Anticancer activity and molecular mechanisms of α‐conidendrin,a polyphenolic compound present in Taxus yunnanensis,on human breast cancer cell lines

α‐Conidendrin is a polyphenolic compound found mainly in Taxus yunnanensis, as the source of chemotherapy drug paclitaxel, which has been used in traditional medicine for treatment of cancer. This study aimed to investigate the anticancer activity and molecular mechanisms of α‐conidendrin on breast cancer cell lines. The results of the present study show that α‐conidendrin possesses potent antiproliferative effects on breast cancer cell lines MCF‐7 and MDA‐MB‐231. α‐Conidendrin significantly induced apoptosis in breast cancer cells via reactive oxygen species generation, upregulation of p53 and Bax, downregulation of Bcl‐2, depolarization of mitochondrial membrane potential (MMP), release of cytochrome c from mitochondria, and activation of caspases‐3 and ‐9. α‐Conidendrin remarkably inhibited the proliferation of breast cancer cells through induction of cell cycle arrest by upregulating p53 and p21 and downregulating cyclin D1 and CDK4. Unlike breast cancer cells, the antiproliferative effect of α‐conidendrin on human foreskin fibroblast cells (normal cells) was very small. In normal cells, reactive oxygen species levels, loss of MMP, release of cytochrome c, mRNA expression of p53, p21, cyclin D1, CDK4, Bax, and Bcl‐2 as well as mRNA expression and activity of caspases‐3 and ‐9 were significantly less affected by α‐conidendrin compared with cancer cells. These results suggest that α‐conidendrin can be a promising agent for treatment of breast cancer with little or no toxicity against normal cells.     

Curcumin Inhibits Proliferation and Induces Apoptosis of Human Colorectal Cancer Cells by Activating the Mitochondria Apoptotic Pathway

Curcumin, a natural plant extract from Curcuma longa, is known for its anti‐carcinogenic and chemopreventive effects on a variety of experimental cancer models. In this study, we evaluated the effects of curcumin and elucidated its mechanism in human colorectal carcinoma cells. Cell viability assay showed that curcumin significantly inhibited the growth of LoVo cells. Curcumin treatment induced the apoptosis accompanied by ultra‐structural changes and release of lactate dehydrogenase in a dose‐dependent manner. Moreover, treatment with 0–30 µg/mL curcumin decreased the mitochondrial membrane potential and activated the caspase‐3 and caspase‐9 in a dose‐ and time‐dependent manner. Nuclear and annexin V/PI staining showed that curcumin induced the apoptosis of LoVo cells. FACS analysis revealed that curcumin could induce the cell cycle arrest of LoVo cells at the S phase. Furthermore, western blotting analysis indicated that curcumin induced the release of cytochrome c, a significant increase of Bax and p53 and a marked reduction of Bcl‐2 and survivin in LoVo cells. Taken together, our results suggested that curcumin inhibited the growth of LoVo cells by inducing apoptosis through a mitochondria‐mediated pathway. Copyright © 2012 John Wiley & Sons, Ltd.     

Cycloartane triterpenoids from Cimicifuga yunnanensis induce apoptosis of breast cancer cells (MCF7) via p53‐dependent mitochondrial signaling pathway

The present study was carried out to investigate the antitumor activity of five cycloartane triterpenoids isolated from Cimicifuga yunnanensis on the breast cancer cell line MCF7 and its corresponding drug resistant subline R‐MCF7, including cimigenol‐3‐O‐β‐d ‐xylopyranoside (compound 1), 25‐O‐acetylcimigenol‐3‐O‐β‐d ‐xylopyranoside (compound 2), 25‐chlorodeoxycimigenol‐3‐O‐β‐d ‐xylopyranoside (compound 3), 25‐O‐acetylcimigenol‐3‐O‐α‐l ‐arabinopyranoside (compound 4) and 23‐O‐acetylcimigenol‐3‐O‐β‐d ‐xylopyranoside (compound 5). The results showed that compounds 2–5 have relatively high antitumor activity on both MCF7 and R‐MCF7 cells. The involvement of apoptosis as a major cause of cycloartane triterpenoids‐induced cell death was further confirmed. The results of RT‐PCR showed that compounds 2–5 increased the expression of p53 and bax, which led to the loss of mitochondrial potential and then resulted in the activation of caspase‐7. These findings collectively demonstrated that compounds 2–5 induced apoptosis of MCF7 via p53‐dependent mitochondrial pathway. Copyright © 2010 John Wiley & Sons, Ltd.     

