. The results of this study illustrate the protective effect of LA on ADR-induced clastogenicity and apoptosis in the erythropoietic system of rats.
... A vast literature is available regarding modulation of apoptosis by NAC. In the large majority of 390 studies on this subject cited in Medline from January 1, 1989, to October 31, 2004, NAC inhibited apoptosis induced by either biological, physical or chemical agents in a variety of test systems (63). These data underlie the ability of NAC to protect the cells by inhibiting upstream mechanisms, such as DNA damage and oxidative stress-induced signalling, that trigger the apoptotic cascade. ...
Several lines of evidence suggest that stem cells are major targets for carcinogens. A normal human breast epithelial cell type was previously shown to possess stem cell characteristics. Further cell lines were derived following sequential transfection with SV40 large T-antigen (immortal, non-tumorigenic M13SV1 cells), exposure to X-rays (weakly tumorigenic M13SV1R2 cells), and ectopic expression of c-erbB2/neu (highly tumorigenic M13SV1R2N1 cells). We evaluated some characteristics of these cells and their susceptibility to the breast carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Compared to M13SV1 cells, the two untreated tumorigenic cell lines displayed higher levels of connexin 43 expression and NF-kappaB nuclear translocation, and a higher frequency of fhit loss. The baseline nuclear translocation of AP-1 and pCREB was particularly evident in M13SV1R2N1 cells and was further enhanced by DMBA treatment, indicating an interaction between c-erbB2/neu and DMBA-induced signalling. Treatment with DMBA did neither affect the baseline fhit loss nor p53 mutation, whereas it increased NF-kappaB nuclear translocation, the proportion of apoptotic cells, and the levels of connexin 43, common 4977-bp mitochondrial DNA deletion, and bulky adducts to nuclear DNA. DMBA-treated M13SV1 cells underwent significant oxidative DNA damage and exhibited the highest DNA adduct levels, while they had the lowest apoptotic rate. Co-treatment of cells with N-acetylcysteine (NAC) attenuated DMBA-induced toxicity and DNA alterations, particularly in M13SV1 cells. Thus, the immortal cell line derived from the normal human adult breast stem cell without further tumorigenic progression is the most susceptible both to DMBA-related alterations and to the protective effects of NAC.
... The involvement in, and activation of, the intrinsic pathway by reactive oxygen species leads to the assumption that antioxidants might prevent fulminant hepatic failure by interfering with the deadly cascade. Different recent studies have demonstrated that treatment with the thiol antioxidant N-acetyl-cysteine (NAC) consistently inhibits the programmed cell death induced by a variety of stimuli [7], blocking apoptosis induced by cytotoxic agents [8][9][10], and protecting from apoptotic death in chronic renal failure [11], chronic obstructive pulmonary disease [12] or exercise in adrenalectomized animals [13]. ...
This work was undertaken to investigate whether treatment with N-acetyl-cysteine (NAC) prevents oxidative stress and inhibits the apoptotic pathways in an animal model of fulminant hepatic failure.
Rabbits were experimentally infected with 2x10(4) hemagglutination units of a rabbit hemorrhagic disease virus isolate.
The spontaneous mortality rate of infected animals was 67% at 36 h post infection (pi) and 90% at 48 h pi. This percentage decreased significantly in animals receiving an i.p. injection of NAC (150 mg/kg body way/daily), for 7 days prior to infection. From 36 h pi marked increases were detected in blood levels of transaminases, lactate dehydrogenase, bilirubin and the oxidised/reduced glutathione ratio. All these effects were significantly prevented by NAC treatment. The Bax to Bcl-2 relative expression, the expression of FasL, cytochrome c and PARP-1, and the activity of caspase 3 were significantly increased at 36 and 48 h pi in infected animals. These changes were markedly reduced in animals treated with NAC, with the exception of FasL.
Our results suggest a potential hepatoprotective role of NAC in fulminant hepatic failure, mediated partially through the modulation of the intrinsic pathway of apoptosis.
... On the other hand, coadministration of AA in the present study reduced the lipid peroxidation level and apoptosis. It is reported that programmed cell death triggered by a variety of stimuli can be inhibited by AA (D'Agostini et al., 2005b). For instance, AA inhibited TRAIL-induced apoptosis in cancer cell lines by suppressing caspase 8 activity (Perez-Cruz et al., 2007). ...
We previously found that administration of ascorbic acid (AA) enhances the liver tumor-promoting activity of kojic acid (KA) in mice. To examine the reproducibility of these results in rats and the underlying mechanism of this effect, we employed a two-stage liver carcinogenesis model using male F344 rats. Two weeks after initiation with diethylnitrosamine (DEN), the animals received a diet containing 2% KA and drinking water with or without 5,000 ppm AA for a period of 7 weeks. A DEN-alone group was also established as a control. One week after the commencement of the administration, the animals were subjected to two-thirds partial hepatectomy. At the end of the experiment, the livers were analyzed immunohistochemically, and the mRNA expression level and extent of lipid peroxidation were measured. AA treatment enhanced the KA-induced tumor-promoting activity in terms of the number and area of liver cell foci that were positive for glutathione-S-transferase placental form. AA coadministration increased the number of hepatocytes positive for proliferating cell nuclear antigen and inversely decreased the number of TUNEL-positive cells. However, the increased level of thiobarbituric acid reactive substances resulting from KA treatment was suppressed by coadministration of AA. Gene expression analyses using low-density microarrays and real-time RT-PCR showed that coadministration of AA resulted in upregulation of genes related to cell proliferation and downregulation of those involved in apoptosis and/or cell cycle arrest. These results indicate that the concerted effects of AA on cell proliferation and apoptosis/cell cycle arrest probably through its antioxidant activity are involved in this enhancement.
Various plants have been used against cancer in the traditional systems of medicine in the South American countries, since many years. The indigenous people, population, or special books have transmitted these applications, which are originally based on verbal traditions through time, and many of these have been experimentally proven. Medicinal plant products have the advantage of having a minimum or no side effects compared to synthetic drugs, and hence, they are largely preferred in recent times. The South America region contains large areas considered with the richest biodiversity worldwide, with very variable geographic and climatic conditions, making the flora of this area an attractive target as a plant resource for the drug development. The phytochemical and pharmacological explorations of this rich biodiversity represent an accessible and affordable way to find new therapeutic treatment from medicinal plant used in the ethnomedicine of these countries. This chapter aims to discuss on the South American plants, their traditional uses, bioactive metabolites, and scientific studies on the anticancer activities. In addition, the information of some plants with anticancer potential used in the folk medicines that still do not have experimental data to validate their properties is also included. This comprehensive compiled data offer an insight into the development of novel anticancer agents in the near future.
Background:
Alveolar type II (ATII) cells in the lung are exposed to mechanical stretch during breathing and mechanical ventilation. Increased mechanical stretch contributes to lung injury by induction of apoptosis and necrosis in ATII cells.
Aim of the study:
In this study, we investigated the intrinsic and the extrinsic apoptosis pathways, and their involvement in our model of stretch-induced apoptosis.
Material and methods:
ATII cells were stretched on elastic membranes for 24 h and apoptosis was determined at different time points. Several factors of the intrinsic and the extrinsic pathway were investigated by FACS, ELISA, and Western blot. We also investigated the inhibition of reactive oxygen species (ROS) expression and cytochrome C release and their influence on induction of apoptosis by stretching.
