Article

Licorice and Licochalcone-A Induce Autophagy in LNCaP Prostate Cancer Cells by Suppression of Bcl-2 Expression and the mTOR Pathway

Authors:
  • Ditmanson Medical Foundation Chia-Yi Christian Hospital
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Abstract

Licorice is a common Chinese medicinal herb with antitumor activity. Some components in licorice root have been shown to induce cell cycle arrest or apoptosis in cancer cells. This paper demonstrates for the first time that licorice Glycyrrhiza glabra and its component licochalcone-A (LA) can induce autophagy in addition to apoptosis in human LNCaP prostate cancer cells. Exposure of cells to licorice or LA resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine (MDC) staining, formation of acidic vesicular organelles (AVOs), and autophagosome membrane association of microtubule-associated protein 1 light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, as well as ultrastructural observation of autophagic vacuoles by transmission electron microscopy. Autophagy induction was accompanied by down-regulation of Bcl-2 and inhibition of the mammalian target of rapamycin (mTOR) pathway. In summary, licorice can induce caspase-dependent and autophagy-related cell death in LNCaP cells.

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... In particular, Glycyrrhiza glabra -enhanced extract (GGE), commonly known as licorice, has been tested in different cancer types showing anti-oxidant, antiproliferative, and antimetastatic properties by triggering cell cycle arrest and apoptosis. Nevertheless, less is known about its potential to interfere with the autophagic machinery of cancer cells (5)(6)(7). ...
... For instance, isoliquiritigenin isolated from Glyccyrhiza glabra extract leads to S and G2/M cycle arrest of DU-145 and LNCaP cells, while in PC-3 cells triggers G2/M cycle arrest and apoptosis (30,31). Furthermore, Glyccyrhiza glabra extract and licochalcone, a flavonoid isolated from licorice extract, cause late G1 and G2 arrest in PC-3 cells, while in LNCaP cells induce both caspasedependent apoptosis and autophagy (7,32). ...
... In addition, GGE treatment alone also induced autophagy interfering with different initiating pathway of autophagy other than ULK1-AMBRA1. Besides, studies in colon, liver, and prostate (LNCaP) cancer lines also suggest the induction of autophagy as one of the mechanisms triggered by GGE and its bioactive compounds, leading to cytotoxicity (7,36,37). Interestingly, the ADR þ GGE cotreatment provoked the enhanced induction of autophagy, compared to ADR treatment alone leading to excessive cell death (12,(38)(39)(40). ...
Article
Prostate cancer is the second most commonly diagnosed cancer in men worldwide, which is almost incurable, once it progresses into the metastatic stage. Adriamycin (ADR) is a known chemotherapeutic agent that causes severe side effects. In recent years, studies in natural plant products have revealed their anticancer activities. In particular, Glycyrrhiza glabra enhanced extract (GGE), commonly known as licorice, has been reported to exert antiproliferative properties against cancer cells. In this study, the cytotoxic potential of GGE was assessed in PC-3 cells, when it is administrated alone or in combination with Adriamycin. PC-3 cells were treated with GGE and/or ADR, and the inhibition of cell proliferation was evaluated by the MTT assay. Cell cycle alterations and apoptosis rate were measured through flow cytometry. Expression levels of autophagy-related genes were evaluated with specific ELISA kits, Western blotting, and real-time PCR, while NMR spectrometry was used to identify the implication of specific metabolites. Our results demonstrated that GGE alone or in co-treatment with ADR shows antiproliferative properties against PC-3 cells, which are mediated by both apoptosis and autophagy mechanisms.
... Licochalcone A (LicA) is a chalcone constituent of licorice, it has anti-inflammatory, antitumor and anti-bacterial ability (4). LicA is a potent inhibitor of Bcl-2 protein expression which is the anti-apoptotic proteins in various tumors, LicA also can induce apoptosis in several cancer cell lines contributing to the antitumor effect (5,6). ...
... LicA has been reported to have antitumor, anti-angiogenesis and anti-inflammatory ability, and considered as Bcl-2 inhibitor (4,5,6,20). LicA can block cell cycle progression through regulating extracellular signal-regulated kinase 1/2 (ERK1/2) and Rb phosphorylation (21). ...
Article
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Chemotherapy is the best choice for the vast majority of hepatocellular carcinoma patients at late stage, but few effective chemotherapy drugs are available in clinic. Licochalcone A (LicA) is a new chemotherapy drug inducing apoptosis as Bcl-2 inhibitor, but few studies report on LicA‑induced autophagy. This study investigated the phenomenon and mechanisms of LicA-induced autophagy looking for a targeted combination drug. Human hepatocellular carcinoma cells (HCCs) were treated with LicA, to detect markers of autophagy and to investigate the mechanisms. In order to investigate the role of reactive oxygen species (ROS) in LicA‑induced autophagy, ROS, glutathione (GSH) and O2- were measured in LicA treated HCCs, and antioxidant N-Acetyl-L-cysteine (NAC) was cotreated with LicA in HCCs, then mechanisms of ROS-induced autophagy was investigated in LicA or LicA combined with NAC treated HCCs. Finally, the LicA-induced apoptosis was detected in LicA combined with NAC treated HCCs. We first report that LicA can induce autophagy through ULK1/Atg13 and ROS pathway in HCCs, suppression of LicA-induced ROS through antioxidant NAC can enhance LicA-induced apoptosis, promoting the function of LicA killing HCCs. LicA can activate the ULK1/Atg13 complex which is upstream of autophagy, additionally, LicA also can promote ROS generation, ROS trigger the expression level of TSC1/2 complex, PRAS40, CTMP, PP2A, PDK1 and Rubicon change, these molecules are upstream of autophagy. In conclusion, LicA can induce autophagy through ULK1/Atg13 and ROS pathway in HCCs, LicA combined with NAC can enhance LicA-induced apoptosis. Our results may provide a novel design for clinical hepatocellular carcinoma therapy trials.
... Licochalcone caused G2/M phase arrest of PC-3 cells by suppressing cyclin B1 and cdc2 [45]. Licochalcone also induced autophagic cell death in LNCaP cells [155]. ...
Article
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Prostate cancer is one of the leading causes of death for men worldwide. The development of resistance, toxicity, and side effects of conventional therapies have made prostate cancer treatment become more intensive and aggressive. Many phytochemicals isolated from plants have shown to be tumor cytotoxic. In vitro laboratory studies have revealed that natural compounds can affect cancer cell proliferation by modulating many crucial cellular signaling pathways frequently dysregulated in prostate cancer. A multitude of natural compounds have been found to induce cell cycle arrest, promote apoptosis, inhibit cancer cell growth, and suppress angiogenesis. In addition, combinatorial use of natural compounds with hormone and/or chemotherapeutic drugs seems to be a promising strategy to enhance the therapeutic effect in a less toxic manner, as suggested by pre-clinical studies. In this context, we systematically reviewed the currently available literature of naturally occurring compounds isolated from vegetables, fruits, teas, and herbs, with their relevant mechanisms of action in prostate cancer. As there is increasing data on how phytochemicals interfere with diverse molecular pathways in prostate cancer, this review discusses and emphasizes the implicated molecular pathways of cell proliferation, cell cycle control, apoptosis, and autophagy as important processes that control tumor angiogenesis, invasion, and metastasis. In conclusion, the elucidation of the natural compounds’ chemical structure-based anti-cancer mechanisms will facilitate drug development and the optimization of drug combinations. Phytochemicals, as anti-cancer agents in the treatment of prostate cancer, can have significant health benefits for humans.
... Licorice and its main constituents decrease cell viability in different types of cancer cells such as gastrointestinal (Khazraei-Moradian et al., 2017;Nourazarian et al., 2016;Wang et al., 2015;Shen et al., 2015;Mehdinejadiani et al., 2015;Khazraei-Moradian et al., 2014;Hibasami et al., 2006;Hibasami et al., 2005), leukemia (Hibasami et al., 2006;Hibasami et al., 2005;Chueh et al., 2012), breast Dong et al., 2007;Jo et al., 2005), prostate (Yo et al., 2009;Thiugnanam et al., 2008;Hawthorne and Gallagher, 2008;Thirugnanam et al., 2008), cervix, uterus (Lee et al., 2008), and melanoma (Abe et al., 1987) cancer cell lines. Besides, their tumor-suppressive effects have been evaluated in vivo (Yang et al., 2015;Lee et al., 2007;Agarwal and Mukhtar, 1991;Shibata et al., 1991;Shiota et al., 1999). ...
Article
Licorice (Glycyrrhiza glabra) has been considered as an herbal drug since ancient time. Nowadays, it is a well-known spice that possesses worth pharmacological effects. However, some relevant articles have revealed negative impacts of licorice in health. By considering the great wishes in using herbal medicine, it is important to show adverse effects of herbal medicine in health. At present, there are misunderstandings toward the safety of herbal medicines. Herein, we gathered scientific research projects on the toxicity effects of licorice and glycyrrhizin to highlight their safety. In this regards, we categorized our findings about the toxicity effects of licorice and glycyrrhizin in acute, sub-acute, sub-chronic, and chronic states. Besides, we discussed on the cytotoxicity, genotoxicity, mutagenicity, and carcinogenicity of licorice and glycyrrhizin as well as their developmental toxicity. This review disclosed that G. glabra and glycyrrhizin salts are moderately toxic. They need to be used with caution during pregnancy. G. glabra and glycyrrhizin possess selective cytotoxic effects on cancerous cells. The most important side effects of licorice and glycyrrhizin are hypertension and hypokalemic-induced secondary disorders. Licorice side effects are increased by hypokalemia, prolonged gastrointestinal transient time, decreased type 2 11-beta-hydroxysteroid dehydrogenase activities, hypertension, anorexia nervosa, old age, and female sex. Copyright © 2017 John Wiley & Sons, Ltd.
... Induction of autophagy is associated with many signaling pathways, and P-AKT is one of the pathways that negatively regulate autophagy [35]. Previously, our group showed that Salmonella alters various signaling pathways such as mitogen-activated protein kinase (MAPK) signaling and AKT/mTOR signaling in many cancer types [36,16]. ...
Article
Thyroid cancer has been known as the most common endocrine malignancy. Although majority of thyroid cancer types respond well to conventional treatment including surgery and radioactive iodine therapy, about 10% of those with differentiated thyroid cancer will present distant metastasis and will have persistent or recurrent disease. Even more serious is a rare type of thyroid cancer called anaplastic thyroid cancer (ATC), which accounts for about 1%, has been demonstrated as the most lethal and aggressive form of human malignancy. Unfortunately, these tumors are also frequently resistant to traditional therapy. Previous study have shown that Salmonella inhibits tumor growth, in part, by inducing autophagy - a cellular process that is important in the innate and adaptive immunity in response to viral or bacterial infection. In our study, we intended to investigate whether Salmonella can inhibit tumor growth by inducing autophagy, specifically in thyroid cancer and elucidate the possible molecular mechanism. In order to determine the signaling pathway involved in tumor cell autophagy, we used Salmonella to treat ATC cells line ASH-3 and KMH-2 in vitro. The autophagic markers, particularly autophagy-related gene 6 (Beclin-1), microtubule-associated protein 1A/1B-light chain 3 (LC3) and p62, were observed to be differentially expressed after infection with Salmonella indicating an activated autophagy in ATC cells. In addition, the protein expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), phospho-p70 ribosomal s6 kinase (P-p70S6K) in tumor cells were decreased after Salmonella infection. In vivo, we also found that substantial cell numbers of Salmonella targeted tumor tissue, and regulated anti-tumor mechanisms. Our findings showed that Salmonella activated autophagic signaling pathway and inhibited ATC tumor growth via downregulation of AKT/mTOR pathway.
