• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.6
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• pp.942-949
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• 2010

Morus alba L., a kind of Oriental medicinal herbs, has been traditionally used to treat pulmonary asthma and congestion. According to recent studies, extracts of M. alba L. have showed anti-inflammatory, anti-oxidant, anti-tumor and hypoglycemic effects. However, the molecular mechanisms on how it acts as a death-inducer in cancer cells have not been fully understood. In this study, we investigated the cell death effects of methylene chloride extracts of M. alba L. (MEMA) in A549 human lung carcinoma cells. It was shown that MEMA induced the apoptotic cell death proved by increased sub-G1 phase cell population, apoptotic body formation and chromatin condensation. MEMA treatment induced the expression of death receptor-related proteins such as death receptor (DR) 4, DR5, Fas and FasL, which further triggered the activation of caspase-8 and the cleavage of Bid in a concentration-dependent manner. However, MEMA reduced anti-apoptotic Bcl-2 and Bcl-xL expression which contributed to the loss of mitochondrial membrane potential (MMP), and the activations of caspase-9 and caspase-3. Meanwhile, the morphological study indicated a characteristic finding of autophagy, such as the formation of autophagosomes in MEMA-treated cells. Furthermore, markers of autophagy, namely, the increased MDC-positive cells, conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II and increased beclin-1 accumulation, were observed. Taken together, these findings demonstrated that MEMA triggered both autophagy and apoptosis in A549 cancer cells. They might suggest that M. alba L. could be a prospective clinical application to treat human lung cancers.

• Tuberculosis and Respiratory Diseases
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• v.64 no.4
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• pp.278-284
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• 2008

Background: TRAIL is a promising anticancer agent which induces selective tumor cell death due to a unique receptor system that includes death receptors and decoy receptors. DR5 TRAIL receptor is an originally identified p53-regulated death receptor gene that was induced, by doxorubicine, only in cells with a wild-type p53 status. We investigated that focused on the correlation between the DR5 and p53 expressions in non-small cell lung cancer (NSCLC). Methods: Immunohistochemical analysis, with using avidin-biotinylated horseradish peroxidase complex, was carried out in 89 surgically resected NSCLC formalin-fixed paraffin-embedded tissue sections. As primary antibodies, we used anti-DR5 polyclonal antibody and anti-p53 monoclonal antibody. A negative control was processed with each slide. The positive tumor cells were quantified twice and these values were expressed as percentage of the total number of tumor cells, and the intensity of immunostaining was expressed. The analysis of the DR5 expression was done separately in tumor area and in a nearby region of normal tissue. Results: The DR5 expression was high in the bronchial epithelium (89% of cases) but this was almost absent in type I & II pneumocytes, lymphocytes and smooth muscle cells. High DR5 expression rate in tumor was seen in 28% (15/53) of squamous cell carcinomas, in 47% (15/32) of adenocarcinomas and, in 50% (2/4) of large cell carcinomas. The DR5 expression did not show any statistical significance relationship with the T stage, N stage, or survival. However, the DR5 expression showed significant inverse correlation with the p53 expression. (p< 0.01). Conclusion: We demonstrated that the DR5 expression in NSCLC via immunohistochemical analysis is relatively tumor-specific except for that in the normal bronchial epithelium and it is significantly dependent on the p53 status. This might be in vivo evidence for the significance of the DR5 gene as a p53 downstream gene.

• Journal of Acupuncture Research
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• v.31 no.2
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• pp.87-98
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• 2014

Objectives : We investigated whether snake venom toxin(SVT) from Vipera lebetina turanica sensitizes HT29 human epithelial colorectal cancer cells to tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) induced apoptosis in cancer cells. Methods : Cell viability assay was used to assess the inhibitory effect of TRAIL on cell growth of HT29 human colorectal cancer cells. And 6-diamidino-2-phenylindole(DAPI), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay(TUNEL) staining assay were used to evaluate cell-apoptosis. Western blot analysis were conducted to observe apoptosis related proteins and death receptor. To assess whether the synergized inhibitory effect of SVT and TRAIL on reactive oxygen species(ROS) generation was reversed by strong anti-oxidative agent. Results : SVT with TRAIL inhibited HT29 cell growth different from TRAIL alone. Consistent with cell growth inhibition, the expression of TRAIL receptors; Expression of death receptor(DR)4 and DR5 was significantly increased and intrinsic pro-apoptotic cleaved caspase-3, -9 was subsequently increased together with increase of Bax/Bcl-2 ratio and extrinsic pro-apototic caspase-8 was also activated. In addition, the expression of anti-apoptotic survival proteins, a marker of TRAIL resistance(eg, cFLIP, survivin, X-linked inhibitor of apoptosis protein(XIAP) and Bcl-2) was suppressed by the combination treatment of SVT and TRAIL. Pretreatment with the ROS scavenger N-acetylcysteine abolished the SVT and TRAIL-induced upregulation of DR4 and DR5 expression and expression of the intrinsic pro-apoptotic caspase-3 and-9. Conclusion : The collective results suggest that SVT facilitates TRAIL-induced apoptosis in $HT_{29}$ human epithelial colorectal cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 and consecutive induction of bilateral apoptosis via regulating apoptosis related proteins.