Results:
In comparison to static cells, ROS generation and cytochrome C release were increased in stretched cells while at the same time, mitochondrial membrane potential was reduced. Both inhibition of ROS generation by tocopherol and inhibition of cytochrome C release by cyclosporine A led to reduction of stretch-induced apoptosis. An increase of caspase 8 and 9 was observed in stretched cells compared to static cells. In contrast, factors of the extrinsic pathway such as TNF-alpha, TRAIL, TNFRI, Fas, FasL, and FADD were not different to static cells at all time points or where not detectable.
Conclusion:
Together, these findings suggest predominance of the intrinsic pathway in stretch-induced apoptosis.
In the present work, a method was developed and validated for the quantification of benzyl isothiocyanate (BITC) in the fruits of Carica papaya. The quantification of this compound was carried out by gas chromatography (GC) with selective detectors - nitrogen phosphorus detector (NPD) and flame photometric detector (FPD). The performance of these detectors showed a higher sensitivity of the NPD with a broader linear range of detection. The LOD/LOQ were 0.038/0.100 mu g/mL for NPD and 5.78/19.29 mu g/mL for FPD. The recovery of the method for BITC was 90,64%. An average value of BITC concentration in all the analyzed samples was 16,23 mu g BITC/g.
It is established that alpha-tocopherol (alpha-TPh) shows cytoprotective effect at the induction of rats' thymocytes apoptosis by endocellular protein kinase inhibitors--staurosporine and phorbol ether in high concentration, and also on necrosis of the cells caused by sphyngosine. The effect of alpha-TPh on thymocytes death caused by protein phosphatase type 2A inhibitor ocadaic acid is much less expressed. The obtained data testify that the known ability of alpha-TPh to the inhibition of PKC and to the activation of protein phosphatase type 2A is not the main mechanism of its cytoprotective action. Partial reproduction of alpha-TPh effects by its analogue alpha-tocopheryl acetate which is not capable to enter in redox reactions, and the absence of influence on the studied processes of an antioxidant of N-acetyl-L-cysteine do not confirm the antioxidant mechanism of alpha-TPh action in this case. The inhibition by alpha-TPh of the release of cytochrome c in the cytosol of cells testifies to the implementation of its cytoprotective effect at the level of mitochondrial membranes. We assume the existence of the universal mechanism of alpha-TPh cytoprotective action that does not depend on the nature of apoptogenes and realized on the general for the majority of them stage of the cells death induction. The prevention by alpha-TPh of mitochondria dysfunction by stabilizing mitochondrial membranes and reduction of their permeabilization is supposed as that.
This work presents an analysis of current data on the investigation into the functional properties of biologically active substances in model systems based on cultivated human cells. The knowledge regarding the practical application of cell cultures for the analysis of functional properties of bioactive substances is summarized, including antioxidant, immunomodulating, pro- and prebiotics, and chemoprevention properties. The most promising directions in cell culture model development for the investigation of functional properties, including three-dimensional models, are discussed.
The aim of this work was to determine the effect of vitamin C, diallyl disulfide (DADS) and dipropyl disulfide (DPDS) towards N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA)-induced apoptosis in human leukemia (HL-60) and hepatoma (HepG2) cell lines using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. None of the vitamin C (5-50 μM), DADS and DPDS (1-5 μM) concentrations selected induced a significant percentage of apoptosis. In simultaneous treatments, vitamin C, DADS and DPDS reduced the apoptosis induced by NPIP and NDBA in HL-60 and HepG2 cells (around 70% of reduction). We also investigated its scavenging activities towards reactive oxygen species (ROS) produced by NPIP and NDBA using 2′,7′-dichlorodihydrofluorescein diacetate in both cell lines. ROS production induced by both N-nitrosamine was reduced to control levels by vitamin C (5-50 μM) in a dose-dependent manner. However, DADS (5 μM) increased ROS levels induced by NPIP and NDBA in HL-60 (40 and 20% increase, respectively) and HepG2 cells (18% increase), whereas DPDS was more efficient scavenger of ROS at the lowest concentration (1 μM) in both HL-60 (52 and 25% reduction, respectively) and HepG2 cells (24% reduction). The data demonstrated that the scavenging ability of vitamin C and DPDS could contribute to inhibition of the NPIP- and NDBA-induced apoptosis. However, more than one mechanism, such as inhibition of phase I and/or induction of phase II enzymes, could be implicated in the protective effect of dietary antioxidants towards NPIP- and NDBA-induced apoptosis in HL-60 and HepG2 cells.
Invasion and metastasis are the major causes of cancer-related death. Pharmacological or therapeutic interventions such as chemoprevention of the progression stages of neoplastic development could result in substantial reduction in the incidence of cancer mortality. (-)-Epigallocatechin-3-gallate (EGCG), a promising chemopreventive agent, has attracted extensive interest for cancer therapy utilizing its antioxidant, anti- proliferative and inhibitory effects on angiogenesis and tumor cell invasion. In this study, we assessed the influence of EGCG on the proliferative potential of HeLa cells by cell viability assay and authenticated the results by nuclear morphological examination, DNA laddering assay and cell cycle analysis. Further we analyzed the anti-invasive properties of EGCG by wound migration assay and gene expression of MMP-9 and TIMP-1 in HeLa cells. Our results indicated that EGCG induced growth inhibition of HeLa cells in a dose- and time- dependent manner. It was observed that cell death mediated by EGCG was through apoptosis. Interestingly, EGCG effectively inhibited invasion and migration of HeLa cells and modulated the expression of related genes (MMP-9 and TIMP-1) . These results indicate that EGCG may effectively suppress promotion and progression stages of cervical cancer development.
Carvacrol is one of the main substances of essential oil which triggers intracellular Ca(2+) mobilization and causes cytotoxicity in diverse cell models. However, the mechanism of carvacrol-induced Ca(2+) movement and cytotoxicity is not fully understood. This study examined the effect of carvacrol on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)), cell viability and apoptosis in OC2 human oral cancer cells. Carvacrol induced a [Ca(2+)](i) rise and the signal was reduced by removal of extracellular Ca(2+). Carvacrol-induced Ca(2+) entry was not altered by store-operated Ca(2+) channel inhibitors and protein kinase C (PKC) activator, but was inhibited by a PKC inhibitor. In Ca(2+) -free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited carvacrol-induced [Ca(2+)](i) rise. Conversely, incubation with carvacrol inhibited TG or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol-induced [Ca(2+)](i) rise. Carvacrol decreased cell viability, which was not reversed when cytosolic Ca(2+) was chelated with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Carvacrol induced apoptosis and activation of reactive oxygen species (ROS) and caspase-3. Together, carvacrol induced a [Ca(2+)](i) rise by inducing PLC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via PKC-sensitive, non store-operated Ca(2+) channels. Carvacrol induced ROS- and caspase-3-associated apoptosis.
Interest in dietary phytochemicals for potential cancer chemoprevention has increased substantially. Screening dietary compounds for chemopreventive activity however, requires a systematic and wide-ranging approach to encompass the complexity of carcinogenesis. We present some of the molecular pathways that underpin the broad biological processes involved in carcinogenesis. Oxidative stress, inflammation, and the evasion of apoptosis are important biological mechanisms by which carcinogenesis occurs. Subsequently, antioxidant, anti-inflammatory, and pro-apoptotic activity represent important activities for preventing, suppressing, or reversing the development of carcinogenesis. Ultimately, these mechanisms of action may provide a useful basis for screening novel phytochemicals for chemopreventive activity. In this review, we identify the important molecular processes that may be targeted in routine screenings of dietary phytochemicals to ultimately select the most effective potential candidates for cancer chemoprevention.