... 9,10 Licochalcone A (LicA) is a characteristic chalcone of licorice, which is the root of Glycyrrhiza inflate. 11 LicA, the most potent component of licorice, has been reported to elicit anti-inflammatory, 12 antiangiogenesis, 13 and antitumor 14,15 effects in various tumor cells. LicA is reported to inhibit the migration and invasion of human lung cancer cells through the deactivation of the Akt signaling pathway and downregulation of MMP-1/-3 expression. ...
Article
To improve the clinical outcome of tumor chemotherapy, more effective combination treatments against tumor metastasis and recurrence are required. Licochalcone A (LicA) is the root of Glycyrrhiza inflata and has been reported to possess anti‐inflammatory, antimicrobial, and antitumor effects. Sorafenib (Sor), a multikinase inhibitor, is used to treat patients with solid tumors such as advanced hepatocellular carcinoma (HCC). However, the synergistic effects of LicA and Sor on the metastasis of human HCC cells have not been reported. We found that LicA and Sor did not have cytotoxic effects or arrest growth in human SK‐Hep‐1 and Huh‐7 cells. In addition, treatment with LicA or Sor alone inhibited migration and invasion in human SK‐Hep‐1 and Huh‐7 HCC cells. Furthermore, cotreatment with LicA and Sor synergistically inhibited the migration and invasion of HCC cells and significantly inhibited uPA protein expression. Notably, cotreatment of LicA and Sor synergistically and significantly downregulated MKK4‐JNK expression. Through tail vein injection in nude mice, the aforementioned cotreatment synergistically suppressed SK‐Hep‐1 cell‐mediated lung metastasis. These findings first revealed the synergistic effects of LicA and Sor cotreatment against human HCC cells, further suggesting that beneficial effects on tumor regression could be confirmed through prospective clinical trials.
... G. uralensis is found in Central Asia to the northeastern part of China and G. glabra is distributed from southern Europe to the northwestern part of China. These species are characterized by various pharmacological activities, including anti-oxidant, antiinflammation, immune improvement, and anti-tumor effects [16][17][18][19]. Previous research has reported that licorice inhibits Aβ -induced neurotoxicity in a cellular model by modulating oxidant stress [20]. ...
Article
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BACKGROUND/OBJECTIVES Neuroinflammation plays critical role in neurodegenerative disorders, such as Alzheimer's disease (AD). We investigated the effect of three licorice varieties, Glycyrhiza uralensis, G. glabra, and Shinwongam (SW) on a mouse model of inflammation-induced memory and cognitive deficit. MATERIALS/METHODS C57BL/6 mice were injected with lipopolysaccharide (LPS; 2.5 mg/kg, intraperitoneally) and orally administrated G. uralensis, G. glabra, and SW extract (150 mg/kg/day). SW, a new species of licorice in Korea, was combined with G. uralensis and G. glabra. Behavioral tests, including the T-maze, novel object recognition and Morris water maze, were carried out to assess learning and memory. In addition, the expressions of inflammation-related proteins in brain tissue were measured by western blotting. RESULTS There was a significant decrease in spatial and objective recognition memory in LPS-induced cognitive impairment group, as measured by the T-maze and novel object recognition test; however, the administration of licorice ameliorated these deficits. In addition, licorice-treated groups exhibited improved learning and memory ability in the Morris water maze. Furthermore, LPS-injected mice had up-regulated pro-inflammatory proteins, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2, interleukin-6, via activation of toll like receptor 4 (TLR4) and nuclear factor-kappa B (NFκB) pathways in the brain. However, these were attenuated by following administration of the three licorice varieties. Interestingly, the SW-administered group showed greater inhibition of iNOS and TLR4 when compared with the other licorice varieties. Furthermore, there was a significant increase in the expression of brain-derived neurotrophic factor (BDNF) in the brain of LPS-induced cognitively impaired mice that were administered licorice, with the greatest effect following SW treatment. CONCLUSIONS The three licorice varieties ameliorated the inflammation-induced cognitive dysfunction by down-regulating inflammatory proteins and up-regulating BDNF. These results suggest that licorice, in particular SW, could be potential therapeutic agents against cognitive impairment.
... The MMP was measured by assessing DiOC6(3) uptake to detect the mitochondrial apoptosis pathway, as previously described [19]. Briefly, different types of HPV-E6/ E7 primary epithelial cells were treated with SR-T100 for 12 h, followed by staining with 40 nM DiOC6(3) for 30 min at 37°C in the dark. ...
Article
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Background The Solanum species have been used for the treatment of warts, tumor and cancer in folk medicine. The S. incanum extract is an important traditional Chinese medicine in Taiwan since 1973. The purpose of the present study was to evaluate the efficacy and safety of Solanum incanum (synonym: Solanum undatum) extract (SR-T100), a water-soluble product primarily composed of alkaloid solamargine, for the treatment of human condyloma and to study the possible underlying anti-condyloma mechanisms. Methods We conducted a pilot study to test the effectiveness and side effects of SR-T100 gel (2.3% solamargine in Solanum incanum plant extract) for the treatment of external genital warts. We produced different types of human papillomavirus (HPV) E6/E7-infected cells by lentiviral technology and studied the differences in apoptosis and autophagy between these cells under the treatment of SR-T100. ResultsNineteen (73%) of 26 patients using the SR-T100 gel exhibited a response, and 16 (61.5%) patients achieved total clearance. Only one patient showed severe (grade 3-4) skin-related side effects. SR-T100 induced mitochondria-dependent apoptosis in HPV-infected cells. Cells expressing the high-risk HPV E6/E7 type were resistant to SR-T100-induced apoptosis. SR-T100 induced a greater autophagic response in HPV 16, 18-E6/E7 cells than in HPV 6b, 11-E6/E7 cells. Autophagy inhibition enhanced SR-T100-induced apoptosis in HPV 16, 18-E6/E7 cells, whereas apoptosis inhibition enhanced SR-T100-induced autophagy in HPV 6b, 11-E6/E7 cells. ConclusionsSR-T100 is effective for the treatment of human vulva condyloma, with few side effects. Compared with those with high-risk HPVs, cells with low-risk HPVs were more sensitive to SR-T100 treatment. Autophagy played a protective role in SR-T100-induced apoptosis in HPV-infected cells. Trial registrationNCT01676792; Registered: August 29, 2012 (retrospectively registered).
... Furthermore, some bulk polyphenolic extracts with high content of flavonoids which are pulled out from different plants, such as Hibiscus sabdariffa, Solanum nigrum or Sideritis scardica, are also able to induce autophagy in some tumor cell lines [94][95][96]. Not only flavonoids can induce autophagy in vitro, but chalcones, which are flavonoid precursors, also trigger autophagy in some in vitro models, for instance, in lung cancer and in colon cancer cells [97][98][99][100][101][102][103][104]. ...
Article
Background: Autophagy is a cellular pathway with the ability to maintain cell homeostasis through the elimination of damaged or useless cellular components, and its deregulation may initiate or aggravate different human diseases. Flavonoids, a group of plant metabolites, are able to modulate different molecular and cellular processes including autophagy. Objective: To review the effects of flavonoids on autophagy pathway in both invasive and noninvasive human diseases, focusing on the global outcomes in their progression. Moreover, the efficacy of the combination of flavonoids with drugs or other natural nontoxic compounds was also reviewed. Methods: A literature search was performed to identify and analyze peer-reviewed publications containingin vitroandin vivostudies focused on autophagy deregulation in different proliferative and non-proliferative pathologies and the potential protective effects of flavonoids. Results: Analyzed publications indicated that imbalance between cell death and survival induced by changes in autophagy play an important role in the pathophysiology of a number of human diseases. The use of different flavonoids as autophagy modulators, alone or in combination with other molecules, might be a worthy strategy in the treatment of cancer, neurodegenerative disorders, cardiovascular diseases, hepatic diseases, leishmaniasis, influenza, gastric ulcers produced byHelicobacter pyloriinfection, diabetes, asthma, age-related macular degeneration or osteoporosis. Conclusion: Flavonoids could potentially constitute important adjuvant agents of conventional therapies in the treatment of autophagy deregulation-related diseases. Moreover, combined therapy may help to diminish the doses of those conventional treatments, leading to reduced drug-derivative side effects and to improved patients' survival.
... Several reports have established that various extracts of licorice (Glycyrrhiza spp.) root exerted anticancer effects. 30−33 Yo et al. 30 demonstrated that G. glabra and licochalcone A induced autophagy and apoptosis in LNCaP cells. Furthermore, the effects of the ethanol extract of roasted licorice, licochalcone A, and isoliquiritigenin were investigated against breast cancermediated bone destruction. ...
Article
Glycyrrhiza glabra cultivation and harvesting produces substantial quantities of aerial parts as waste. With the aim to prospect an innovative valorization of these byproducts, the aerial parts were harvested in May and October and analyzed for their chemical profile, antioxidant properties, and effects on viability of five cancer cell lines. Pinocembrin was the main constituent. A significant protection of lipid peroxidation was observed with the May total extract (IC50 of 4.2 ± 0.4 μg/mL at 30 min of incubation). The effects on viability of HeLa, MCF-7, MDA-MB-231, Caco-2, and PC3 human cancer cells were investigated. All samples shown a remarkable activity with IC50 values below 25 μg/mL. Samples from plants harvested in May exhibited greater activity than those harvested in October. MCF-7 and HeLa were the most sensitive cells with IC50 in the range 2.73–3.01 and 3.28–5.53 μg/mL, respectively. G. glabra aerial parts represent a good source of valuable products.
... Various antioxidant properties of Glycyrrhiza glabra roots has also been reported [1][2][3] which are attributed to the phenolic compounds such as Glabridin, Licoisoflavone B, Licochalcone and Liquirtigenin 4 . These flavonoids of Glycyrrhiza glabra have also been reported to exhibit multiple pharmacological properties like antiparasitic, anti-inflammatory, antibacterial and anti-tumor and choleretic properties [5][6][7][8][9] . However, the extraction of these compounds from field grown plants requires uprooting of the plant leading to complete loss of the plant. ...
... However, the pharmacological basis underlying the effects of MSJZT on asthma still needs to be elucidated. Recently, A. membranaceus and Glycyrrhiza, key herbs in MSJZT, were showed to have inhibitory effects on the mTOR signaling pathway (Yo et al., 2009;Zhou et al., 2018). Therefore, the goal of this study was to confirm the anti-asthmatic effects of MSJZT during the remission phase in a murine model of established asthma, and to investigate the possible mechanisms of underlying the action of MSJZT, with particular emphasis on the mTOR signaling pathway. ...