Aloe‐emodin Induces Apoptosis in Human Liver HL‐7702 Cells through Fas Death Pathway and the Mitochondrial Pathway by Generating Reactive Oxygen Species

Aloe‐emodin (1,8‐dihydroxy‐3‐hydroxymethyl‐anthraquinone) is one of the primary active compounds in total rhubarb anthraquinones isolated from some traditional medicinal plants such as Rheum palmatum L. and Cassia occidentalis, which induce hepatotoxicity in rats. Thus, the aim of this study was to determine the potential cytotoxic effects and the underlying mechanism of aloe‐emodin on human normal liver HL‐7702 cells. The CCK‐8 assays demonstrated that aloe‐emodin decreased the viability of HL‐7702 cells in a dose‐dependent and time‐dependent manner. Aloe‐emodin induced S and G2/M phase cell cycle arrest in HL‐7702 cells. This apoptosis was further investigated by flow cytometry and nuclear morphological changes by DAPI staining, respectively. Moreover, aloe‐emodin provoked the production of intracellular reactive oxygen species and the depolarization of mitochondrial membrane potential (MMP). Further studies by western blot indicated that aloe‐emodin dose‐dependently up‐regulated the levels of Fas, p53, p21, Bax/Bcl‐2 ratio, and cleaved caspase‐3, ‐8, ‐9, and subsequent cleavage of poly(ADP‐ribose)polymerase (PARP). Taken together, these results suggest that aloe‐emodin inhibits cell proliferation of HL‐7702 cells and induces cell cycle arrest and caspase‐dependent apoptosis via both Fas death pathway and the mitochondrial pathway by generating reactive oxygen species, indicating that aloe‐emodin should be taken into account in the risk assessment for human exposure. Copyright © 2017 John Wiley & Sons, Ltd.     

Morin enhances auranofin anticancer activity by up‐regulation of DR4 and DR5 and modulation of Bcl‐2 through reactive oxygen species generation in Hep3B human hepatocellular carcinoma cells

Evidence suggests that auranofin (AF) exhibits anticancer activity by inhibiting thioredoxin reductase (TrxR). Here, in this study, we have investigated the synergistic effects of AF and morin and their mechanism for the anticancer effects focusing on apoptosis in Hep3B human hepatocellular carcinoma cells. We assessed the anticancer activities by annexin V/PI double staining, caspase, and TrxR activity assay. Morin enhances the inhibitory effects on TrxR activity of AF as well as reducing cell viability. Annexin V/PI double staining revealed that morin/AF cotreatment induced apoptotic cell death. Morin enhances AF‐induced mitochondrial membrane potential (ΔΨm) loss and cytochrome c release. Further, morin/AF cotreatment upregulated death receptor DR4/DR5, modulated Bcl‐2 family members (upregulation of Bax and downregulation of Bcl‐2), and activated caspase‐3, ‐8, and ‐9. Morin also enhances AF‐induced reactive oxygen species (ROS) generation. The anticancer effects results from caspase‐dependent apoptosis, which was triggered via extrinsic pathway by upregulating TRAIL receptors (DR4/DR5) and enhanced via intrinsic pathway by modulating Bcl‐2 and inhibitor of apoptosis protein family members. These are related to ROS generation. In conclusion, this study provides evidence that morin can enhance the anticancer activity of AF in Hep3B human hepatocellular carcinoma cells, indicating that its combination could be an alternative treatment strategy for the hepatocellular carcinoma.     

Apigenin as an anticancer agent

Apigenin is an edible plant‐derived flavonoid that has been reported as an anticancer agent in several experimental and biological studies. It exhibits cell growth arrest and apoptosis in different types of tumors such as breast, lung, liver, skin, blood, colon, prostate, pancreatic, cervical, oral, and stomach, by modulating several signaling pathways. Apigenin induces apoptosis by the activation of extrinsic caspase‐dependent pathway by upregulating the mRNA expressions of caspase‐3, caspase‐8, and TNF‐α. It induces intrinsic apoptosis pathway as evidenced by the induction of cytochrome c, Bax, and caspase‐3, while caspase‐8, TNF‐α, and B‐cell lymphoma 2 levels remained unchanged in human prostate cancer PC‐3 cells. Apigenin treatment leads to significant downregulation of matrix metallopeptidases‐2, ?9, Snail, and Slug, suppressing invasion. The expressions of NF‐κB p105/p50, PI3K, Akt, and the phosphorylation of p‐Akt decreases after treatment with apigenin. However, apigenin‐mediated treatment significantly reduces pluripotency marker Oct3/4 protein expression which might be associated with the downregulation of PI3K/Akt/NF‐κB signaling.     