• Journal of Life Science
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• v.26 no.7
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• pp.847-854
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• 2016

Cordycepin, a derivative of the nucleoside adenosine, is one of the active components extracted from fungi of genus Cordyceps, and has been shown to have many pharmacological activities. In this study, we investigated the effects of cordycepin on proliferation and apoptosis of human gastric cancer AGS cells, and its possible mechanism of action. Treatment of cordycepin resulted in significant decrease in cell viability of AGS cells in a concentration-dependent manner. A concentration-dependent apoptotic cell death was also measured by agarose gel electrophoresis and flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled cordycepin treatment resulted in an enhanced expression of tumor necrosis factor-related apoptosis-inducing ligand, death receptor 5 and Fas ligand. Furthermore, up-regulation of pro-apoptotic Bax, and down-regulation of anti-apoptotic Bcl-2 and Bcl-xL expression were also observed in cordycepin-treated AGS cells. These were followed by activation of caspases (caspase-9, -8 and -3), subsequently leading to poly (ADP-ribose) polymerase cleavage. Taken together, these findings indicate that cordycepin induces apoptosis in AGS cells through regulation of multiple apoptotic pathways, including death receptor and mitochondria. Although further mechanical studies are needed, our results revealed that cordycepin can be regarded as a new effective and chemopreventive compound for human gastric cancer treatment.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1665-1672
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• 2012

An 80% ethanol extract of Hizikia fusiforme was obtained and followed by successive fractionation using the organic solvents n-hexane, ethyl acetate, and n-butanol to identify the antioxidative substance. The aqueous part of the nbutanol fractionation step, showing high antioxidative activity, was subjected to reverse-phase liquid chromatography. As a result, a substance purified from a BB-2 fraction showed high antioxidative activity. The m/z 419 [M+H] molecular ion peak in the fraction was observed by the analysis of the ESI-LC/MS spectrum. By the analysis of 1H NMR (500 MHz, DMSO-$d_6$) and $^{13}C$ NMR (125 MHz, DMSO-$d_6$) spectra, a unique compound of the fraction was biochemically identified as a 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5HHMF). We also investigated the effect of 5HHMF on human gastric AGS carcinoma cells. Western blot analysis suggested that the flavone substantially increased the levels of the death receptor-associated apoptosis mediators Fas, Fas L, FADD, TRADD, and DR4 in a concentration-dependent manner. The levels of Fas, Fas L, TRADD, and DR4 in the cells treated with 5HHMF ($5{\mu}g/ml$) were approximately 26.4-, 12.8-, 6.7-, and 9.8-times higher than those of non-treated cells, respectively. Of note, the level of FADD protein in the cells exposed to 5HHMF ($1{\mu}g/ml$) increased approximately 9.6-times. In addition, the cleavage of caspase-3, -8, and -9 in cultured AGS cells treated with 5HHMF was significantly confirmed. Therefore, our results suggest that 5HHMF from H. fusiforme is involved in the induction of death receptor-associated apoptosis mediators in human gastric AGS carcinoma cells.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.5
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• pp.2637-2641
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• 2016

Tumor necrosis factor ($TNF-{\alpha}$), an inflammatory cytokine that plays an important role in the control of cell proliferation, differentiation, and apoptosis, has previously been used in anti-cancer therapy. However, the therapeutic applications of $TNF-{\alpha}$ are largely limited due to its general toxicity and anti-apoptotic influence. To overcome this problem, the present study focused on the effect of active constituents isolated from a medicinal plant on $TNF-{\alpha}$-induced apoptosis in human colon adenocarcinoma (HT-29) cells. Nimbolide from Azadirachta indica was evaluated for cytotoxicity by methyl tetrazolium 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and phase contrast microscopy. Effects on apoptotic signaling proteins were investigated using Western blot analysis. Nimbolide showed cytotoxicity against HT-29 cells that was significantly different from the control group (p<0.01), a concentration of $10{\mu}M$ significantly inducing cell death (p<0.01). In combination with $TNF-{\alpha}$, nimbolide significantly enhanced-induced cell death. In apoptotic pathway, nimbolide activated c-Jun N-terminal kinase (JNK) phosphorylation, BH3 interacting-domain death agonist (Bid) and up-regulated the death receptor 5 (DR5) level. In the combination group, nimbolide markedly sensitized $TNF-{\alpha}$-induced JNK, Bid, caspase-3 activation and the up-regulation of DR5. Our findings overall indicate that nimbolide may enhance $TNF-{\alpha}$-mediated cellular proliferation inhibition through increasing cell apoptosis of HT-29 cells by up-reglation of DR5 expression via the JNK pathway.