Article
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Background: Modified Si–Jun–Zi–Tang (MSJZT), a multi-herb formulation, is frequently used in traditional Chinese medicine for patients during the remission stage of asthma. However, the pharmacological basis underlying the effects of MSJZT on asthma has yet to be elucidated. This study aims at evaluating the anti-asthmatic effects of MSJZT and investigating its possible mechanism. Methods: A chronic murine model of asthma was established by sensitization and repeated challenge with ovalbumin (OVA) in female BALB/c mice, followed with oral administration of MSJZT during remission, and then mouse were re-challenged by OVA. The chemical profile of MSJZT was analyzed by high-performance liquid chromatography. The characteristic features of allergic asthma, including airway hyperreactivity, histopathology, cytokine levels (IL-4, -5, -13, -17, and INF-γ), T regulatory (Treg) lymphocytes (Foxp3+CD4+CD25+), and T effector (Teff) lymphocytes (Foxp3-CD25+CD4+) in bronchoalveolar lavage fluid (BALF), and downstream proteins of mTORC1/2 signaling pathway were examined. Results: MSJZT markedly suppressed airway hyper-responsiveness to aerosolized methacholine, and reduced levels of IL-4, IL-5, and IL-13 in the BALF. Histological studies showed that MSJZT significantly reduced inflammatory infiltration in lung tissues. The percentage and absolute number of Teff cells were suppressed to a remarkable level by MSJZT without affecting Treg cells. Furthermore, MSJZT effectively inhibited the mTORC1 activity, but exerted limited effects on mTORC2, as assessed by the phosphorylation of the mTORC1 and mTORC2 substrates, S6 ribosomal protein, p70 S6 kinase, mTOR S2481, and Akt, respectively. Conclusion: MSJZT attenuated chronic airway inflammation in a mouse model of asthma by inhibiting Teff cells, which occurred, at least in part, via modulation of the mTORC1 signaling pathway.
... Licochalcone-A, a chalconoid and a type of natural phenol, has been isolated from the root of the licorice plant and has exhibited various pharmacological effects, including antimalarial, anticancer, antibacterial and antiviral properties (7). Furthermore, treatment with licochalcone-A induced apoptosis in various cancer cell types, including oral (36), bladder (37,38), lung (39), gastric (40) and prostate cancer cells (41). In addition, Lico-E, a retrochalone, with various pharmacological effects, including antiparasitic, antibacterial, antioxidative and superoxide-scavenging properties, has been isolated from the root of licorice (24). ...
Article
The aim of the present study was to investigate licochalcone-E (Lico-E)-induced apoptosis and the associated apoptotic signaling pathway in FaDu cells, a human pharyngeal squamous carcinoma cell line. Treatment with Lico-E exhibited significant cytotoxicity on FaDu cells in a concentration-dependent manner. The IC50 value of Lico-E in FaDu cells was ~50 µM. Treatment with Lico-E increased the number of dead FaDu cells. Furthermore, chromatin condensation, which is associated with apoptotic cell death, was observed in FaDu cells treated with Lico-E for 24 h. By contrast, Lico-E did not produce cytotoxicity or increase the number of dead cells when applied to human normal oral keratinocytes (hNOKs). Furthermore, chromatin condensation was not observed in hNOKs treated with Lico-E. Treatment with Lico-E increased the expression of Fas ligand and the cleaved form of caspase-8 in FaDu cells. Furthermore, treatment with Lico-E increased the expression of pro-apoptotic factors, including apoptosis regulator BAX, Bcl-2-associated agonist of cell death, apoptotic protease-activating factor 1, caspase-9 and tumor suppressor p53, while decreasing the expression of anti-apoptotic factors, including apoptosis regulator Bcl-2 and Bcl-2-like protein 1 in FaDu cells. The expression of cleaved caspases-3 and poly (ADP-ribose) polymerase was significantly upregulated following treatment with Lico-E in FaDu cells, while Lico-E-induced apoptotic FaDu cell death was partially suppressed by treatment with Z-VAD-FMK, a pan caspase inhibitor. Therefore, Lico-E-induced oral cancer (OC) cell-specific apoptosis is mediated by the death receptor-dependent extrinsic and mitochondrial-dependent intrinsic apoptotic signaling pathways. In conclusion, these data suggested that Lico-E exhibits potential chemopreventive effects and warrants further developed as a chemotherapeutic agent against OC.
... In a study of gastric cancer, LCA was shown to affect gastric cancer cell viability by blocking cell cycle progression and inducing apoptosis. In addition, LCA induces autophagy through the mTOR pathway in prostate cancer cells (Yo et al., 2009). Furthermore, LCA was determined to induce apoptosis via the FasL signaling pathway . ...
Article
Ethnopharmacological relevance: Licochalcone A (LCA) is a characteristic chalcone that is found in licorice, which is a traditional medicinal plant. In traditional medicine, LCA possesses many potential biological activities, including anti-parasitic, anti-inflammatory and antitumor activities. Aim of the study: To determine the antioxidant activity of LCA and, on this basis, to investigate the role of its anticancer activity. Materials and methods: To validate the antioxidant activity of LCA, the proteins SOD, CAT and GPx1 were analyzed using western blotting and cellular antioxidant activity (CAA) assays. Oxidative free radicals are associated with cancer cells. Therefore, the anticancer activity of LCA was also evaluated. To assess the anticancer activity, cell viability assays were performed and apoptosis was evaluated. In addition, MAPK-related proteins were analyzed using western blotting. Results: The experimental data showed that the EC50 of LCA is 58.79 ± 0.05μg/mL and 46.29 ± 0.05μg/mL under the two conditions tested, with or without PBS. In addition, LCA at a concentration of approximately 2-8μg/mL can induce the expression of SOD, CAT and GPx1 proteins. Further, LCA inhibits the growth of HepG2 cells through cell proliferation arrest and the subsequent induction of apoptosis, and LCA attenuated the p38/JNK/ERK signaling pathway in a dose-dependent manner. Conclusion: The results showed that LCA suppresses the oxidation of cells and markedly inhibits the proliferation of cancer cells. These findings confirm the traditional use of LCA in folk medicine.
... The use of natural products in gastric cancer therapy has been the focus of a number of studies (5)(6)(7)(8)(9); licorice is a traditional herbal medicine that originates from the licorice root, and is one of the most commonly prescribed herbs in China for the treatment of various diseases, including microbial infections and cancer (10,11). Licochalcone A (LCA; C 21 H 22 O 4 ; molecular weight, 338.4) is the primary active compound in the licorice species Glycyrrhiza glabra and is an estrogenic flavonoid with antiparasitic, antibacterial and antitumor properties (12)(13)(14). It has been demonstrated previously that LCA is the most cytotoxic licorice compound, and is able to inhibit the growth of gastric cancer cells by arresting cell cycle progression and inducing apoptosis (15). ...
Article
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Licochalcone A (LCA) is a flavonoid extracted from licorice root that has antiparasitic, antibacterial and antitumor properties. Previous studies have revealed that LCA may be a novel treatment for gastric cancer. The present study further assessed the potential antitumor effects of LCA alone or in combination with 5-fluorouracil (5-FU), and the underlying mechanisms responsible for those effects in gastric cancer cells. The effects of LCA alone or in combination with 5-FU on SGC7901 and MKN-45 gastric cancer cell lines were studied using Cell Counting Kit-8, cell cycle, apoptosis and western blot analyses of cell check points and apoptosis-associated proteins. The results revealed that LCA inhibited cell proliferation, blocked cell cycle progression at the G2/M transition and induced apoptosis. Western blot analysis demonstrated that LCA treatment increased the levels of tumor proteins 21 and 27, as well as mouse double minute 2 homolog in gastric cancer cells. In addition, LCA treatment increased the expression levels of Bax, cleaved-poly ADP ribose polymerase, tumor protein 53 and caspase 3, and decreased the expression levels of Bcl-2. Therefore, the present study demonstrated that LCA alone or in combination with 5-FU may have significant anticancer effects on gastric cancer cells, and may be a novel therapeutic for the treatment of gastric cancer in the future.
... The Bcl-2 protein family includes major regulators of cell survival, including Bax, which promotes or suppresses apoptosis progression. Additionally, the caspase signaling pathway was partly regulated by Bcl-2 (19). The data above indicated that restoration of miR-370 could suppress the growth and proliferation of gastric cancer cells and PTEN might be a potential target of miR-370. ...
Article
Growing evidence suggests that microRNA plays an essential role in the development and metastasis of many tumors, including gastric cancer. Aberrant miR‑370 expression has been indicated in tumor growth, but the mechanism of miR‑370 inhibits both the proliferation and metastatic ability for gastric cancer remains unclear. Accumulating evidence reported that PTEN signaling pathway plays an important role in the cellular processes, such as apoptosis, cell growth and proliferation. The goal of this study was to identify whether miR‑370 could inhibit the growth, migration, invasion, proliferation and metastasis of gastric cancer through targeting PTEN. Real-time PCR (RT-PCR) was used to quantify miR-370 expression in vitro experiments. The biological functions of miR‑370 were determined via cell proliferation. Our study indicated that miR‑370 targeted PTEN leading to activation of apoptosis signaling and the cell proliferation of cervical cancer cells, ameliorating gastric cancer growth and progression. In addition, the combination of miR‑370 and PTEN inactivated AKT, MDM2 and mTOR while stimulated caspase-3, p53 and GSK3β expression, promoting apoptosis and suppressing proliferation of gastric cancer cells. Therefore, our study revealed the mechanistic links between miR‑370 and PTEN in the pathogenesis of gastric cancer through modulation of cell apoptosis and proliferation. Additionally, targeting miR‑370 could serve as a novel strategy for future gastric cancer therapy clinically.
... For instance, a number of different kinds of cancer have been treated with TCM drugs either alone or in combination with conventional therapies [11,12]. Glycyrrhiza uralensis Fisch. is one of the oldest prescribed herb in Chinese traditional medicine for the treatment of various disease syndromes including cancer, by arresting tumorigenesis and metastasis [13][14][15][16][17][18]. Glycyrrhiza uralensis belongs to the Family Fabaceae (Leguminosae), genus Glycyrrhiza and species Glycyrrhiza glabra, Glycyrrhiza lepidota, and Glycyrrhiza uralensis found in Europe, Asia, Russia and Turkey where its roots are commonly used. ...
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Background Chinese licorice, (Glycyrrhiza uralensis Fisch.) is one of the commonly prescribed herbs in Traditional Chinese Medicine (TCM). Gancao, as commonly known in China, is associated with immune-modulating and anti-tumor potential though the mechanism of action is not well known. In this study, we investigated the in vitro immunomodulatory and antitumor potential of Glycyrrhiza uralensis polysaccharides fractions of high molecular weight (fraction A), low molecular weight (fraction B) and crude extract (fraction C). Methods Cell proliferation and cytotoxicity was investigated using Cell Counting kit 8 (CCK-8) on Intestinal epithelial cell line (IEC-6) and Colon carcinoma cell line (CT-26). IL-7 gene expression relative to GAPDH was analysed using Real time PCR. The stimulation and viability of T lymphocytes was determined by Trypan blue exclusion assay. Results G.uralensis polysaccharides did not inhibit proliferation of IEC-6 cells even at high concentration. The ED50 was found to be 100 μg/ml. On the other hand, the polysaccharides inhibited the proliferation of cancer cells (CT-26) at a concentration of ≤50 μg/ml. Within 72 h of treatment with the polysaccharides, expression of IL-7 gene was up-regulated over 2 times. It was also noted that, IEC-6 cells secrete IL-7 cytokine into media when treated with G.uralensis polysaccharides. The secreted IL-7 stimulated proliferation of freshly isolated T lymphocytes within 6 h. The effect of the polysaccharides were found to be molecular weight depended, with low molecular weight having a profound effect compared to high molecular weight and total crude extract. Conclusion Our findings indicate that G.uralensis polysaccharides especially those of low molecular weight have a potential as anticancer agents. Of great importance, is the ability of the polysaccharides to up-regulate anticancer cytokine IL-7, which is important in proliferation and maturation of immune cells and it is associated with better prognosis in cancer. Therefore, immunomodulation is a possible mode of action of the polysaccharides in cancer therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1171-4) contains supplementary material, which is available to authorized users.