Apoptosis is one of the most critical forms of defense against cancer, and the induction of apoptosis by dietary polyphenols represents significant potential for cancer preventive activity. The present study examined polyphenols extracted from selected native Australian fruits--Illawarra plum (Podocarpus elatus Endl., Podocarpaceae), Kakadu plum (Terminalia ferdinandiana Exell, Combretaceae), muntries (Kunzea pomifera F. Muell., Myrtaceae), and native currant (Acrotriche depressa R.Br., Epacridaceae)--for antiproliferative activity against a panel of cancer and normal cell lines. Each fruit selectively inhibited the growth of cancer cell lines in a dose-dependent manner. The mechanism of growth inhibition of the human promyelocytic leukaemia cells (HL-60) was determined to be apoptosis by morphological assessment, DNA fragmentation, flow cytometry, and caspase-3 induction. Furthermore, Kakadu plum was found to activate caspase-7, -9, and poly (ADP-ribose) polymerase (PARP), suggesting it acts via the intrinsic apoptosis pathway. The same fruit also caused direct DNA damage in colon adenocarcinoma cells (HT-29) as detected using the cytokinesis-block micronucleus cytome (CBMN Cyt) assay.
Generation of reactive oxygen species (ROS) has been suggested as a mechanism of fetal membrane (FM) weakening leading to rupture, particularly with preterm premature rupture of the fetal membranes (PROM). In vitro, FM incubation with tumor necrosis factor (TNF) mimics physiological FM weakening, concomitant with generation of ROS and collagen remodeling. Proinflammatory cytokines are also postulated to have a role in the development of the FM physiological weak zone where rupture normally initiates in-term gestations. We hypothesized that antioxidant treatment may block ROS development and resultant FM weakening. Two studies examining antioxidant effects upon FM strength were conducted, one in vivo and the other in vitro. Fetal membrane of patients enrolled in a multicenter placebo-controlled trial to determine the effect of vitamin C (1 g/day) and vitamin E (400 IU/day) upon complications of pre-eclampsia were examined for FM biomechanical properties and biochemical remodeling at birth. Separately, biomechanics and biochemical markers of remodeling were determined in FM fragments incubated with TNF with or without vitamin C preincubation. Supplemental dietary vitamin C in combination with vitamin E did not modify rupture strength, work to rupture, or matrix metalloproteinase-9 (MMP9; protein or activity) either within or outside the term FM physiological weak zone. In vitro, TNF decreased FM rupture strength by 50% while increasing MMP9 protein. Vitamin C did not inhibit these TNF-induced effects. Vitamin C alone had a weakening effect on FM in vitro. We speculate that vitamin C supplementation during pregnancy will not be useful in the prevention of preterm PROM.
This study investigated the ability of NAC to prevent cerebral vasospasm in a rabbit model of SAH.
Twenty-one, male New Zealand white rabbits were randomly divided into 3 groups of 7 rabbits each: group 1 (control), group 2 (SAH only), group 3 (SAH + NAC treatment). NAC (150 mg/kg, single dose, IP) was administered just before SAH and continued until 72 hours after SAH in group 3. Animals were killed 72 hours after SAH. Tissue MDA levels, SOD, and GSH-Px activities were measured, and basilar artery cross-sectional areas, arterial wall thickness, and endothelial apoptosis in a cross section of basillary artery were determined in all groups.
Intraperitoneal administration of NAC was found to be markedly effective against developing a cerebral vasospasm following a SAH in rabbits. It could significantly reduce elevated lipid peroxidation and increase the level of tissue GSH-Px and SOD enzymatic activities. Also, NAC treatment was found to be effective in increasing the luminal area and reducing wall thickness of the basilar artery. The morphology of arteries in the NAC treatment group was well protected. NAC markedly reduced apoptotic index and protects the endothelial integrity.
This study demonstrates, for the first time, that NAC treatment attenuates cerebral vasospasm in a rabbit SAH model. NAC treatment has significant neuroprotective effect and markedly prevents cerebral vasospasm after SAH. In conclusion, the NAC treatment might be beneficial in preventing cerebral vasospasm after SAH, thus showing potential for clinical implications.
Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) is of particular interest due to the very high production and widespread environmental contamination. We recently demonstrated that the oral administration of BPA to mice results in the formation of DNA adducts not only in liver but also in mammary tissue. The present study aimed at evaluating the modulation of BPA-related DNA adducts and proteome alterations by the chemopreventive agents budesonide (BUD) and phenethyl isothiocyanate (PEITC). Swiss ICR (CD-1) mice received, for 8 days, BPA with the drinking water and either chemopreventive agent with the diet. We measured DNA adducts by (32)P postlabeling and 656 proteins by antibody microarray. BPA induced the formation, with similar patterns, of DNA adducts in liver and in mammary tissue. Moreover, BPA dysregulated 13 proteins in mammary tissue, mostly in the sense of upregulation, including estrogen receptor-beta and proteins involved in cell proliferation, inhibition of apoptosis, tissue remodeling, inflammation, stress response, and glutathione synthesis. PEITC significantly inhibited the formation of BPA-induced DNA adducts, but only at the highest dose tested, and BUD was totally ineffective. The chemopreventive agents modulated a variety of BPA-induced changes in proteome profiles. However, as shown by both hierarchical cluster analysis and principal component analysis, BUD and especially PEITC were not able to restore the physiological situation in BPA-treated mice. Therefore, the in vivo use of proteome analysis proves to be a sensitive tool for the early prediction not only of protective effects but also of adverse effects of chemopreventive agents.
We previously showed that exposure to environmental cigarette smoke (ECS) for 28 days causes extensive downregulation of microRNA expression in the lungs of rats, resulting in the overexpression of multiple genes and proteins. In the present study, we evaluated by microarray the expression of 484 microRNAs in the lungs of either ECS-free or ECS-exposed rats treated with the orally administered chemopreventive agents N-acetylcysteine, oltipraz, indole-3-carbinol, 5,6-benzoflavone, and phenethyl isothiocyanate (as single agents or in combinations). This is the first study of microRNA modulation by chemopreventive agents in nonmalignant tissues. Scatterplot, hierarchical cluster, and principal component analyses of microarray and quantitative PCR data showed that none of the above chemopreventive regimens appreciably affected the baseline microRNA expression, indicating potential safety. On the other hand, all of them attenuated ECS-induced alterations but to a variable extent and with different patterns, indicating potential preventive efficacy. The main ECS-altered functions that were modulated by chemopreventive agents included cell proliferation, apoptosis, differentiation, Ras activation, P53 functions, NF-kappaB pathway, transforming growth factor-related stress response, and angiogenesis. Some microRNAs known to be polymorphic in humans were downregulated by ECS and were protected by chemopreventive agents. This study provides proof-of-concept and validation of technology that we are further refining to screen and prioritize potential agents for continued development and to help elucidate their biological effects and mechanisms. Therefore, microRNA analysis may provide a new tool for predicting at early carcinogenesis stages both the potential safety and efficacy of cancer chemopreventive agents.