Neferine,an alkaloid from lotus seed embryo targets HeLa and SiHa cervical cancer cells via pro‐oxidant anticancer mechanism

Apoptosis and autophagy are important processes that control cellular homeostasis and have been highlighted as promising targets for novel anticancer drugs. This study aims to investigate the inhibitory effects and mechanisms of Neferine (Nef), an alkaloid from the lotus seed embryos of Nelumbo nucifera (N. nucifera), as a dual inducer of apoptosis and autophagy through the reactive oxygen species (ROS) activation in cervical cancer cells. Nef and N. nucifera extract suppressed the cell viability of HeLa and SiHa cells in a dose‐dependent manner. Importantly, Nef showed minimal toxicity to normal cells. Furthermore, Nef inhibited anchorage‐independent growth, colony formation and migration ability of cervical cancer cells. Nef induces mitochondrial apoptosis by increasing pro‐apoptotic protein bax, cytochrome‐c, cleaved caspase‐3 and caspase‐9, poly‐ADP ribose polymerase (PARP) cleavage, DNA damage (pH₂AX) while downregulating Bcl‐2, procaspase‐3 and procaspase‐9, and TCTP. Of note, apoptotic effect by Nef was significantly attenuated in the presence of N‐acetylcysteine (NAC), suggesting pro‐oxidant activity of this compound. Nef also promoted autophagy induction through increasing beclin‐1, atg‐4, atg‐5 and atg‐12, LC‐3 activation, and _P62/SQSTM1 as determined by western blot analysis. Collectively, these results demonstrate that Nef is a potent anticancer compound against cervical cancer cells through inducing apoptosis and autophagic pathway involving ROS.     

Corosolic Acid Triggers Mitochondria and Caspase‐dependent Apoptotic Cell Death in Osteosarcoma MG‐63 Cells

The response of osteosarcoma MG‐63 cells to corosolic acid treatment has been investigated. The results showed that corosolic acid significantly inhibited cell viability in both a dose and a time dependent manner. It was found that corosolic acid increased the Bax/Bcl‐2 ratio by up‐regulating Bax expression, disrupted mitochondrial membrane potential and triggered the release of cytochrome c from mitochondria into the cytoplasm. Corosolic acid treatment triggered the activation of caspase‐8, 9 and 3. The apoptosis was obviously inhibited by pretreatment with a general caspase inhibitor, z‐VAD‐FMK. Moreover, pretreatment of CsA, a cyclophilin D ligand that inhibits mitochondria potential uncoupling, prevented the activation of caspase‐9 and caspase‐3, but not caspase‐8, and the apoptosis of MG‐63 cells, triggered by corosolic acid. All these results indicated that corosolic acid‐induced apoptosis was associated with the activation of caspases via a mitochondrial pathway. Copyright © 2011 John Wiley & Sons, Ltd.     

β‐Eudesmol Induces JNK‐Dependent Apoptosis through the Mitochondrial Pathway in HL60 Cells

β‐eudesmol, a natural sesquiterpenol present in a variety of Chinese herbs, is known to inhibit the proliferation of human tumor cells. However, the molecular mechanisms of the effect of β‐eudesmol on human tumor cells are unknown. In the present study, we report the cytotoxic effect of β‐eudesmol on the human leukemia HL60 cells and its molecular mechanisms. The cytotoxic effect of β‐eudesmol on HL60 cells was associated with apoptosis, which was characterized by the presence of DNA fragmentation. β‐eudesmol‐induced apoptosis was accompanied by cleavage of caspase‐3, caspase‐9, and poly (ADP‐ribose) polymerase; downregulation of Bcl‐2 expression; release of cytochrome c from mitochondria; and decrease in mitochondrial membrane potential (MMP). Activation of c‐Jun N‐terminal kinases (JNK) mitogen‐activated protein kinases was observed in β‐eudesmol‐treated HL60 cells, and the inhibitor of JNK blocked the β‐eudesmol‐induced apoptosis, downregulation of Bcl‐2, and the loss of MMP. These data suggest that β‐eudesmol induces apoptosis in HL60 cells via the mitochondrial apoptotic pathway, which is controlled through JNK signaling. Copyright © 2012 John Wiley & Sons, Ltd.     