... We found that miR-224 significantly stimulated Bcl-2 expression via mTOR upregulation in vivo and in vitro experiments, inducing Bax, caspase-9 and caspase-3 down-regulation, inhibiting apoptosis in gastric cancer development. Caspase-3 is known as an essential activator for apoptosis development in tissues and cells via Bcl-2 and its family members, which are involved in cancer progression based on previous studies (35)(36)(37). Notably, miR-224 suppression could reverse expression of these proteins with mTOR and Bcl-2 downregulation while with Bax, caspase-9 and caspase-3 were upregulated, thus, leading to apoptosis in gastric cancer and promoting cancer cell attenuating gastric cancer progression. Collectively, the results above elucidated that miR-224 might be linked to gastric cancer progression via mTOR-regulated caspase-3 signaling pathway. ...
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Growing evidence suggests that microRNA plays an essential role in the development and metastasis of many tumors, including gastric cancer. Aberrant miR-224 expression has been indicated in tumor growth, the mechanism of miR-224 promoting the proliferation and metastatic ability for gastric cancer remains unclear. Accumulating evidence reports that mTOR signal pathway plays an important role in the cellular process, such as apoptosis, cell growth and proliferation. The goal of the present study was to identify whether miR-224 could inhibit the growth, migration, invasion, proliferation and metastasis of gastric cancer through targeting mTOR expression. Real-time PCR (RT-PCR) was used to quantify miR-224 expression in vitro and in vivo experiments. Luciferase reporter assays were performed to confirm the activity of mTOR pathway, and immunofluorescence staining assay was conducted to observe apoptosis and cell proliferation ability. Bioinformatics as well as cell luciferase function studies distinguished the direct modulation of miR-224 on the 3'-UTR of the mTOR, which leads to the inactivation of apoptosis signaling and the activation of cell proliferation. In addition, inhibition of miR-224 significantly reduced the expression of mTOR and improved caspase-9/3 expression while decreased cyclin D1/2 levels, attenuating gastric cancer cell proliferation. Therefore, the present study revealed the mechanistic links between miR-224 and mTOR in the pathogenesis of gastric cancer through modulation of caspase-9/3 and cyclin D1/2. In addition, targeting miR-224 could serve as a novel strategy for future gastric cancer therapy.
... V posledních dvou desetiletích byla zjištìna øada dalších úèinkù lékoøice v experimentech in vitro i in vivo. Zjištìny byly úèinky antibakteriální (Astafeva a Sukhenko, 2014 ), antivirové (Laconi et al., 2014), antifungální (Fatima et al., 2009), antiprotozoální (Sangian et al., 2013), hepatoprotektivní a antioxidaèní (Yin et al., 2011) a protinádorové (Sheela et al., 2006; Yo et al., 2009). Lékoøice pùsobí také na centrální nervový systém, ovlivòuje srážlivost krve a imunitní systém, pùsobí i jako antioxidans a scavenger volných radikálù (Asl a Hosseinzadeh, 2008; Olukoga a Donaldson, 2000; Jahodáø, 2010; Kaur et al., 2013). ...
Article
The licorice (liquorice) plant (Glycyrrhiza glabra) is a legume native to southern Europe, India, and parts of Asia. It is a herbaceous perennial, growing to 1 m in height, also known as „sweet root.“ Licorice root contains compounds that are about 50 times sweeter than sugar. Much of the sweetness in licorice comes from glycyrrhizin. Licorice root has been used in both Eastern and Western medicine to treat a variety of illnesses, ranging from the common cold to liver disease. It is still used today for several conditions, although not all its uses are supported by scientific evidence. In the past few years a neuroprotective effect of substances present in licorice has been shown. Mainly liquiritigenin, one of the active ingredients licorice roots, could indicate the development of a new drug for neurodegenerative diseases. Interesting is also its antidepressive and anxiolytic effect
... Previous studies indicated that LCA induced autophagy in prostate cancer LNCaP cells as evidenced by the increasing of the LC3-II expression and autophagosome accumulation 34 . However, up-regulation of LC3-II expression or autophagosome accumulation might be an autophagic inducer (increasing the autophagic flux) or inhibitor (suppressing the fusion of autophagosome with lysosome or the function of lysosome) 27,28 . ...
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Licochalcone A (LCA), a flavonoid isolated from the famous Chinese medicinal herb Glycyrrhiza uralensis Fisch, presents obvious anti-cancer effects. In this study, the anti-cancer effects and potential mechanisms of LCA in non-small cell lung cancer (NSCLC) cells were studied. LCA decreased cell viability, increased lactate dehydrogenase release, and induced apoptosis in a concentration-dependent manner in NSCLC cells while not in human embryonic lung fibroblast cells. The expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II) and formation of GFP-LC3 punta, two autophagic markers, were increased after treatment with LCA. LCA-induced LC3-II expression was increased when combined with chloroquine (CQ), while knock-down of autophagy related protein (ATG) 7 or ATG5 reversed LCA-induced LC3-II expression and GFP-LC3 punta formation, suggesting that LCA induced autophagy in NSCLC cells. Inhibition of autophagy could not reverse the LCA-induced cell viability decrease and apoptosis. In addition, LCA increased the expression of endoplasmic reticulum stress related proteins, such as binding immunoglobulin protein and C/EBP homologous protein (CHOP). Knock-down of CHOP reversed LCA-induced cell viability decrease, apoptosis, and autophagy. Taken together, LCA-induced autophagic effect is an accompanied phenomenon in NSCLC cells, and CHOP is critical for LCA-induced cell viability decrease, apoptosis, and autophagy.
... And licochalcone a (LCA) showed the best score in three compounds. According to previous study, LCA can inhibit BCL2 for inducing autophagy and promoting apoptosis in cancer cells [49]. Another study reported that LCA induced autophagy effect in NSCLC cells [50]. ...
Article
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Background: Chinese traditional herbal medicine Fuzhengkangai (FZKA) formulation combination with gefitinib can overcome drug resistance and improve the prognosis of lung adenocarcinoma patients. However, the pharmacological and molecular mechanisms underlying the active ingredients, potential targets, and overcome drug resistance of the drug are still unclear. Therefore, it is necessary to explore the molecular mechanism of FZKA. Methods: A systems pharmacology and bioinformatics-based approach was employed to investigate the molecular pathogenesis of EGFR-TKI resistance with clinically effective herb formula. The differential gene expressions between EGFR-TKI sensitive and resistance cell lines were calculated and used to find overlap from targets as core targets. The prognosis of core targets was validated from the cancer genome atlas (TCGA) database by Cox regression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment is applied to analysis core targets for revealing mechanism in biology. Results: The results showed that 35 active compounds of FZKA can interact with eight core targets proteins (ADRB2, BCL2, CDKN1A, HTR2C, KCNMA1, PLA2G4A, PRKCA and LYZ). The risk score of them were associated with overall survival and relapse free time (HR = 6.604, 95% CI: 2.314-18.850; HR = 5.132, 95% CI: 1.531-17.220). The pathway enrichment suggested that they involved in EGFR-TKI resistance and non-small cell lung cancer pathways, which directly affect EGFR-TKI resistance. The molecular docking showed that licochalcone a and beta-sitosterol can closely bind two targets (BCL2 and PRKCA) that involved in EGFR-TKI resistance pathway. Conclusions: This study provided a workflow for understanding mechanism of CHM for against drug resistance.
... LA induced autophagy via PI3K/Akt/mTOR signaling which repressed cervical cancer growth [283]. Androgen-sensitive prostate adenocarcinoma and adenoid cystic carcinoma were sensitive to LA-and ISL-induced autophagic cell death mediated by mTOR inhibition [146,284]. ISL also suppressed breast cancer progression through autophagy induction [285]. Therefore, Radix glycyrrhizae has the chemotherapeutic potential to function as an effective cancer therapeutic. ...
Article
Autophagy is a universal catabolic cellular process for quality control of cytoplasm and maintenance of cellular homeostasis upon nutrient deprivation and environmental stimulus. It involves the lysosomal degradation of cellular components such as misfolded proteins or damaged organelles. Defects in autophagy are implicated in the pathogenesis of diseases including cancers, myopathy, neurodegenerations, infections and cardiovascular diseases. In the recent decade, traditional drugs with new clinical applications are not only commonly found in Western medicines, but also highlighted in Chinese herbal medicines (CHM). For instance, pharmacological studies have revealed that active components or fractions from Chaihu (Radix bupleuri), Hu Zhang (Rhizoma polygoni cuspidati), Donglingcao (Rabdosia rubesens), Hou po (Cortex magnoliae officinalis) and Chuan xiong (Rhizoma chuanxiong) modulate cancers, neurodegeneration and cardiovascular disease via autophagy. These findings shed light on the potential new applications and formulation of CHM decoctions via regulation of autophagy. This article reviews the roles of autophagy in the pharmacological actions of CHM and discusses their new potential clinical applications in various human diseases.
... Licochalcone A inhibits the proliferation of human colon (Lee et al., 2008), prostate (Fu et al., 2004;Yo et al., 2009), liver (Niu et al., 2018;Wang et al., 2018), breast (Bortolotto et al., 2017;Kang et al., 2017;Xue et al., 2018), and lung (Chen et al., 2018;Qiu et al., 2017;Tang et al., 2016) cancer cells. In agreement with previous studies (Chen et al., 2018;Qiu et al., 2017;Tang et al., 2016), we found that licochalcone A inhibited A549 cell proliferation by inducing apoptosis, cell cycle arrest, and autophagy. ...
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Background Chinese herbal medicines (CHMs) are a resource of natural compounds (ingredients) and their potential chemical derivatives with anticancer properties, some of which are already in clinical use. Bei–Mu (BM), Jie–Geng (JG), and Mai–Men–Dong–Tang (MMDT) are important CHMs prescribed for patients with lung cancer that have improved the survival rate. Hypothesis/Purpose The aim of this study was to systemically investigate the mechanisms of action of these CHM products in lung cancer cells. Methods We used a network pharmacology approach to study CHM product-related natural compounds and their lung cancer targets. In addition, the underlying anti-lung cancer effects of the natural compounds on apoptosis, cell cycle progression, autophagy, and the expression of related proteins was investigated in vitro. Results Ingredient-lung cancer target network analysis identified 20 natural compounds. Three of these compounds, ursolic acid, 2-(3R)-8,8-dimethyl-3,4-dihydro-2H-pyrano(6,5-f)chromen-3-yl)-5-methoxyphenol, and licochalcone A, inhibited the proliferation of A549 lung cancer cells in a dose-dependent manner. Signal pathway analyses suggested that these three ingredients may target cellular apoptosis, anti-apoptosis, and cell cycle-related proteins. These three ingredients induced apoptosis through the regulation of the expression of apoptotic and anti-apoptotic proteins, including B-cell lymphoma-2 and full-length and cleaved poly(ADP-ribose) polymerase proteins. They also induced cell cycle arrest in S and G2/M phases and autophagy in A549 cells. Conclusion The pharmacological mechanisms of ingredients from MMDT on lung cancer may be strongly associated with their modulatory effects on apoptosis, autophagy, cell cycle progression, and cell proliferation.
... Autophagy plays a dual role during the physiological process and tumorigenesis including maintenance of cell survival and the defeat of cell malfunction (Hippert et al. 2006;Mizushima 2007). It was reported that the inducing (Yo et al. 2009) and suppression (Law et al. 2014) of autophagy could both inhibit tumor cell viability. In the present study, we further investigated the effect of (GlcN) 5 on autophagy in HepG2 cells. ...