• Journal of Periodontal and Implant Science
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• v.41 no.1
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• pp.17-22
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• 2011

Purpose: Nitric oxide (NO) has been known as an important regulator of osteoblasts and periodontal ligament cell activity. This study was performed to investigate the relationship between NO-mediated cell death of human periodontal ligament fibroblasts (PDLFs) and N-methyl-D-aspartic acid (NMDA) receptor antagonist (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801). Methods: Human PDLFs were treated with various concentrations (0 to 4 mM) of sodium nitroprusside (SNP) with or without $200\;{\mu}M$ MK801 in culture media for 16 hours and the cell medium was then removed and replaced by fresh medium containing MTS reagent for cell proliferation assay. Western blot analysis was performed to investigate the effects of SNP on the expression of Bax, cytochrome c, and caspase-3 proteins. The differences for each value among the sample groups were compared using analysis of variance with 95% confidence intervals. Results: In the case of SNP treatment, as a NO donor, cell viability was significantly decreased in a concentration-dependent manner. In addition, a synergistic effect was shown when both SNP and NMDA receptor antagonist was added to the medium. SNP treated PDLFs exhibited a round shape in culture conditions and were dramatically reduced in cell number. SNP treatment also increased levels of apoptotic marker protein, such as Bax and cytochrome c, and reduced caspase-3 in PDLFs. Mitogen-activated protein kinase signaling was activated by treatment of SNP and NMDA receptor antagonist. Conclusions: These results suggest that excessive production of NO may induce apoptosis and that NMDA receptor may modulate NO-induced apoptosis in PDLFs.

• Journal of Gastric Cancer
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• v.3 no.4
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• pp.186-190
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• 2003

Purpose: The balance between cell proliferation and apoptosis is crucial for homeostatic maintenance in a cell population. Decreased apoptosis or uncontrolled proliferation can lead to cancer. The Fas receptor signal through a cytoplasmic death domain is very important in the apoptotic pathway. To identify the effect of the death domain of the Fas gene in the development and/or progression of gastric cancer, we examined the apoptotic potential of five known Fas mutants detected in gastric cancers. Materials and Methods: A wild-type Fas gene was cloned with cDNA from normal liver tissue and full length Fas was sequenced. Mutants of the gene were generated with sitedirected mutagenesis by using the wild-type gene and specific primers. Wild- and mutant-type genes were transfected to HEK293 cells. Forty-eight hours after transfection the cells were stained with DAPI and cell death was counted under fluorescent microscopy. Results: In wild-type Fas-transfected cells, the percentage of apoptotic cells was $85.9\pm3.6\%$, and significant cell death and classic morphologic signs of apoptosis were observed. However, the percentages of apoptotic cells transfected with N239D, E240G, D244V, and R263H of tumor-derived mutant Fas were $29.5\pm2.08\%,\;28.5\pm3.34\%,\;25.225\pm2.06\%,\;and\;36.625\pm4.49\%$, respectively. Conclusion: These results suggest that inactivation of Fas caused by mutations in the death domain of the Fas gene may be one of the possible escape mechanisms against Fas-mediated apoptosis and that inactivating mutation of the Fas may contribute to the development or progression of gastric cancers.

• Molecules and Cells
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• v.38 no.2
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• pp.138-144
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• 2015

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

• Nutrition Research and Practice
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• v.8 no.2
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• pp.132-137
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• 2014

BACKGROUND/OBJECTIVES: In this study, the apoptogenic activity and mechanisms of cell death induced by hexane extract of aged black garlic (HEABG) were investigated in human leukemic U937 cells. MATERIALS/METHODS: Cytotoxicity was evaluated by MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide) assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay. RESULTS: Exposure to HEABG was found to result in a concentration- and time-dependent growth inhibition by induction of apoptosis, which was associated with an up-regulation of death receptor 4 and Fas legend, and an increase in the ratio of Bax/Bcl-2 protein expression. Apoptosis-inducing concentrations of HEABG induced the activation of caspase-9, an initiator caspase of the mitochodrial mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. HEABG also induced apoptosis via a death receptor mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid, and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. However, pre-treatment of U937 cells with the caspase-3 inhibitor, z-DEVD-fmk, significantly blocked the HEABG-induced apoptosis of these cells, and increased the survival rate of HEABG-treated cells, confirming that HEABG-induced apoptosis is mediated through activation of caspase cascade. CONCLUSIONS: Based on the overall results, we suggest that HEABG reduces leukemic cell growth by inducing caspase-dependent apoptosis through both intrinsic and extrinsic pathways, implying its potential therapeutic value in the treatment of leukemia.