3,4,5‐Tricaffeoylquinic Acid Attenuates TRAIL‐induced Apoptosis in Human Keratinocytes by Suppressing Apoptosis‐related Protein Activation

Caffeoyl derivatives exhibit antiinflammatory and antioxidant effects. However, the effect of 3,4,5‐tricaffeoylquinic acid on the tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptosis in keratinocytes that may be involved in skin diseases has not been studied. In this respect, we investigated the effect of 3,4,5‐tricaffeoylquinic acid on TRAIL‐induced apoptosis in human keratinocytes. 3,4,5‐Tricaffeoylquinic acid and oxidant scavengers attenuated the decrease in the cytosolic levels of Bid, Bcl‐2, and survivin proteins; the increase in the levels of cytosolic Bax, p53, and phosphorylated p53; the increase in the levels of phosphorylated p38; the increase in the mitochondrial levels of the voltage‐dependent anion channel; loss of the mitochondrial transmembrane potential; the release of cytochrome c; activation of caspases (8, 9, and 3); cleavage of poly [ADP‐ribose] polymerase‐1; production of reactive oxygen species; the depletion of glutathione (GSH); nuclear damage; and cell death in keratinocytes treated with TRAIL. These results suggest that 3,4,5‐tricaffeoylquinic acid may reduce TRAIL‐induced apoptosis in human keratinocytes by suppressing the activation of the caspase‐8 and Bid pathways and the mitochondria‐mediated cell death pathway. The effect appears to be associated with the inhibitory effect on the production of reactive oxygen species and depletion of GSH. 3,4,5‐Tricaffeoylquinic acid appears to be effective in the prevention of TRAIL‐induced apoptosis‐mediated skin diseases. Copyright © 2015 John Wiley & Sons, Ltd.     

Antiproliferative activity and apoptosis induction by trijuganone C isolated from the root of Salvia miltiorrhiza Bunge (Danshen)

In this study, we found that the hexane fraction of Danshen, the dried root of Salvia miltiorrhiza (Lamiaceae), exerted antiproliferative effects on human leukemia cells. Phytochemical investigation of the hexane fraction achieved the isolation of the tanshinone diterpenes: dihydrotanshinone I ( 1 ), trijuganone C ( 2 ), trijuganone B ( 3 ), cryptotanshinone ( 4 ), tanshinone IIA ( 5 ), and tanshinone I ( 6 ). Compound 2 showed significant antiproliferative activities against human leukemia cells HL‐60, Jurkat, and U937. The antiproliferative activities of 2 against human cancer and normal cells indicated that 2 exhibited potent antiproliferative activities with IC₅₀ values less than 10 μM against HL‐60 and Jurkat cells as well as on the colon cancer cells DLD‐1, COLO 205, and Caco‐2. Compound 2 induced chromatin condensation, DNA fragmentation, activation of caspase‐3, ‐8, and ‐9, and the cleavage of poly (ADP‐ribose) polymerase (PARP) in HL‐60 cells. Moreover, 2 activated Bid and Bax, leading to the loss of mitochondrial membrane potential, and 2 induced the cytochrome c release from mitochondria into cytosol. In contrast, Bcl‐2 and Bcl‐xL were unaffected by 2 . These results suggest that 2 exerts antiproliferative effects via apoptosis induction mediated by mitochondrial dysfunction and caspase activation. Compound 2 may serve as a candidate of potential chemotherapeutic agent for human leukemia.     

Ramalin‐Mediated Apoptosis Is Enhanced by Autophagy Inhibition in Human Breast Cancer Cells