Article
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Hepatocellular carcinoma (HCC) is one of the most prevalent and deadliest cancers. In this study, the anti-tumor effect of singular degree of polymerization (DP) chitooligosaccharides (COS) (DP 2–5) and the underlay molecular mechanisms were investigated on HCC cell line HepG2. MTT assay showed that (GlcN) 5 have the best anti-proliferation effect among the different DP of COS (DP2-5). Furthermore, the administration of (GlcN) 5 could decrease mitochondrial membrane potential, release cytochrome c into cytoplasm, activate the cleavage of Caspases9/3, thus inducing mitochondrial-mediated apoptosis in HepG2 cells (accounting for 24.57 ± 2.25%). In addition, (GlcN) 5 treatment could increase the accumulation of autophagosomes. Further investigation showed that (GlcN) 5 suppressed protective autophagy at the fusion of autophagosomes and lysosomes. Moreover, the inhibition of protective autophagy flux by (GlcN) 5 could further decrease cell viability and increase the apoptosis rate. Our findings suggested that (GlcN) 5 suppressed HepG2 proliferation through inducing apoptosis via the intrinsic pathway and impairing cell-protective autophagy. COS might have the potential to be an agent for lowering the risk of HCC.
... ese results indicated that overexpression of Rab23 and LCA could inhibit the proliferation, migration, and invasion of glioma U251 cells, and the combined treatment had a better effect. Studies revealed that LCA can activate mitochondrialdependent endogenous apoptosis, and can downregulate the level of antiapoptotic protein Bcl-2 and upregulate the level of proapoptotic protein Bax [16,17]. e balance of Bcl-2 and Bax protein expression levels determines the state of cell survival or apoptosis [18]. ...
Article
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This research aimed to explore the effect of Licochalcone-A (LCA) combined with Rab23 gene on the proliferation, migration, and invasion of glioma U251 cells through the Wnt/β-catenin signaling pathway. The glioma U251 cell line was taken as the research object, and the Rab23 overexpression plasmid was constructed. According to the treatment method, U251 cells were rolled into blank control group (BC), Rab23 overexpression plasmid transfection group (Rab23), 25 μmol·L−1 LCA treatment group (LCA), and Rab23 overexpression plasmid transfection combined with 25 μmol·L−1 LCA treatment group (Rab23 + LCA). Subsequently, the ability of cell proliferation, migration, and invasion of each group was detected by methyl thiazolyl tetrazolium (MTT) assay, scratch healing test, and Transwell cell invasion test, respectively. Western blot was implemented to detect the expression differences of cell proliferation antigen Ki-67, apoptosis-related proteins Bcl-2 and Bax, and Wnt/β-catenin pathway-related proteins β-catenin, glycogen synthase kinase-3 (GSK3β), Axin2, and c-myc. The results showed the successful construction of Rab23 overexpression and stable transfection U251 cell line. After grouping and treatments, the cell proliferation, migration, and invasion ability of the Rab23 group, LCA group, and Rab23 + LCA group was substantially reduced relative to BC group (P
... Licochalcone A (Lico A) is a flavonoid extracted from licorice root, demonstrating a wide range of pharmacological effects, including the anti-inflammatory and antitumor activities [16,17]. The antitumor effects of Lico A have been documented in various types of tumors, including gastric, [18] prostate, [19] ovarian, [20] liver [21], and lung cancers [16,22]. The Lico A promotes cell cycle arrest, induces apoptosis, and suppresses angiogenesis and metastasis [23][24][25]. ...
Article
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Met proto-oncogene (MET) amplification and tyrosine-protein kinase Met (c-Met) overexpression confer gefitinib resistance in non-small cell lung cancer (NSCLC). The natural product Licochalcone A (Lico A) exhibits a broad range of inhibitory effects against various tumors. However, the effects of Lico A on c-Met signaling and gefitinib resistance in NSCLC remain unclear. In the present study, Lico A efficiently overcame gefitinib-acquired resistance in NSCLC cells by suppressing c-Met signaling. Lico A decreased cell viability and colony formation dose-dependently and impaired in vivo tumorigenesis of gefitinib-resistant HCC827 and PC-9 cells. Furthermore, Lico A induced intrinsic apoptosis and upregulated the protein expression levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3. Lico A promoted the interaction between c-Met and E3 ligase c-Casitas B-lineage lymphoma (Cbl), which enhanced c-Cbl-mediated c-Met ubiquitination and degradation. Depletion of c-Cbl compromised Lico A-induced c-Met ubiquitination and its inhibitory efficacy in gefitinib-resistant NSCLC cells. Taken together, the results suggest that Lico A is a promising antitumor agent that might be used to overcome c-Met overexpression-mediated gefitinib resistance in NSCLC cells.
... Licochalcone A can induce apoptosis in the human hepatoma [80], lung cancer [86], osteosarcoma [129], bladder cancer [125], and prostate cancer [123] cells. It triggered the apoptotic cascade in HepG2 cells and human bladder cancer cells by upregulating Bcl-2, Bax, caspase-3, and caspase-8. ...
Article
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Glycyrrhizae Radix et Rhizoma is the most frequently prescribed natural medicine in China and has been used for more than 2,000 years. The flavonoids of licorice have garnered considerable attention in recent decades due to their structural diversity and myriad pharmacological effects, especially as novel therapeutic agents against inflammation and cancer. Although many articles have been published to summarize different pharmacological activities of licorice in recent years, the systematic summary for flavonoid components is not comprehensive. Therefore, in this review, we summarized the pharmacological and mechanistic data from recent researches on licorice flavonoids and their bioactive components.
... Moreover, the cytotoxic effects of licochalcone A have also been investigated against the head and neck squamous cell carcinoma (HNSCC) FaDu cell line and it was found to exhibit an apoptotic effect via both the extrinsic and intrinsic pathways triggered by the ERK1/2 and p38 phosphorylation regulated Tumor Necrosis Factor (TNF)related apoptosis-inducing ligand (TRAIL) expression [105]. The other cancer cells against which licochalcone A exerts anti-cancer activity through the induction of apoptosis include the human LNCaP and androgen-insensitive DU145 prostate cancer cells, the NSCLC cells (H460, A549, H292 and H1299) and the cervical cancer cells [106][107][108][109][110][111][112][113]. In a more recent study [114], it was demonstrated that licochalcone A abrogated interferon-gamma (IFN-γ)-induced programmed death ligand-1 (PD-L1) expression in lung cancer cells, A549, NCI-H1650 and NCI-H1299, and exerted an immunotherapy effect. ...
Article
In the search for novel drugs for health preservation, the active ingredients of medicinal herbs have been used since time immemorial. Liquorice, a potent medicinal herb derived from the root and rhizomes of Glycyrrhiza species, has been used worldwide for the treatment of various diseases. A number of compounds have been isolated from the liquorice root, including retrochalcones, viz. echinatin, licochalcone A, B, C, D and E, having a diverse range of pharmacological applications such as antioxidant, anti-inflammatory, anti-cancer, anti-bacterial, anti-diabetic, anti-obesity, anti-parasitic, to name a few. This review highlights the different synthetic procedures for retrochalcones and their in vitro, in vivo, ex vivo and in silico studies, along with the underlying molecular mechanisms. In addition, the total syntheses of licochalcone F and H, the regioisomers of licochalcone E and C, respectively, have also been included. Herein, the maximum possible biological applications of retrochalcones cited so far in the literature have been reviewed. The insights from this review will further advance the role and application of retrochalcones in the field of medicinal chemistry.
... Isoliguiritigenin is a member of the flavonoids, which evokes obvious tracheal relaxation effects Liu et al. (2008), and the traditional use of licorice for asthma treating is due to antiinflammation (Kuang et al., 2018;Huang et al., 2019;Upadhyay et al., 2020) related to inhibiting inflammatory cells' infiltration, decreasing oxidative stress, and reducing pro-inflammatory mediators' production, such as TNF-α and IL-1β (Yu et al., 2018;Hou et al., 2019). In addition, licorice and its component licochalcone-A can induce autophagic cell death though inhibition of the mTOR pathway in cancer cells (Yo et al., 2009). These findings shed light on the development of the new usage of licorice in disease treatment through induction of autophagy. ...
Article
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Asthma has become a global health issue, suffering more than 300 million people in the world, which is a heterogeneous disease, usually characterized by chronic airway inflammation and airway hyperreactivity. Combination of inhaled corticosteroids (ICS) and long acting β-agonists (LABA) can relieve asthma symptoms and reduce the frequency of exacerbations, especially for patients with refractory asthma, but there are limited treatment options for people who do not gain control on combination ICS/ LABA. The increase in ICS dose generally provides little additional benefit, and there is an increased risk of side effects. Therefore, therapeutic interventions integrating the use of different agents that focus on different targets are needed to overcome this set of diseases. Some findings suggest autophagy is closely correlated with the severity of asthma through eosinophilic inflammation, and its modulation may provide novel therapeutic approaches for severe allergic asthma. The chinese herbal medicine (CHM) have been demonstrated clinically as potent therapeutic interventions for asthma. Moreover some reports have found that the bioactive components isolated from CHM could modulate autophagy, and exhibit potent Anti-inflammatory activity. These findings have implied the potential for CHMs in asthma or allergic inflammation therapy via the modulation of autophagy. In this review, we discuss the basic pathomechanisms underpinning asthma, and the potential role of CHMs in treating asthma with modulating autophagy.
... Moreover, our study showed that 18β Glycyrrhetinic 30 methyl ester (ME GA) was highly selective which is in accordance with the previous reports on glycyrrhetinic acid derivatives and recommended them as an alternative or complementary medicine for liver cancer [22,29]. It has been reported that other glycyrrhizin derivatives induced apoptosis in prostate cancer cells via downregu lation of Bcl 2 expression and inhibition of mammalian target of rapamycin (mTOR) pathway [30]. Numerous reports on the chemo preventive and anti genotoxic effects of triterpenoids have been published [31] which showed that triterpenoids are highly m lti functional. ...
Background Liver cancer is a life threating disease as it occupies the fifth most common cancers from incidences and the third cause of death worldwide and with no available safe, efficient, economic drug for treatment. Method Therefore, this study intended to investigate different glycyrrhizin and their derivatives for possible use as a cytotoxic agent and as a possible drug for liver cancer treatment. Thus, after treatment against liver cancer cell line HepG-2 with 50 µM of each compounds cell viability was determined. Results The cytotoxicity assay showed that glycyrrhizin derivative ME-GA (18β-Glycyrrhetinic-30-methyl ester) and AKBA (3-acetyl-11- keto-β-Boswellic acid) were most potent against liver cancer cell line HepG-2 with IC50 values 25.50 ± 1.06 and 19.73 ± 0.89 µM, respectively. Both compounds showed superior selectivity towards hepatocellular carcinoma rather than the normal lung fibroblast cell line WI-38. The presence of methyl ester at C-30 greatly increased the cytotoxicity of ME-GA and may attribute for its superior activity and selectivity. Both ME-GA and AKBA contribute the ability to inhibit the cancer cell migration in the wound healing assay and inhibited colony formation. Using flow cytometry to determine cell cycle analysis and determination the possible mechanisms of action for apoptosis revealed that ME-GA arrested the cell cycle at G2/M that lead to inhibition of hepatocellular carcinoma and induced apoptosis via the extrinsic pathway and its ability to increase p53 transactivation. Conclusions This work highlights the cytotoxicity of glycyrrhizin and its derivative for possible use as a chemotherapeutic agent against hepatocellular carcinoma cells HepG-2. The most cytotoxic compound was ME-GA (18β-Glycyrrhetinic-30-methyl ester) with no cytotoxic effect on the normal cell line. In summary this new derivative may be used as an alternative or complementary medicine for liver cancer.