Using the Ames bacterial mutagenicity test, the comet assay, and an in vivo micronucleus test, we investigated the effect of the chemoprotective substance phenethyl isothiocyanate (PEITC) on the mutagenic activity of indirect-acting mutagens and carcinogens aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and direct-acting mutagen and carcinogen N-nitroso-N-methylurea (MNU). In the Ames test, the antimutagenic activity of PEITC was studied in the concentration range 0.3-300 microg/plate. PEITC at concentrations of 0.3, 3 and 30 microg/plate reduced dose-dependently mutagenicity of AFB1 and IQ in both S. typhimurium TA98 and TA100 strains. In the case of the direct mutagen MNU, the antimutagenic effect of PEITC was detected only at concentration of 30 microg/plate in the strain TA100. The PEITC concentration 300 microg/plate was toxic in the Ames test. The 24 h pre-treatment of HepG2 cells with PEITC at concentration 0.15 microg/ml resulted in a significant decrease of DNA breaks induced by MNU at concentrations 0.25 and 0.5 mM. Although a trend towards reduced strand break level were determined also at PEITC concentrations 0.035 and 0.07 microg/ml it did not reach the statistical significance. No effect, however, of PEITC on IQ-induced DNA breaks was observed. Chemopreventive effect of PEITC was revealed also in vivo. Pretreatment of mice with PEITC concentrations of 25 and 12.5 mg/kg b.w. administered to mice in three daily doses resulted in reduction of micronucleus formation in mice exposed to all three mutagens under study, with statistically significant effect at concentration of 25 mg/kg. Results of this study indicate that the strong PEITC antimutagenic properties may have an important role in the prevention of carcinogenesis and other chronic degenerative diseases that share some common pathogenetic mechanisms.
Our discovery that the perinatal period involves nucleotide modifications and gene overexpression in mouse lung prompted us to evaluate whether mice may become more susceptible to cigarette smoke when exposure starts immediately after birth. We previously showed that mainstream cigarette smoke is a quite potent carcinogen in neonatal mice. Further on, we showed that exposure of mice to environmental cigarette smoke (ECS), starting at birth, results in alterations of a variety of intermediate biomarkers. However, after 4 months of exposure to ECS followed by 7 months of recovery in filtered air, the lung tumor yield was rather low. In the present study, we evaluated the protective effects of the glucocorticoid budesonide and of the dietary agent phenethyl isothiocyanate in mice exposed to ECS for 9 months followed by 2 months of recovery. After weanling, the mice exposed to ECS since birth underwent a variety of alterations of molecular and cytogenetical end points, and 11 months after birth, they exhibited significant histopathologic changes, such as pulmonary anthracosis, emphysema, hemorrhagic areas, alveolar bronchiolarization, bronchial hyperplasia, and tumors, both benign and malignant. The carcinogenic response was less evident in dams exposed to ECS under identical conditions. Both phenethyl isothiocyanate and budesonide, administered daily with the diet after weanling, attenuated several alterations of ECS-related biomarkers and moderately protected the lungs from histopathologic alterations, including tumors. Thus, although not as efficiently as the bioassay in mainstream cigarette smoke-exposed mice, the model in neonatal mice is suitable to evaluate both ECS carcinogenicity and its modulation by chemopreventive agents.
Chemoprevention by dietary and pharmacological means provides a strategy for attenuating the health risks resulting from cigarette smoking and in particular from passive exposure to environmental cigarette smoke (ECS). We evaluated the ability of the glucocorticoid budesonide and of the natural agent phenethyl isothiocyanate (PEITC) to affect DNA damage in bronchoalveolar lavage (BAL) cells of CD-1 mice exposed to ECS, starting within 12 h after birth and continuing until the end of the experiment. After weanling, based on a preliminary subchronic toxicity study, groups of mice received daily either budesonide (24 mg/kg diet) or PEITC (1,000 mg/kg diet). After 2 weeks of treatment, all mice were sacrificed and subjected to BAL, mainly recovering pulmonary alveolar macrophages. Evaluation of single-cell DNA strand breaks was made by using the alkaline-halo test, a modification of the comet assay. The analysis of 481 BAL cells yielded the following results (expressed as nuclear spread factor): (a) Sham-exposed mice: mean 0.84 (lower-upper 95% confidence intervals 0.74-0.94); (b) ECS-exposed mice: 2.77 (2.46-3.09); (c) ECS-exposed mice treated with PEITC: 1.15 (1.05-1.26); (d) ECS-exposed mice treated with budesonide: 1.37 (1.25-1.49). Thus, exposure to ECS resulted in a significant increase of DNA damage as compared with sham, and both PEITC and budesonide significantly attenuated this damage. In conclusion, the analysis of sentinel cells collected by BAL, a semi-invasive technique that is commonly used in humans for diagnostic purposes, showed that the investigated chemopreventive agents are able to revert the DNA damage produced by passive exposure to cigarette smoke.
The aim of this study was to evaluate the effect of vitamin C towards N-nitrosopyrrolidine (NPYR)- and N-nitrosodimethylamine (NDMA)-induced apoptosis in human hepatoma (HepG2) and leukemia (HL-60) cell lines using flow cytometry analysis and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay (TUNEL). None of the vitamin C concentrations tested (1-100 microM) caused cytotoxicity in HepG2 cells. However, there were significant losses of HL-60 cells viability, measured by MTT assay, 72 h after treatment with 50 and 100 microM vitamin C (29 and 46%, respectively). Moreover, an increase of lactate dehydrogenase release was significant with 50 microM at 72 h (28%) and with 100 microM of vitamin C at 48 and 72 h (27 and 36%, respectively). Also, the percentage of apoptotic HL-60 cells found in TUNEL assay increased to 21% when they were treated with 100 microM vitamin C for 72 h. Thus, in subsequent simultaneous treatments with NPYR (30 and 50 mM) or NDMA (27 and 68 mM) and vitamin C, concentrations of 5-50 microM vitamin C were used. Our results revealed that vitamin C, at all concentrations and times tested, reduced the apoptosis induced by NPYR and NDMA in both cell lines, showing a similar effect in HepG2 and HL-60 cells towards NPYR (50 mM)--65 and 63% of reduction, respectively--whereas towards NDMA (27 mM) the inhibition was higher in HL-60 than in HepG2 cells--75 and 57%, respectively. Therefore, our findings suggest that inhibition of apoptosis may be one of the mechanisms by which vitamin C exerts its protective effect.
Chemoprevention opens new perspectives in the prevention of cancer and other chronic degenerative diseases associated with tobacco smoking, exploitable in current smokers and, even more, in exsmokers and passive smokers. Evaluation of biomarkers in animal models is an essential step for the preclinical assessment of efficacy and safety of potential chemo- preventive agents. Groups of Sprague Dawley rats were exposed whole body to a mixture of mainstream and sidestream cigarette smoke for 28 consecutive days. Five chemopreventive agents were given either with drinking water (N-acetyl-L-cysteine, 1 g/kg body weight/day) or with the diet (1,2-dithiole-3-thione, 400 mg; Oltipraz, 400 mg; phenethyl isothio- cyanate, 500 mg; and 5,6-benzoflavone, 500 mg/kg diet). The monitored biomarkers included: DNA adducts in bronchoalveolar lavage cells, tra- cheal epithelium, lung and heart; oxidative damage to pulmonary DNA; hemoglobin adducts of 4-aminobiphenyl and benzo(a)pyrene-7,8-diol- 9,10-epoxide; micronucleated and polynucleated alveolar macrophages and micronucleated polychromatic erythrocytes in bone marrow. Expo- sure of rats to smoke resulted in dramatic alterations of all investigated parameters. N-Acetyl-L-cysteine, phenylethyl isothiocyanate, and 5,6- benzoflavone exerted a significant protective effect on all alterations. 1,2-Dithiole-3-thione was a less effective inhibitor and exhibited both a systemic toxicity and genotoxicity in alveolar macrophages, whereas its substituted analogue Oltipraz showed limited protective effects in this model. Interestingly, combination of N-acetyl-L-cysteine with Oltipraz was the most potent treatment, resulting in an additive or more than additive inhibition of smoke-related DNA adducts in the lung and hemoglobin adducts. These results provide evidence for the differential ability of test agents to modulate smoke-related biomarkers in the respiratory tract and other body compartments and highlight the potential advantages in com- bining chemopreventive agents working with distinctive mechanisms.
TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70-80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.
Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis.
7,12-Dimethylbenz(a)anthracene (DMBA) is a prototype carcinogen that induces a high yield of mammary tumors in rats after a single feeding. We investigated the induction and chemoprevention of DNA adducts in female Sprague Dawley rats receiving DMBA by gavage according to a variety of treatment schedules. The patterns of 32P-postlabeled DNA adducts in liver and mammary epithelial cells were similar to those produced by the in vitro reaction of metabolically activated DMBA with calf thymus DNA. There was a high and statistically significant correlation between dose of DMBA administered to rats (0, 0.6, 2.4, and 12 mg/kg body weight) and levels of DNA adducts in both types of cells. The regression lines relating DMBA doses to total DNA adduct levels were significantly divergent and crossed at 1.5 mg/kg body weight, indicating that, at lower doses, the formation of DNA adducts is more intense in target mammary cells, whereas at higher doses, DNA adduct levels are more elevated in liver cells, presumably due to the greater metabolic capacity of this organ. When the rats were sacrificed 7 days rather than 2 days after DMBA administration, DNA adduct levels were approximately halved in both liver and mammary cells. The observed patterns can be interpreted based on toxicokinetic factors, local and distant metabolism, removal of DNA adducts by excision repair, and cell proliferation rate. Of three chemopreventive agents given with the diet to rats treated with 12 mg of DMBA, 5,6-benzoflavone (1650 ppm) was the most effective, inhibiting DNA adduct formation in liver and mammary cells by 96.5 and 83.5%, respectively. Feeding of 1,2-dithiole-3-thione (600 ppm) inhibited this biomarker by 68.5 and 50.2%, whereas butyl hydroxyanisole (BHA; 5000 ppm) showed a significant inhibition in the liver (46.5%) but was ineffective in mammary cells (29.0%, not significant). These data correlate nicely with the results of a parallel study in which 5,6-benzoflavone, 1,2-dithiole-3-thione, and BHA inhibited formation of hemoglobin adducts by 80.0, 44.0, and 0%, respectively; the incidence of mammary tumors by 82.4, 47.1, and 5.9%, respectively; and their multiplicity by 92.6, 80.0, and 7.4%, respectively. Therefore, biomarkers of biologically effective dose are highly predictive of the efficacy of chemopreventive agents in the DMBA rat mammary model. The selective inhibition by BHA of DNA adducts in the liver but not in mammary cells is consistent with the finding that this phenolic antioxidant stimulated phase II activities in the liver but not in the mammary gland (L. L. Song et al., manuscript in preparation). In any case, the broad-spectrum inducer 5,6-BF appears to be more effective than the two monofunctional phase II inducers, presumably because an enhanced activation of DMBA to reactive metabolites is coordinated with their blocking, detoxification, and excretion.
Cyclooxygenase-2 (COX-2) is up-regulated in many cancers and is a rate-limiting step in colon carcinogenesis. Nonsteroidal antiinflammatory drugs, which inhibit COX-2, prevent colon cancer and cause apoptosis. The mechanism for this response is not clear, but it might result from an accumulation of the substrate, arachidonic acid, an absence of a prostaglandin product, or diversion of the substrate into another pathway. We found that colon adenocarcinomas overexpress another arachidonic acid-utilizing enzyme, fatty acid-CoA ligase (FACL) 4, in addition to COX-2. Exogenous arachidonic acid caused apoptosis in colon cancer and other cell lines, as did triacsin C, a FACL inhibitor. In addition, indomethacin and sulindac significantly enhanced the apoptosis-inducing effect of triacsin C. These findings suggested that unesterified arachidonic acid in cells is a signal for induction of apoptosis. To test this hypothesis, we engineered cells with inducible overexpression of COX-2 and FACL4 as "sinks" for unesterified arachidonic acid. Activation of the enzymatic sinks blocked apoptosis, and the reduction of cell death was inversely correlated with the cellular level of arachidonic acid. Inhibition of the COX-2 component by nonsteroidal antiinflammatory drugs restored the apoptotic response. Cell death caused by exposure to tumor necrosis factor alpha or to calcium ionophore also was prevented by removal of unesterified arachidonic acid. We conclude that the cellular level of unesterified arachidonic acid is a general mechanism by which apoptosis is regulated and that COX-2 and FACL4 promote carcinogenesis by lowering this level.
Epidemiological and clinical data suggest that selenium may prevent prostate cancer, but the biological effects of selenium on normal or malignant prostate cells are not well known. We evaluated the effects of sodium selenite (Na2SeO3) or l-selenomethionine (SeMet) on monolayer and anchorage-independent growth in a series of normal primary prostate cultures (epithelial, stromal, and smooth muscle) and prostate cancer cell lines (LNCaP, PC-3, and DU145). We observed differential, dose-dependent growth inhibition and apoptosis within prostate cancer cells (compared with normal prostate cells) treated with 1-500 microM of Na2SeO3 or SeMet. Na2SeO3 more potently inhibited growth at any given concentration. The androgen-responsive LNCaP cells were the most sensitive to selenium growth suppression (IC50s at 72 h for Na2SeO3 and SeMet were 0.2 and 1.0 microM, respectively). Growth of the primary prostate cells virtually was not suppressed (IC50s at 72 h for Na2SeO3 and SeMet were 22-38 and >500 microM, respectively). We also observed that DNA condensation and DNA fragmentation (terminal deoxynucleotidyltransferase dUTP nick end labeling/fluorescence-activated cell sorting) were elevated in selenium-treated cells and that activated caspase-3 colocalized with terminal deoxynucleotidyltransferase dUTP nick end labeling-stained cells by immunofluorescence. Higher basal poly(ADP-ribose) polymerase (PARP) expression levels and PARP cleavage (a substrate for caspase-3) were observed during apoptosis in tumor cells, compared with normal cells. Selective tumor cell death was associated with an increase in sub-G0-G1 cells after propidium iodide staining and fluorescence-activated cell sorting analysis. SeMet caused an increase in arrest in the G2-M phase of the cell cycle selectively in cancer cells. Inhibition of cancer cell growth by SeMet was associated with phosphorylation of P-Tyr15-p34/cdc2, which caused growth arrest in the G2-M phase. Anchorage-independent growth of prostate cancer cells in soft agar was sensitive to selenium. Our results suggest that Na2SeO3 is the more potent inducer of apoptosis in normal and cancer prostate cells. Our SeMet results involving PARP and G2-M cell-cycle arrest (cited above) indicate that SeMet selectively induces apoptosis in cancer but not primary cells of the human prostate. Our overall findings are relevant to the molecular mechanisms of selenium actions on prostate carcinogenesis and help demonstrate the selective, dose-dependent effects of selenium (especially SeMet) on prostate cancer cell death and growth inhibition.