Breast cancer, the most commonly diagnosed cancer in women worldwide, is treated in various ways. Ramalin is a chemical compound derived from the Antarctic lichen Ramalina terebrata and is known to exhibit antioxidant and antiinflammatory activities. However, its effect on breast cancer cells remains unknown. We examined the ability of ramalin to induce apoptosis and its mechanisms in MCF‐7 and MDA‐MB‐231 human breast cancer cell lines. Ramalin inhibited cell growth and induced apoptosis in both cell lines in a concentration‐dependent manner. By upregulating Bax and downregulating Bcl‐2, ramalin caused cytochrome c and apoptosis‐inducing factor to be released from the mitochondria into the cytosol, thus activating the mitochondrial apoptotic pathway. In addition, activated caspase‐8 and caspase‐9 were detected in both types of cells exposed to ramalin, whereas ramalin activated caspase‐3 only in the MDA‐MB‐231 cells. Ramalin treatment also increased the levels of LC3‐II and p62. Moreover, the inhibition of autophagy by 3‐methyladenine or Atg5 siRNA significantly enhanced ramalin‐induced apoptosis, which was accompanied by a decrease in Bcl‐2 levels and an increase in Bax levels. Therefore, autophagy appears to be activated as a protective mechanism against apoptosis in cancer cells exposed to ramalin. These findings suggest that ramalin is a potential anticancer agent for the treatment of patients with non‐invasive or invasive breast cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

Xiao-Xu-Ming decoction preserves mitochondrial integrity and reduces apoptosis after focal cerebral ischemia and reperfusion via the mitochondrial p53 pathway

Ethnopharmacological relevance

Xiao-Xu-Ming decoction (XXMD) has been used to treat stroke and other neurological diseases for more than 1000 years. The purpose of this study was to investigate the effects of XXMD on mitochondrial damage and apoptosis after cerebral ischemia and reperfusion.

Materials and methods

Male Sprague-Dawley rats were randomly divided into 3 groups: sham, cerebral ischemia and reperfusion (I/R), and cerebral ischemia and reperfusion plus XXMD (60 g/kg/day) (XXMD60). Focal cerebral ischemia and reperfusion models were induced by middle cerebral artery occlusion. Cerebral ischemic injury was evaluated by hematoxylin and eosin staining. Ultrastructural features of mitochondria in the penumbra of the ischemic cortex were analyzed by transmission electron microscopy. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) staining and cleaved caspase 3 immunohistochemistry. Proteins in the mitochondrial p53 pathway were detected by western blot and immunofluorescence.

Results

The results showed that XXMD treatment markedly attenuated ischemic changes, preserved mitochondrial integrity, and significantly reduced apoptosis. In addition, we found that XXMD treatment reduced p53 and Bax levels and increased Bcl-2 levels in mitochondrial fractions. XXMD significantly blocked the release of cytochrome c and Smac/Diablo from mitochondria, and inhibited activation of caspase 9 and caspase 3 in cytoplasmic fractions. Increased expression of c-IAP1 was observed in the XXMD60 group.

Conclusions

The findings demonstrated that XXMD protected mitochondria from ischemic injury and inhibited apoptosis. The mitochondrial p53 pathway could be partially involved in the protective effects.     

Pongapin and Karanjin,furanoflavanoids of Pongamia pinnata,induce G2/M arrest and apoptosis in cervical cancer cells by differential reactive oxygen species modulation,DNA damage,and nuclear factor kappa‐light‐chain‐enhancer of activated B cell signaling

In this study, the antitumor activity of two furanoflavanoid derivatives, Pongapin and Karanjin, was evaluated in comparison with Plumbagin, a plant‐derived polyphenol with proven antitumor activity. The compounds differentially inhibit the growth of different cancer cell lines (most effective on HeLa cells), with very low inhibitory effect on the growth of normal mouse embryonic fibroblast cell line. Pongapin like Plumbagin could significantly increase the intracellular reactive oxygen species (ROS) in the HeLa cells by stabilization of nuclear factor of kappa light polypeptide gene enhancer in B‐cells inhibitor (I‐κB) expression and reduction of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) expression. In contrast, Karanjin could decrease ROS level by inhibition of I‐κB degradation resulting restriction of NF‐κB nuclear translocation. Pongapin and Plumbagin significantly increased DNA damage‐induced p53 expression and p21 nuclear expression. However, Karanjin treatment showed low DNA damage with increased p53 expression. The compounds induced G2/M arrest and increase in SubG1 population, indicating induction of apoptosis. Apoptosis was further validated by acridine orange/ethidium bromide dual staining and terminal deoxynucleotidyl transferase dUTP nick‐end labeling assay in HeLa cells after treatment with the compounds. The compounds induced caspase‐dependent apoptosis through induction of Bax/Bcl‐2 ratio either through increased expression of Bax by Pongapin and Plumbagin or low expression of Bcl‐2 by Karanjin. Thus, Pongapin and Karanjin may be potential natural anticancer agents in the future, like Plumbagin.     