... Compared with 3-MA for the early inhibition of autophagy, which attenuated the cytotoxicity induced by imatinib, the autophagy inhibition of baffinomycin A1 in the late stage enhanced imatinib-induced cytotoxicity [144]. In addition, autophagy inhibition in the late stage (inhibitor CQ) rather than in the early stage (inhibitor 3-MA or shRNA Beclin1) also enhanced the therapeutic effect of malignant gliomas with quercetin [145]. The reason for this may be that CQ leads to the accumulation of autophagy vacuoles and induces caspase-3-dependent apoptosis. ...
Article
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Autophagy, which is a conserved biological process and essential mechanism in maintaining homeostasis and metabolic balance, enables cells to degrade cytoplasmic constituents through lysosomes, recycle nutrients, and survive during starvation. Autophagy exerts an anticarcinogenic role in normal cells and inhibits the malignant transformation of cells. On the other hand, aberrations in autophagy are involved in gene derangements, cell metabolism, the process of tumor immune surveillance, invasion and metastasis, and tumor drug-resistance. Therefore, autophagy-targeted drugs may function as anti-tumor agents. Accumulating evidence suggests that flavonoids have anticarcinogenic properties, including those relating to cellular proliferation inhibition, the induction of apoptosis, autophagy, necrosis, cell cycle arrest, senescence, the impairment of cell migration, invasion, tumor angiogenesis, and the reduction of multidrug resistance in tumor cells. Flavonoids, which are a group of natural polyphenolic compounds characterized by multiple targets that participate in multiple pathways, have been widely studied in different models for autophagy modulation. However, flavonoid-induced autophagy commonly interacts with other mechanisms, comprehensively influencing the anticancer effect. Accordingly, targeted autophagy may become the core mechanism of flavonoids in the treatment of tumors. This paper reviews the flavonoid-induced autophagy of tumor cells and their interaction with other mechanisms, so as to provide a comprehensive and in-depth account on how flavonoids exert tumor-suppressive effects through autophagy.
... Quite a lot of researchers have proven that non-protein ingredients obtained from the licorice root such as flavonoid, and polysaccharides have the ability to induce apoptosis in cancer cells, consequently being able to reverse the growth of cancer cells [28]. Aside from that, a number of investigations revealed that licorice and its bioactive constituents restrain cancer cell growth through induction of apoptosis, cell cycle arrest, and autophagy [29,30]. In brief, in this research, we characterized three proteins from the licorice root. ...
... Autophagy plays a dual role during physiological process and tumorigenesis including maintenance of cell survival and defeat of cell malfunction (Hippert, O'Toole, & Thorburn, 2006;Mizushima, 2007). It was reported that the inducing (Yo, Shieh, & Hsu, 2009) and suppression (Law et al., 2014) of autophagy could both inhibit tumor cell viability. In the present study, we further investigated the effect of (GlcN) 5 on autophagy in HepG2 cells. ...
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Hepatocellular carcinoma (HCC) is one of the most prevalent and deadliest cancers. In this study, the anti-tumor effect of singular degree of polymerization (DP) chitooligosaccharides (COS) (DP 2-5) and the underlay molecular mechanisms were investigated on HCC cell line HepG2. MTT assay showed that (GlcN) 5 have the best anti-proliferation effect among the different DP of COS (DP2-5). Furthermore, the administration of (GlcN) 5 could decrease mitochondrial membrane potential, release cytochrome c into cytoplasm, activate the cleavage of Caspases9/3, thus inducing mitochondrial-mediated apoptosis. Additionally, (GlcN) 5 treatment could increase the accumulation of autophagosomes. Further investigation showed that (GlcN) 5 suppressed protective autophagy at the fusion of autophagosomes and lysosomes. Moreover, the inhibition of protective autophagy flux by (GlcN) 5 could further decrease cell viability and increase apoptosis rate. Our findings suggested that (GlcN) 5 suppressed HepG2 proliferation through inducing apoptosis via intrinsic pathway and impairing cell protective autophagy. COS might have the potential to be an agent for lowering the risk of HCC.
... For assaying decontrolled gene expression, the differentially expressed genes (DEGs) were identified using the "limma" package of R software [37], and the DEGs in each microarray were also filtered using the same package. Target integration of the DEGs discriminated from four datasets (GSE19804, GSE18842, GSE43458, and GSE62113) was performed using Robus-tRankAggreg [38]. Genes with a log2-fold change |log 2 FC| ≥1 and an FDR-adjusted P value < 0.05 were considered DEGs [39]. ...
Article
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Background: Javanica oil emulsion injection (JOEI) is an effective therapeutic option for patients with non-small cell lung cancer (NSCLC), but its mechanisms have not been fully elucidated. Methods: In this study, we utilized network pharmacology to systematically investigate the bioactive components and targets of JOEI, identify common targets in NSCLC, and understand and evaluate the underlying mechanism of JOEI in the treatment of NSCLC through expression level, correlation, enrichment, Cox, survival and molecular docking analyses. The results indicated that five compounds of JOEI interact with five pivotal targets (LDLR, FABP4, ABCB1, PTGS2, and SDC4) that might be strongly correlated with the JOEI-mediated treatment of NSCLC. Results: The expression level analysis demonstrated that NSCLC tissues exhibit low expression of FABP4, ABCB1, LDLR and PTGS2 and high SDC4 expression. According to the correlation analysis, a decrease in FABP4 expression was strongly correlated with decreases in LDLR and ABCB1, and a decrease in LDLR was strongly correlated with decreased PTGS2 and increased in SDC4 expression. Cox and survival analyses showed that the survival rate of the high-risk group was significantly lower than that of the low-risk group (p = 0.00388). In the survival analysis, the area under the curve (AUC) showed that the pivotal gene model exhibited the best predictive capacity over 4 years (AUC = 0.613). Moreover, the molecular docking analysis indicated that LDLR, FABP4, ABCB1, PTGS2 and SDC4 exhibit good binding activity with the corresponding compounds. Conclusion: In conclusion, this study predicted and verified that the mechanism of JOEI against NSCLC involves multiple targets and signaling pathways. Furthermore, this study provides candidate targets for the treatment of NSCLC, lays a good foundation for further experimental research and promotes the reasonable application of JOEI in clinical treatment.
... Quite a lot of researchers have proven that non-protein ingredients obtained from the licorice root such as flavonoid, and polysaccharides have the ability to induce apoptosis in cancer cells, consequently being able to reverse the growth of cancer cells [28]. Aside from that, a number of investigations revealed that licorice and its bioactive constituents restrain cancer cell growth through induction of apoptosis, cell cycle arrest, and autophagy [29,30]. In brief, in this research, we characterized three proteins from the licorice root. ...
Article
Glycyrrhiza glabra is a perennial herb of Fabaceae family containing wide range of biologically active substances including flavonoids. The medicinal properties of the plant are mainly attributed to the presence of flavonoids. Extraction is an important step in isolation of these flavonoids, as it is a crucial process requiring intense standardization. The aim of the current research was to determine the optimized conditions for efficient and simultaneous extraction of major flavonoids from Glycyrrhiza glabra callus. Callus extracts were prepared with ethanol using Heat stirred extraction (HSE) method. Optimization was done by using L16 orthogonal design of experiment. The effect of factors such as Temperature, extraction time, solvent concentration material ratio, number of extractions on content of flavonoids was investigated. The maximum flavonoid content (10.2 mg/g) was obtained under optimum conditions of 1:30 material ratio, using 70% ethanol as solvent for 4 hrs extraction duration at temperature 85 ºC in three cycles of extractions. Ethanol concentration and extraction time were found to be the most significant factors influencing the flavonoid yield, and had a positive effect on flavonoid contents.
Article
Overexpression or activation of Yes-associated protein (YAP) is common in cancer cells. Thus, targeting YAP may be a strategy for cancer therapy. Licochalcone A (LicA) is a primary active compound of licorice root and is known to have medicinal effects, such as antioxidant, antibacterial, antiviral, and anticancer effects. However, the anticancer pharmacological mechanism of LicA has not been investigated in cholangiocarcinoma. In this study, we investigated the antiproliferative effect of LicA and the underlying molecular mechanism in HCCC-9810 and RBE human cholangiocarcinoma cells. Our experiments indicated that LicA suppressed the growth of cholangiocarcinoma cells through inactivation of the Hippo pathway. Pescadillo ribosomal biogenesis factor 1 (PES1) was notably upregulated and related to carcinogenesis. We also found that LicA suppressed the expression and nuclear localization of PES1, which was associated with the inhibition of YAP expression and transcriptional activity.
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Quinazoline scaffold has shown excellent antitumor activity and has aroused public attention because of its low toxicity, high efficiency and unique mode of action. In this paper, five quinazoline analogs D1-D5 were preliminary designed through scaffold shopping from mTOR inhibitors and synthesized in four steps. Five compounds exhibited potent antitumor activity against the HepG2 cell line by MTT assay. Compound D2 (IC50= 4.06 μM) was found as the most potent analog and showed better antiproliferative ability than sorafenib (IC50= 6.14 μM). The result of the wound healing assay and transwell migration assay indicated D2 strong potential to suppress HepG2 cell migration in a dose- and time-dependent manner. The underlying mechanism of its cytotoxicity was also investigated and the results of western blotting confirmed that D2 exposure could block the cell cycle, promote apoptosis and inhibit AKT and mTOR phosphorylation in HepG2 cells. Molecular docking further supported that D2 showed a high affinity to mTOR kinase. The results favored our rational design intention and hinted the new quinazolines might be helpful in the further explorations of potent agents.
Autophagy, an intricate response to nutrient deprivation, pathogen infection, Endoplasmic Reticulum (ER)-stress and drugs, is crucial for homeostatic maintenance in living cells. This highly regulated, multi-step process has been involved in several diseases including cardiovascular and neurodegenerative diseases, especially in cancer. It can function as either a promoter or a suppressor in cancer, which underlining the potential utility as a therapeutic target. In recent years, increasing evidence has suggested that many natural products could modulate autophagy through diverse signaling pathways, either inducing or inhibiting. In this review, we briefly introduce autophagy and systematically describe several classes of natural products that implicated in autophagy modulation. These compounds are of great interest for their potential activity against many types of cancer, such as ovarian, breast, cervical, pancreatic, and so on, hoping to provide valuable information for the development of cancer treatments based on autophagy.
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Allyl isothiocyanate (AITC), one of the most widely studied phytochemicals, inhibits the survival of human prostate cancer cells while minimally affecting normal prostate epithelial cells. Our study demonstrates the mechanism of AITC-induced cell death in prostate cancer cells. AITC induces autophagy in RV1 and PC3 cells, judging from the increased level of LC3-II protein in a dose- and time-dependent manner, but not in the normal prostate epithelial cell (PrEC). Inhibition of autophagy in AITC-treated cells decreased cell viability and enhanced apoptosis, suggesting that the autophagy played a protective role. There are several pathways activated in ATIC-treated cells. We detected the phosphorylation forms of mTOR, ERK, AMPK, JNK and p38, and ERK AMPK and JNK activation were also detected. However, inhibition of AITC-activated ERK, AMPK and JNK by pre-treatment of specific inhibitors did not alter autophagy induction. Finally, increased beclin-1 expression was detected in AITC-treated cells, and inhibition of AITC-induced beclin-1 attanuated autophagy induction, indicating that AITC-induced autophagy occurs through upregulating beclin-1. Overall, our data show for the first time that AITC induces protective autophagy in Rv1 and PC3 cells through upregulation of beclin-1. Our results could potentially contribute to a therapeutic application of AITC in prostate cancer patients.