Preclinical studies may elucidate the meaning of biomarkers applicable to epidemiologic studies and to clinical trials for cancer prevention. No study has explored so far the effect of cigarette smoke on apoptosis in vivo. We evaluated modulation of apoptosis in cells of the respiratory tract of smoke-exposed Sprague-Dawley rats both by morphological analysis and TUNEL method. In a first study, exposure of rats to mainstream cigarette smoke for either 18 or 100 consecutive days produced a significant and time-dependent increase in the proportion of apoptotic cells in the bronchial and bronchiolar epithelium. Oral N:-acetylcysteine did not affect the background frequency of apoptosis but significantly and sharply decreased smoke-induced apoptosis. In a second study, exposure of rats to a mixture of sidestream and mainstream smoke for 28 consecutive days resulted in a >10-fold increase in the frequency of pulmonary alveolar macrophages undergoing apoptosis. Dietary administration of either 5,6-benzoflavone, 1,2-dithiole-3-thione or oltipraz did not affect the frequency of smoke-induced apoptosis, whereas phenethyl isothiocyanate produced a further significant enhancement. Again, N-acetylcysteine and its combination with oltipraz significantly decreased smoke-induced apoptosis. In both studies exposure to smoke resulted in a sharp increase of cells positive for proliferating cell nuclear antigen (PCNA), which was unaffected by the examined chemopreventive agents. These findings highlight the concept that modulation of apoptosis has diversified meanings. Different meanings (as explained in the following lines). First, the apoptotic process is triggered as a defense system against genotoxic agents, such as the components of cigarette smoke. The further induction produced by phenethyl isothiocyanate, favoring removal of damaged cells, represents an example of a detoxification mechanism. Inhibition of smoke-induced apoptosis by N:-acetylcysteine should be interpreted as an epiphenomenon of antigenotoxic mechanisms, as shown in parallel studies evaluating modulation of DNA alterations in the respiratory tract of the same animals. Thus, it is important to discriminate between whether the opposite modulation of apoptosis is per se a protective mechanism or the beneficial outcome of other mechanisms inhibiting genotoxicity.
Although smoking cessation is the primary goal for the control of cancer and other smoking-related diseases, chemoprevention provides a complementary approach applicable to high risk individuals such as current smokers and ex-smokers. The thiol N-acetylcysteine (NAC) works per se in the extracellular environment, and is a precursor of intracellular cysteine and glutathione (GSH). Almost 40 years of experience in the prophylaxis and therapy of a variety of clinical conditions, mostly involving GSH depletion and alterations of the redox status, have established the safety of this drug, even at very high doses and for long-term treatments. A number of studies performed since 1984 have indicated that NAC has the potential to prevent cancer and other mutation-related diseases. N-Acetylcysteine has an impressive array of mechanisms and protective effects towards DNA damage and carcinogenesis, which are related to its nucleophilicity, antioxidant activity, modulation of metabolism, effects in mitochondria, decrease of the biologically effective dose of carcinogens, modulation of DNA repair, inhibition of genotoxicity and cell transformation, modulation of gene expression and signal transduction pathways, regulation of cell survival and apoptosis, anti-inflammatory activity, anti-angiogenetic activity, immunological effects, inhibition of progression to malignancy, influence on cell cycle progression, inhibition of pre-neoplastic and neoplastic lesions, inhibition of invasion and metastasis, and protection towards adverse effects of other chemopreventive agents or chemotherapeutical agents. These mechanisms are herein reviewed and commented on with special reference to smoking-related end-points, as evaluated in in vitro test systems, experimental animals and clinical trials. It is important that all protective effects of NAC were observed under a range of conditions produced by a variety of treatments or imbalances of homeostasis. However, our recent data show that, at least in mouse lung, under physiological conditions NAC does not alter per se the expression of multiple genes detected by cDNA array technology. On the whole, there is overwhelming evidence that NAC has the ability to modulate a variety of DNA damage- and cancer-related end-points.
Alveolar macrophages (AMs) may play a critical role in cigarette smoke (CS)-related pulmonary diseases. This study was designed to determine whether CS induces apoptosis of AMs. In in vitro studies, mouse, rat, and human AMs and human blood monocyte-derived macrophages cultured with aqueous whole CS extracts underwent apoptosis that was detected by light and electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. The gas phase of CSE did not cause apoptosis. The CS-induced apoptosis was associated with increased oxidative stress, Bax protein accumulation, mitochondrial dysfunction, and mitochondrial cytochrome c release but was independent of p53, Fas, and caspase activation. This apoptosis was inhibited by antioxidants such as glutathione, ascorbic acid, and alpha-tocopherol. In in vivo studies where rats were exposed to the smoke from 10 cigarettes over 5 h in an exposure chamber, approximately 3% of AMs obtained by bronchoalveolar lavage after 24 h showed apoptosis. These results suggest that acute CS exposure is capable of inducing apoptosis of AMs.
Indole-3-carbinol (I-3-C) is among the most widely and popularly known antiestrogens. Due to its putative chemopreventive action, I-3-C is being marketed to the general public in health food establishments. Although it has been demonstrated to prevent cancer in animal bioassays, I-3-C also acts as a promoter in the liver and colon. Because of this potential dual biological activity, it is important to investigate both the inhibitory and promotional activities of I-3-C in multi-organ tumorigenesis animal models. 7,12-Dimethylbenz[a]anthracene, aflatoxin B1 and azoxymethane were used to initiate mammary, liver and colon carcinogenesis, respectively in female Sprague-Dawley rats. The rats were fed continuously on a diet containing I-3-C for 25 weeks after initiation. I-3-C treatment was begun one week after the last carcinogen treatment had been administered. I-3-C treatment resulted in a delay in latency of mammary tumor formation, but did not alter tumor incidence or multiplicity among survivors. In the colon, the protocol produced a 40% decrease in aberrant colon crypt foci. However, in the liver, it strongly-induced GST-P foci in carcinogen-treated (a four-fold increase in volume percent foci) and in the vehicle controls (a 69-fold increase). These data support previous findings in other rodent and fish tumor models that I-3-C both inhibits and promotes carcinogenesis. The results of this study clearly demonstrate that I-3-C is not an appropriate chemoprotective agent for human use, in spite of its effects in the breast and colon in this rat animal model.
We have found previously that ascorbic acid (vitamin C), as well as acting as a radical scavenger, may modulate the expression of several genes [i.e. fra-1, glutathione S-transferase Pi (GSTpi) and Mut L homologue-1 (MLH1)] in human keratinocytes. In the present paper, we demonstrate that MLH1, as well as its downstream target p73, can be positively modulated by this antioxidant vitamin, indeed, up-regulation of the two mRNAs was observed after just 2 h, and increased further up to 16 h of treatment. Modulation of MLH1 and p73 gene expression improves cellular susceptibility to apoptosis triggered by the DNA-damaging agent cisplatin. Indeed, in ascorbate-supplemented cells, increased cisplatin-induced apoptosis was seen, involving activation of the MLH1/c-Abl/p73 signalling cascade. Our results were further confirmed by studies performed on genetically defined mutants, i.e. mouse embryo fibroblasts derived from knock-out animals for c-Abl or p53, as well as human colon carcinoma cell lines deficient in MLH1. The increased sensitivity to cisplatin observed in ascorbate-loaded cells appeared to be dependent exclusively on MLH1 and c-Abl expression, and independent of p53. These data suggest a potential mechanism accounting for the anti-carcinogenic and anti-cancer activities of vitamin C.
We have studied the effect of N-(4-hydroxyphenyl)retinamide on either malignant human leukaemia cells or normal cells and investigated its mechanism of action. We demonstrate that 4HPR induces reactive oxygen species increase on mitochondria at a target between mitochondrial respiratory chain complex I and II. Such oxidative stress causes cardiolipin peroxidation which in turn allows cytochrome c release to cytosol, caspase-3 activation and therefore apoptotic consumption. Moreover, this apoptotic pathway seems to be bcl-2/bax independent and count only on malignant cells but not normal nor activated lymphocytes.
British Journal of Cancer (2002) 86, 1951–1956. doi:10.1038/sj.bjc.6600356 www.bjcancer.com
© 2002 Cancer Research UK
The transplacental exposure of fetuses to maternal cigarette smoke may increase the risk of developmental impairments, congenital diseases, and childhood cancer. The whole-body exposure of Swiss mice to environmental cigarette smoke (ECS) during pregnancy decreased the number of fetuses per dam, placenta weight, and fetus weight. ECS increased DNA adducts, oxidative nucleotide alterations, and cytogenetic damage in fetus liver. Evaluation by cDNA array of 746 genes showed that 61 of them were expressed in fetus liver under basal conditions. The oral administration of N-acetylcysteine (NAC) during pregnancy enhanced the expression of three genes only, including two glutathione S-transferases and alpha1-antitrypsin precursor, whose deficiency plays a pathogenetic role in congenital emphysema. Transplacental ECS upregulated the expression of 116 genes involved in metabolism, response to oxidative stress, DNA and protein repair, and signal transduction. NAC inhibited the ECS-related genetic damage and upregulation of most genes. ECS stimulated pro-apoptotic genes and genes downregulating the cell cycle, which may justify growth impairments in the developing fetus. Thus, both genetic and epigenetic mechanisms were modulated by ECS. Moreover, hypoxia-related genes and several oncogenes and receptors involved in proliferation and differentiation of leukocytes were induced in the fetal liver, which also bears hematopoietic functions.
No information is available on the interaction between cigarette smoke, the most important man-made carcinogen, and light, the most widespread natural carcinogen. In order to clarify this issue, SKH-1 hairless mice were exposed to environmental smoke and/or to the light emitted by sunlight-simulating halogen quartz bulbs. After 28 days, intermediate biomarkers were evaluated in skin, respiratory tract, bone marrow and peripheral blood. The results showed that, individually, the light produced extensive alterations not only in the skin but even at a systemic level, as shown by formation of bulky DNA adducts in both lung and bone marrow and induction of cytogenetic damage in bone marrow and peripheral blood erythrocytes. Smoke damaged the respiratory tract and produced significant alterations in the skin as well as an evident cytogenetic damage in both bone marrow and peripheral blood. Interestingly, as compared with exposure to smoke only, alternate daily cycles of exposure to both light and smoke significantly increased malondialdehyde concentrations and DNA adduct levels in lung and the frequency of micronuclei in pulmonary alveolar macrophages. The oral administration of sulindac, a non-steroidal anti-inflammatory drug, attenuated several biomarker alterations due to the combined exposure of mice to light and smoke. In conclusion, the light induces a systemic genotoxic damage, which is presumably due to the UV-mediated formation in the skin of long-lived derivatives, such as aldehydes. This damage may mechanistically be involved in light-related hematopoietic malignancies. In addition, the light displayed an insofar unsuspected synergism with smoke in the induction of DNA damage in the respiratory tract.
A study of MCF-7 human breast cancer cells was undertaken to ascertain the degree of apoptosis induction by paclitaxel and if the induction of apoptosis could be enhanced by caffeine. Paclitaxel (0–20 ng/ml) caused concentration-dependent increases in morphologically identifiable apoptotic cells (up to 43% of cell population) and cells with DNA strand breaks (up to 38%), a commonly cited marker of apoptosis. Maximal DNA strand breakage occurred after 16 hr of exposure to paclitaxel and maximal apoptotic-appearing cells occurred after 24 hr. The remaining non-apoptotic paclitaxel-exposed cells were growth arrested in G2. A 4-hr exposure to caffeine concentration-dependently (0–20 mM) increased apoptosis to 88% of the cell population. Our results show induction of apoptosis in breast cancer cells by paclitaxel, and enhancement of this process by caffeine. Int. J. Cancer, 70:214–220, 1997. © 1997 Wiley-Liss Inc.
Alpha-tocopheryl succinate (alpha-TOS), a redox-inactive analogue of vitamin E, is a strong inducer of apoptosis, whereas alpha-tocopherol (alpha-TOH) lacks apoptogenic activity (J. Neuzil et al., FASEB J., 15: 403-415, 2001). Here we investigated the possible antineoplastic activities of alpha-TOH and alpha-TOS and further explored the potential of alpha-TOS as an antitumor agent. Using nude mice with colon cancer xenografts, we found that alpha-TOH exerted modest antitumor activity and acted by inhibiting tumor cell proliferation. In contrast, alpha-TOS showed a more profound antitumor effect, at both the level of inhibition of proliferation and induction of tumor cell apoptosis. alpha-TOS was nontoxic to normal cells and tissues, triggered apoptosis in p53(-/-) and p21(Waf1/Cip1(-/-)) cancer cells, and exerted a cooperative proapoptotic activity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) due to differences in proapoptotic signaling. Finally, alpha-TOS cooperated with tumor necrosis factor-related apoptosis-inducing ligand in suppression of tumor growth in vivo. Vitamin E succinate is thus a potent and highly specific anticancer agent and/or adjuvant of considerable therapeutic potential.
The aminothiol N-acetyl-l-cysteine (NAC) is an analog and precursor of reduced glutathione (GSH). During the last four decades, it has been extensively used as a mucolytic agent. In addition, because of its multiple protective mechanisms, NAC has been proposed for a broad array of applications, both preventive and therapeutic. The scientific community has a continuously growing interest in this molecule, which is being used with increasing frequency in both clinical investigations and experimental studies. As of February 1, 2002, a total of 5153 scientific papers were available in MEDLINE under the query term “acetylcysteine,” with an impressive growth during the last 10 years. In one month alone (January 2002), 134 new papers were added to this database.
Preclinical studies may elucidate the meaning of biomarkers applicable to epidemiologic studies and to clinical trials for cancer prevention. No study has explored so far the effect of cigarette smoke on apoptosis in vivo .W e evaluated modulation of apoptosis in cells of the respiratory tract of smoke-exposed Sprague–Dawley rats both by morphological analysis and TUNEL method. In a first study, exposure of rats to mainstream cigarette smoke for either 18 or 100 consecutive days produced a significant and time-dependent increase in the proportion of apoptotic cells in the bronchial and bronchiolar epithelium. Oral N-acetylcysteine did not affect the background frequency of apoptosis but significantly and sharply decreased smokeinduced apoptosis. In a second study, exposure of rats to a mixture of sidestream and mainstream smoke for 28 consecutive days resulted in a >10-fold increase in the frequency of pulmonary alveolar macrophages undergoing apoptosis. Dietary administration of either 5,6-benzoflavone, 1,2-dithiole-3-thione or oltipraz did not affect the frequency of smoke-induced apoptosis, whereas phenethyl isothiocyanate produced a further significant enhancement. Again, N-acetylcysteine and its combination with oltipraz significantly decreased smoke-induced apoptosis. In both studies exposure to smoke resulted in a sharp increase of cells positive for proliferating cell nuclear antigen (PCNA), which was unaffected by the examined chemopreventive agents. These findings highlight the concept that modulation of apoptosis has diversified meanings. Different meanings (as explained in the following lines). First, the apoptotic process is triggered as a defense system against genotoxic agents, such as the components of cigarette smoke. The further induction produced by phenethyl isothiocyanate, favoring removal of damaged cells, represents an example of a detoxification mechanism. Inhibition of smoke-induced
Purpose:
To evaluate the in vitro effects of an aerosolized cyclooxygenase-2 (COX-2) inhibitor, nimesulide, on the cytotoxicity and apoptotic response of doxorubicin against the human lung adenocarcinoma cell line A549.