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(E)-3-(3-Aryl-1-phenyl-1H-pyrazol-4-yl)-1-(pyridin- 3-yl)prop-2-en-1-ones 4a–i have been synthesized and evaluated for their in vitro cytotoxicity against a panel of three human cancer cell lines Caco-2, MIA PaCa-2, MCF-7 and a normal NIH-3T3 cell line. Compound 4g is cytotoxic with the IC50 value of 15.32±0.62 μm against the Caco-2 cell line.
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Licochalcone A (LCA) was found to possess anticancer effects. This study aimed to investigate the anticancer effects and mechanisms of LCA in melanoma. A375 and B16 melanoma cells were stimulated with LCA, MTT assay was used to assess cell proliferation. Expression of miR‐142‐3p, microphthalmia‐associated transcription factor (MITF, which regulates melanin production) and autophagy‐related genes was determined by Real‐time PCR or western blot. The apoptosis was analyzed by flow cytometry and caspase‐3 activity. The roles of miR‐142‐3p and Ras homolog enriched in brain (Rheb) in LCA‐affected cells were investigated by gain‐ and loss‐of functions. LCA inhibited proliferation and MITF expression, but increased apoptosis and autophagy of melanoma cells. Moreover, LCA elevated miR‐142‐3p expression, but decreased its target gene Rheb expression. The effects of LCA on melanoma cells were abrogated by miR‐142‐3p inhibitor or Rheb overexpression. LCA suppressed mTOR signaling activation via Rheb. Additionally, rapamycin (a mTOR antagonist) notably attenuated the effects of Rheb on the autophagy, proliferation, apoptosis, and MITF expression in LCA‐treated melanoma cells. In conclusion, LCA restrained MITF expression and growth by activating autophagy in melanoma cells via miR‐142‐3p/Rheb/mTOR pathway. This study suggested that LCA might be a potential therapeutic candidate for prevention and treatment of melanoma.
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Dysfunction of epidermal growth factor receptor (EGFR) signalling plays a critical role in the oncogenesis of non–small‐cell lung cancer (NSCLC). Here, we reported the natural product, licochalcone A, exhibited a profound anti‐tumour efficacy through directly targeting EGFR signalling. Licochalcone A inhibited in vitro cell growth, colony formation and in vivo tumour growth of either wild‐type (WT) or activating mutation EGFR‐expressed NSCLC cells. Licochalcone A bound with L858R single‐site mutation, exon 19 deletion, L858R/T790M mutation and WT EGFR ex vivo, and impaired EGFR kinase activity both in vitro and in NSCLC cells. The in silico docking study further indicated that licochalcone A interacted with both WT and mutant EGFRs. Moreover, licochalcone A induced apoptosis and decreased survivin protein robustly in NSCLC cells. Mechanistically, we found that treatment with licochalcone A translationally suppressed survivin through inhibiting EGFR downstream kinases ERK1/2 and Akt. Depletion of the translation initiation complex by eIF4E knockdown effectively inhibited survivin expression. In contrast, knockdown of 4E‐BP1 showed the opposite effect and dramatically enhanced survivin protein level. Overall, our data indicate that targeting survivin might be an alternative strategy to sensitize EGFR‐targeted therapy.
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Objectives: This study aimed to characterize the glucuronidation pathway of licochalcone A (LCA) in human liver microsomes (HLM). Methods: HLM incubation systems were employed to catalyze the formation of LCA glucuronide. The glucuronidation activity of commercially recombinant UDP-glucuronosyltransferase (UGT) isoforms toward LCA was screened. Kinetic analysis was used to identify the UGT isoforms involved in the glucuronidation of LCA in HLM. Key findings: LCA could be metabolized to two monoglucuronides in HLM, including a major monoglucuronide, namely, 4-O-glucuronide, and a minor monoglucuronide, namely, 4’-O-glucuronide. Species-dependent differences were observed among the glucuronidation profiles of LCA in liver microsomes from different species. UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 participated in the formation of 4-O-glucuronide, with UGT1A9 exhibiting the highest catalytic activity in this biotransformation. Only UGT1A1 and UGT1A3 were involved in the formation of 4’-O-glucuronide, exhibiting similar reaction rates. Kinetic analysis demonstrated that UGT1A9 was the major contributor to LCA-4-O-glucuronidation, while UGT1A1 played important roles in the formation of both LCA-4-O- and 4’-O-glucuronide. Conclusion: UGT1A9 was the major contributor to the formation of LCA-4-O-glucuronide, while UGT1A1 played important roles in both LCA-4-O- and 4’-O-glucuronidation.
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Urinary bladder cancer is known as a common cancer diagnosed across the world and results in significant mortality and morbidity rates among patients. The retinoblastoma (Rb) protein, as a main tumor suppressor, controls cellular responses to potentially oncogenic stimulation. Rb phosphorylation could disrupt E2F complex formation, resulting in diverse transcription factor dysfunction. In our study, we investigated how Rb is involved in controlling urinary bladder cancer progression. The results indicate that Rb expression is reduced in mice with urinary bladder tumor, and its suppression leads to urinary bladder cancer progression in vivo and in vitro. Rb mutation directly results in tumor size with lower survival rate in vivo. Rb knockdown in vitro promoted bladder tumor cell proliferation, migration and invasion. Interestingly, Rb knockout and knockdown result in autophagy and apoptosis inhibition via suppressing p53 and caspase-3 signaling pathways, enhancing bladder cancer development in vitro and in vivo. These findings reveal that Rb deficiency accelerated urinary bladder cancer progression, exposing an important role of Rb in suppressing urinary bladder cancer for treatment in the future.
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Phytochemicals, especially flavonoids, have been widely investigated for their diversified pharmacological activities including anticancer activities. Previously we identified isoangustone A from licorice-derived compounds as a potent inducer of cell death. In the present study, the exact mechanism by which isoangustone A induced cell death was further investigated, with autophagy as an indispensible part of this process. Isoangustone A treatment activated autophagic signaling and induced a complete autophagic flux in colorectal cancer cells. Knockdown of ATG5 or pre-treatment with autophagy inhibitors significantly reversed isoangustone A-induced apoptotic signaling and loss of cell viability, suggesting autophagy plays an important role in isoangustone A-induced cell death. Isoangustone A inhibited Akt/mTOR signaling, and overexpressing of a constitutively activated Akt mildly suppressed isoangustone A-induced cell death. More importantly, isoangustone A inhibited cellular ATP level and activated AMPK, and pre-treatment with AMPK inhibitor or overexpression of dominant negative AMPKα2 significantly reversed isoangustone A-induced autophagy and cell death. Further study shows isoangustone A dose-dependently inhibited mitochondrial respiration, which could be responsible for isoangustone A-induced activation of AMPK. Finally, isoangustone A at a dosage of 10 mg/kg potently activated AMPK and autophagic signaling in and inhibited the growth of SW480 human colorectal xenograft in vivo. Taken together, induction of autophagy through activation of AMPK is an important mechanism by which isoangustone A inhibits tumor growth, and isoangustone A deserves further investigation as a promising anti-cancer agent.
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Licorice shows a variety of pharmacological activities. This work aims to discover bioactive natural products from one botanical source of licorice, Glycyrrhiza inflata. A total of 67 free phenolics were isolated to form a compound library. Based on the bioactivities of licorice, these compounds were screened using cell- or enzyme-based bioassay methods. A total of 11 compounds exhibited potent cytotoxic activities against three human cancer cell lines (HepG2, SW480 and MCF7), while showed little toxicity on human normal cell lines LO2 and HEK293T. A number of chalcones showed remarkable anti-inflammatory activities. Among them, 2 (licochalcone B, IC50 8.78 μM), 10 (licoagrochalcone C, IC50 9.35 μM) and 13 (licochalcone E, IC50 9.09 μM) exhibited the most potent inhibitory activities on LPS-induced NO production, whereas 1, 8, 10, 12 and 13 (IC50 13.9, 7.27, 2.44, 6.67 and 3.83 μM) showed potent inhibitory activities on NF-κB transcription. Nine prenylated phenolics were found to be PTP1B inhibitors. Particularly, licoagrochalcone A (4), kanzonol C (7), 2′-hydroxyisolupalbigenin (35), gancaonin Q (45), glisoflavanone (50) and glabrol (53) showed IC50 values of 0.31-0.97 μM. Compounds 24 (semilicoisoflavone B, IC50 0.25 μM), 26 (allolicoisoflavone B, IC50 0.80 μM) and 64 (glabridin, IC50 0.10 μM) showed noticeable tyrosinase inhibitory activities. Most of the above bioactive compounds were reported for the first time.
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Herbal medicine can be used to overcome the side effects of conventional treatments. This study aimed to evaluate the anticancer activities of ginger and licorice extracts, as well as the synergistic effects of their combination. Ginger ethanolic extract (GEE) and licorice methanolic extract (LME) were isolated by a Soxhlet extractor. Next, the anti-proliferative activity of the extracts, apoptosis induction, tumor growth inhibition, and tumor-infiltrating T lymphocytes were investigated. The MTT (3-[4, 5-dimethylthiazol-2-yl]-2, five diphenyl tetrazolium bromide) assay showed that GEE and LME decreased the CT26 cell viability in a dose-dependent manner; however, the GEE + LME combination was more effective (P < 0.05). The CT26 cells treated with each extract showed a significant increase in Bax/Bcl-2 ratio and caspase-3 gene expression, especially in the GEE + LME group (P < 0.001). Tumor volume significantly reduced in the GEE + LME group, compared to the negative controls. Finally, mice treated with GEE + LME showed a significant increase in the CTL/Treg cell ratio (P < 0.001) and Bax/Bcl2 ratio (P < 0.05). The study results revealed that GEE + LME can suppress cancer cell growth, increase apoptosis, and improve CTL infiltrating to the tumor site in a synergetic manner in-vivo and in-vitro. Therefore, the prepared mixture can be used in future clinical trials.
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Licochalcone A (LicA) has been reported to possess antitumor properties. However, its effect on human glioma cells remains unknown. In this study, we observed that LicA significantly suppressed ADAM9 expression and cell migration and invasion activities of human glioma cells (M059K, U-251 MG, and GBM8901) and exhibited no cell cytotoxicity. Human proteinase antibody array and immunoblot analysis indicated that the LicA treatment inhibited the expression of ADAM9 protein in human glioma cells. Recombinant human ADAM-9 (Rh-ADAM9) treatment significantly reversed the LicA-induced reduction in the ADAM9 level and the cell migration and invasion activities of human glioma cells. Additionally, the phosphorylation/activation of the mitogen-activated protein kinase kinase (MEK)-extracellularly responsive kinases (ERK) signaling pathway was significantly suppressed in LicA-treated human glioma cells. Cotreatment with LicA and PD98059 synergistically inhibited ADAM9 expression, cell migration, and cell invasion, which suggested that the MEK-ERK signaling pathway was involved in LicA-induced inhibition of ADAM9 expression and invasion activity of human glioma cells. These findings are the first evidence of the LicA’s anti-invasive properties against human glioma cells.