Methods:
Nimesulide was formulated into a metered dose inhaler (MDI) formulation and characterized for aerodynamic particle size and medication delivery. The in vitro cytotoxicity of nimesulide-MDI in the presence or absence of doxorubicin was assessed by using the six-stage viable impactor by an already standardized method. Induction of apoptosis in A549 cells by nimesulide (nonaerosolized or aerosolized) in combination with doxorubicin was evaluated by established techniques such as caspase-3 estimation and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. Finally, to understand the mechanism of action, the influence of different treatments on the expression of COX-2 and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in A549 cells was studied by immunoblotting.
Results:
The nimesulide-MDI formulation had a mass median aerodynamic diameter (MMAD) of 1.1 microm, (GSD = 2.8) and a medication delivery of 51 microg/shot. Nimesulide-MDI (40 shots) in combination with doxorubicin (0.01 microg/ml) had a cell kill of more than 60% as determined by in vitro cytotoxicity assay. The specific caspase-3 activity in A549 cells treated with nimesulide (40 microg/ml) and doxorubicin (0.25 microg/ml) in combination was 3 and 5 times higher than doxorubicin and nimesulide, respectively. Further, TUNEL staining showed apoptosis in over 30% of A549 cells treated with aerosolized nimesulide and doxorubicin combination vs. negligible as seen in cells treated individually. The expression of COX-2 was not altered in control or treatments, whereas PPAR-gamma was expressed only in the combination treatment.
Conclusion:
Our results indicate that aerosolized nimesulide significantly enhances doxorubicin activity against A549 cells, and the enhanced cytotoxicity was probably mediated via a COX-2-independent mechanism.
A study of MCF-7 human breast cancer cells was undertaken to ascertain the degree of apoptosis induction by paclitaxel and if the induction of apoptosis could be enhanced by caffeine. Paclitaxel (0–20 ng/ml) caused concentration-dependent increases in morphologically identifiable apoptotic cells (up to 43% of cell population) and cells with DNA strand breaks (up to 38%), a commonly cited marker of apoptosis. Maximal DNA strand breakage occurred after 16 hr of exposure to paclitaxel and maximal apoptotic-appearing cells occurred after 24 hr. The remaining non-apoptotic paclitaxel-exposed cells were growth arrested in G2. A 4-hr exposure to caffeine concentration-dependently (0–20 mM) increased apoptosis to 88% of the cell population. Our results show induction of apoptosis in breast cancer cells by paclitaxel, and enhancement of this process by caffeine. Int. J. Cancer, 70:214–220, 1997. © 1997 Wiley-Liss Inc.
Hepatocellular carcinoma (HCC) is characterized by high drug resistance to currently available chemotherapeutic agents. In a prospective clinical study, we have demonstrated that high-dose tamoxifen significantly enhanced the therapeutic efficacy of doxorubicin in patients with far-advanced HCC. In a search for a possible mechanism, we found that tamoxifen at a clinically achievable concentration (2.5 microM) significantly enhanced doxorubicin-induced cytotoxicity and apoptosis of Hep-3B cells, a multidrug resistance (MDR)-1 expressing HCC cell line. This synergistic cytotoxic effect of tamoxifen, at this concentration, however, was not mediated by MDR inhibition. Instead, as evidenced by both western blot and immunofluorescence studies, tamoxifen inhibited the cytoplasmic-membrane translocation of protein kinase C (PKC)-alpha. 12-O-Tetradecanoylphorbol-13-acetate (TPA) restored the membrane translocation of PKC-alpha and abrogated the synergistic cytotoxicity of tamoxifen. We also showed that tamoxifen, at this concentration, did not directly affect the enzyme activity of PKC. Further, membrane translocation of other membrane-bound proteins, such as Ras protein, was similarly inhibited by tamoxifen, but could not be restored by the addition of TPA. Together, these data suggested that tamoxifen may act on the cytoplasmic membrane, and thereby inhibit PKC-alpha translocation to the membrane where it is activated. We hypothesize that high-dose tamoxifen may be an effective modulator of doxorubicin in the treatment of HCC, and suggest that biochemical modulation of PKC as a measure to improve systemic chemotherapy for HCC deserves further investigation.
Most cancers are associated to some degree with environmental factors. Unfortunately, people often assume that a similar proportion of cancers are preventable now through simple regulatory control directed predominantly at industrial pollution and man-made chemicals. Consequently, scientists and public health authorities are critized both for failure to develop effective prevention measures and for inadequate and misdirected research on environmental carcinogenesis, because the scientific and nonscientific limitations to effective control of many human cancers are not always recognized. Thus we have thought it worthwhile to summarize our view on the current state of environmental carcinogenesis and the possibilities of primary cancer prevention. The objectives of this paper are to stress that extensive information is already available on cancer causation in man and to summarize additional inferences concerning possible carcinogenic mechanisms and etiology that may be drawn from epidemiologic data; to emphasize the complexities inherent in the concept of 'environment', particularly, 'carcinogenic risk factors' and 'life-style' and their significance for cancer prevention; and to propose that more research is needed in human cancer on the role of cocarcinogenesis, including promotion.
The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations. Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological.
The structural changes take place in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments. In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells.
Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development. It occurs spontaneously in untreated malignant neoplasms, and participates in at least some types of therapeutically induced tumour regression. It is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal.
ImagesFig. 8-10Fig. 1Fig. 2Fig. 3Fig. 4Fig. 6Fig. 7Fig. 11-14Fig. 15-18Fig. 19Fig. 20-22Fig. 23 and 24
In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death. Although
much is known about the control of cell proliferation, less is known about the control of cell death. Physiologic cell death
occurs primarily through an evolutionarily conserved form of cell suicide termed apoptosis. The decision of a cell to undergo
apoptosis can be influenced by a wide variety of regulatory stimuli. Recent evidence suggests that alterations in cell survival
contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative
disorders, and AIDS (acquired immunodeficiency syndrome). Treatments designed to specifically alter the apoptotic threshold
may have the potential to change the natural progression of some of these diseases.
Naturally occurring and synthetic isothiocyanates are among the most effective chemopreventive agents known. A wide variety of isothiocyanates prevent cancer of various tissues including the rat lung, mammary gland, esophagus, liver, small intestine, colon, and bladder. Mechanistic studies have shown that the chemopreventive activity of isothiocyanates is due to favorable modification of Phase I and Phase II carcinogen metabolism, resulting in increased carcinogen excretion or detoxification and decreased carcinogen DNA interactions. In the majority of studies reported, the isothiocyanate must be present at the time of carcinogen exposure in order to observe inhibition of tumorigenesis. Our studies have focused on the naturally occurring isothiocyanates phenethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) as inhibitors of lung cancer. The carcinogens employed in these studies have been the major lung carcinogens in tobacco smoke- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP). Combinations of chemopreventive agents that inhibit tumorigenesis by NNK and BaP in rodents may be effective in addicted smokers. PEITC is an effective inhibitor of lung tumor induction by NNK in F-344 rats and A/J mice. BITC but not PEITC inhibits BaP induced lung tumorigenesis in A/J mice. PEITC is a selective inhibitor of the metabolic activation of NNK in the rodent lung, and studies in smokers who consumed watercress, a source of PEITC, indicate that the metabolic activation of NNK is also inhibited by PEITC in humans. Combinations of chemopreventive agents active against different carcinogens in tobacco smoke may be useful in the chemoprevention of lung cancer.