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In several different cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant ter-butylhydroperoxide (t-BHP) but has no cytoprotective effect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid his 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential delta psi(m) that is not inhibited by the broad spectrum caspase inhibitor z-VAD.fmk, although z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the delta psi(m) of t-BHP-treated cells but has no inhibitory effect on the delta psi(m) collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of delta psi(m) disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamide-triggered delta psi(m) collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-purified PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-X(L) proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PT-modulatory capacity. Again, Bcl-2 fails to confer protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their effects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.
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Colorectal cancer (CRC) is a leading cause of cancer death, yet primary prevention remains the best approach to reducing overall morbidity and mortality. Studies have shown that COX-2-derived PGE2 promotes CRC progression, and both nonselective COX inhibitors (NSAIDs) and selective COX-2 inhibitors (such as glucocorticoids) reduce the number and size of colonic adenomas. However, increased gastrointestinal side effects of NSAIDs and increased cardiovascular risks of selective COX-2 inhibitors limit their use in chemoprevention of CRC. We found that expression of 11beta-hydroxysteroid dehydrogenase type II (11betaHSD2), which converts active glucocorticoids to inactive keto-forms, increased in human colonic and Apc+/min mouse intestinal adenomas and correlated with increased COX-2 expression and activity. Furthermore, pharmacologic inhibition or gene silencing of 11betaHSD2 inhibited COX-2-mediated PGE2 production in tumors and prevented adenoma formation, tumor growth, and metastasis in mice. Inhibition of 11betaHSD2 did not reduce systemic prostacyclin production or accelerate atherosclerosis in mice, thereby avoiding the major cardiovascular side effects seen with systemic COX-2 inhibitors. Therefore, 11betaHSD2 inhibition represents what we believe to be a novel approach for CRC chemoprevention and therapy by increasing tumor glucocorticoid activity, which in turn selectively blocks local COX-2 activity.
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Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C(2)-ceramide and C(6)-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr(69), Ser(70), and Ser(87), located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress.
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To determine whether a low pH intracellular "sorting" step is required to route peptides into secretory granules, the effects of pH altering drugs on the biosynthesis and secretion of peptides by AtT-20 mouse corticotrope tumor cells and rat intermediate pituitary cells were examined. Doses of each drug maintaining normal protein synthesis and cell morphology, while obliterating the intracellular pH gradients detected by acridine orange fluorescence, were experimentally determined. Regions of the cell rich in secretory granules were localized by immunocytochemistry and were found to coincide with organelles with a low internal pH. Biosynthetic labeling experiments were coupled with immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel analyses to examine the biosynthesis and secretion of corticotropin (ACTH(1-39], alpha-melanotropin, ACTH(18-39), beta-endorphin, gamma-melanotropin, alpha-amidated joining peptide, and the NH2-terminal region of pro-ACTH/endorphin. Chloroquine (20-40 microM) and a mixture of NH4Cl and methylamine (2-5 mM each) dissipated pH gradients but had no effect on the synthetic rate of pro-ACTH/endorphin, the extent and rate of precursor processing to smaller peptides, the rate of basal secretion of the various peptides, or the extent to which secretion of each of the peptides could be stimulated by secretagogues. Monensin (0.1-1 microM) had no discernible effect on intracellular pH gradients yet totally blocked proteolytic processing of pro-ACTH/endorphin. Thus, a monensin-blockable step occurs in peptide processing, presumably in the trans Golgi region; however, a low pH chloroquine-sensitive sorting step is not required for processing or for routing peptides to a stable storage form which can be released in response to secretagogues.
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A water extract of licorice root inhibits granuloma angiogenesis in adjuvant-induced chronic inflammation (Phytother. Res., 5, 195. 1991). The present study has investigated the effects of licorice-derived compounds on granuloma angiogenesis. Isoliquiritin (0.31-3.1 mg/kg), a licorice-derived flavonoid, inhibited the carmine content of granuloma tissue 50-fold greater than licorice extract. Glyeyrrhizin (20-80 mg/kg), a licorice-derived saponin, inhibited carmine content with a weak potency. The licorice extract (0.01-1 mg/ml) also inhibited tube formation from vascular endothelial cells in a concentration-dependent manner. From the chemical structure-activities of used licorice-derived flavonoids (0.1-100 microM), their potencies for anti-tube formation were in the order isoliquiritigenin > isoliquiritin > liquiritigenin > isoliquiritin-apioside. Glycyrrhizin (0.1-100 microM) and glycyrrhetinic acid (0.1-10 microM) increased tube formation. A glycyrrhizin (82 micrograms/ml)-induced increase in tube formation was inhibited by isoliquiritin. The combined effect of a mixture of 82 micrograms/ml glycyrrhizin and 4.2 micrograms/ml isoliquiritin, a similar concentration ratio to their yield ratio in the licorice extract, corresponded to the effect of 100 micrograms/ml extract. In conclusion, the anti-angiogenic effect of licorice extract depended on the anti-tube formation effect of isoliquiritin.
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In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.
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In a number of experimental systems, the early stage of the apoptotic process, i.e., the stage that precedes nuclear disintegration, is characterized by the breakdown of the inner mitochondrial transmembrane potential (delta psi m). This delta psi m disruption is mediated by the opening of permeability transition (PT) pores and appears to be critical for the apoptotic cascade, since it is directly regulated by Bcl-2 and since mitochondria induced to undergo PT in vitro become capable of inducing nuclear chromatinolysis in a cell-free system of apoptosis. Here, we addressed the question of which apoptotic events are secondary to mitochondrial PT. We tested the effect of a specific inhibitor of PT, bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, on a prototypic model of apoptosis glucocorticoid-induced thymocyte death. In addition to abolishing the apoptotic delta psi m disruption, BA prevents a number of phenomena linked to apoptosis: depletion of nonoxidized glutathione, generation of reactive oxygen species, translocation of NF kappa B, exposure of phosphatidylserine residues on the outer plasma membrane, cytoplasmic vacuolization, chromatin condensation, and oligonucleosomal DNA fragmentation. BA is also an efficient inhibitor of p53-dependent thymocyte apoptosis induced by DNA damage. These data suggest that a number of apoptotic phenomena are secondary to PT. In addition, we present data indicating that apoptotic delta psi m disruption is secondary to transcriptional events. These data connect the PT control point to the p53- and ICE/ Ced 3-regulated control points of apoptosis and place PT upstream of nuclear and plasma membrane features of PCD.
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The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.
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Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.
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To understand the roles of bcl-2 for the survival of leukemic cells, we constructed human leukemic HL60 transformant lines in which full length bcl-2 antisense message was conditionally expressed by a tetracycline-regulatable expression system. Cell growth was completely inhibited after antisense message induction and massive cell death was induced. Electron microscopic examinations show that cells died by autophagy, but not by apoptosis. The morphology and the function of mitochondria remained intact: neither the reduction in mitochondrial membrane potential nor the nuclear translocation of AIF, a mitochondrial protein that translocates to nuclei in cases of apoptosis, was observed. Caspase inhibitors did not rescue bcl-2-antisense-mediated autophagy. Thus, bcl-2 is essential for leukemic cell survival and its down-regulation results in autophagy. Cell Death and Differentiation (2000) 7, 1263 - 1269.
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Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.
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The herbal mixture PC-SPES, used to manage advanced prostate cancer, has proven thrombogenic and highly estrogenic in clinical trials. However, attempts to identify the active compounds in PC-SPES have yielded incongruous results. Moreover, warfarin was identified in the serum of a patient taking PC-SPES who experienced a bleeding disorder. To determine the active components in PC-SPES potentially responsible for these effects, we analyzed PC-SPES lots manufactured from l996 through mid-2001. Antineoplastic activity of PC-SPES and its individual component extracts was determined by colony-forming assays with several prostate cancer cell lines, and estrogenicity was determined by analyzing expression of an estrogen-responsive reporter gene in breast cancer cells. High-pressure liquid chromatography was used to isolate, identify, and quantify components of PC-SPES. Components were also identified by proton nuclear magnetic resonance, gas chromatography/mass spectrometry, and mass spectra analysis. PC-SPES lots manufactured from 1996 through mid-1999 contained the synthetic compounds indomethacin (range = 1.07-13.19 mg/g) and diethylstilbestrol (range = 107.28-159.27 micro g/g) and were two to six times more antineoplastic and up to 50 times more estrogenic than lots manufactured after the spring of 1999. In lots manufactured after mid-1999, gradual declines in the concentrations of indomethacin (from 1.56 to 0.70 mg/g), diethylstilbestrol (from 46.36 to 0.00 micro g/g), and total phytosterols (from 0.586 to 0.085 mg/g) were observed. Warfarin was identified for the first time in lots manufactured after July 1998 (range = 341-560 micro g/g). In the August 2001 lot, increases were found in concentrations of the natural products licochalcone A (from 27.6 to 289.2 micro g/g) and baicalin (from 12.5 to 38.8 mg/g). The phytochemical composition of PC-SPES varied by lot, and chemical analyses detected various amounts of the synthetic drugs diethylstilbestrol, indomethacin, and warfarin and several natural products. To qualify for clinical pharmacologic exploration, nutritional supplements including herbal mixtures should meet standards of quality control under the Good Manufacturing Practice system, and the manufacturers of such supplements should provide reliable analytical quality assurance.
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Autophagy is originally named as a process of protein recycling. It begins with sequestering cytoplasmic organelles in a membrane vacuole called autophagosome. Autophagosomes then fuse with lysosomes, where the materials inside are degraded and recycled. To date, however, little is known about the role of autophagy in cancer therapy. In this study, we present that temozolomide (TMZ), a new alkylating agent, inhibited the viability of malignant glioma cells in a dose-dependent manner and induced G2/M arrest. At a clinically achievable dose (100 microM), TMZ induced autophagy, but not apoptosis in malignant glioma cells. After the treatment with TMZ, microtubule-associated protein light-chain 3 (LC3), a mammalian homologue of Apg8p/Aut7p essential for amino-acid starvation-induced autophagy in yeast, was recruited on autophagosome membranes. When autophagy was prevented at an early stage by 3-methyladenine, a phosphatidylinositol 3-phosphate kinase inhibitor, not only the characteristic pattern of LC3 localization, but also the antitumor effect of TMZ was suppressed. On the other hand, bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, that prevents autophagy at a late stage by inhibiting fusion between autophagosomes and lysosomes, sensitized tumor cells to TMZ by inducing apoptosis through activation of caspase-3 with mitochondrial and lysosomal membrane permeabilization, while LC3 localization pattern stayed the same. These results indicate that TMZ induces autophagy in malignant glioma cells. Application of an autophagy inhibitor that works after the association of LC3 with autophagosome membrane, such as bafilomycin A1, is expected to enhance the cytotoxicity of TMZ for malignant gliomas.
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We have previously reported that the microtubule stabilizing agents (MSAs) paclitaxel, epothilone B and discodermolide induce caspase-independent cell death in non-small cell lung cancer (NSCLC) cells. Here we present two lines of evidence indicating a central role for the lysosomal protease cathepsin B in mediating cell death. First, inhibition of cathepsin B, and not of caspases or other proteases, such as cathepsin D or calpains, results in a strong protection against drug-induced cell death in several NSCLC cells. Second, MSAs trigger disruption of lysosomes and release and activation of cathepsin B. Interestingly, inhibition of cathepsin B prevents the appearance of multinucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in this novel cell death pathway.
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