α‐Hederin induces the apoptosis of gastric cancer cells accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway

α‐Hederin, a monodesmosidic triterpenoid saponin, exhibited promising antitumor potential against a variety of human cancer cell lines. However, few related studies about effects of α‐hederin on gastric cancer are available. Herein, our results showed that α‐hederin significantly inhibited the proliferation of gastric cancer cells and arrested the cell cycle in G1 phase in vitro (p < .05). Further research of the potential mechanism reflected that α‐hederin could induce intracellular glutathione decrement, adenosine triphosphate level, and mitochondrial membrane potential variation via inducing reactive oxygen species accumulation during the apoptosis of gastric cancer cells. Moreover, the detection of mitochondrial and cytosol proteins with apoptosis‐inducing factor, apoptosis protease activating factor‐1, and cytochrome C showed an increase in the cytosol, followed by a decrease of Bcl‐2 levels and increases of caspase‐3, caspase‐8, caspase‐9, and Bax, which revealed that α‐hederin induced apoptosis via triggering activation of the mitochondrial pathway. Furthermore, the above changes were amplified when pretreated with buthionine sulfoximine, whereas attenuated in the group pretreated with NAC than α‐hederin alone (p < .05). In addition, α‐hederin significantly inhibited the growth of xenografted gastric tumors with favorable safety. In conclusion, α‐hederin could inhibit the proliferation and induce apoptosis of gastric cancer accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway.     

Paris saponin VII induces cell cycle arrest and apoptosis by regulating Akt/MAPK pathway and inhibition of P‐glycoprotein in K562/ADR cells

Paris saponinVII (PSVII) is a steroidal saponin isolated from the roots and rhizomes of Trillium tschonoskii Maxim. We found that PSVII could inhibit the growth of adriamycin‐resistant human leukemia cells (K562/ADR) in a dose‐dependent manner. Furthermore, the molecular mechanism underlying the cytotoxicity and downregulation of P‐glycoprotein (P‐gp) expression by PSVII was clarified. PSVII significantly suppressed cell proliferation by cell cycle arrest in the G0/G1 phase, which was associated with an obvious decrease in cyclin B1/D1 and CDK2/4/6 protein expression. Moreover, PSVII could attenuate mitochondrial membrane potential, increase the expression of apoptosis‐related proteins, such as Bax and cytochrome c, and decrease the protein expression levels of Bcl‐2, caspase‐9, caspase‐3, PARP‐1, and p‐Akt. We also found that JNK, ERK1/2, and p38 were regulated by PSVII in K562/ADR cells. And further studies indicated that the decrease in the reactive oxygen species level inhibited intrinsic P‐gp expression. Therefore, PSVII‐induced apoptosis in K562/ADR cells was associated with Akt/MAPK and the inhibition of P‐gp. In addition, PSVII induced a robust autophagy in K562/ADR cells as demonstrated by the degradation of LC3‐I. These results provide a biochemical basis for possible clinical applications of PSVII in the treatment of leukemia.     

土贝母苷甲在人宫颈癌HeLa细胞内对线粒体的影响

目的:探讨线粒体在土贝母苷甲(TBMS1)诱导人宫颈癌HeLa细胞凋亡过程中的作用。方法:流式细胞仪测定线粒体膜电位(ΔΨm)、琼脂糖凝胶电泳分析DNA断裂片和蛋白质免疫印迹法检测细胞色素c(cyt c)的表达。结果:TBMS1引起人宫颈癌HeLa细胞凋亡,使得ΔΨm下降,释放入胞质的cyt c增加,并具有时间、剂量依赖性;TBMS1还可直接作用于分离的鼠肝脏线粒体,使之释放cyt c;TBMS1的这种作用部分能够被线粒体膜保护剂环孢菌素A(CsA)阻断。结论:线粒体途径是TBMS1诱导的HeLa细胞凋亡的途径之一。     

Pachymic Acid Induces Apoptosis of EJ Bladder Cancer Cells by DR5 Up‐Regulation,ROS Generation,Modulation of Bcl‐2 and IAP Family Members

Pachymic acid (PA) is a lanostane‐type triterpenoid derived from Poria cocos mushroom that possess various biological effects such as anti‐cancer, antiinflammatory and anti‐metastasis effects. In this study, we investigated the anti‐cancer effects of PA in EJ bladder cancer cells. The results showed that PA significantly inhibited proliferation of EJ cells in a dose‐dependent manner. PA induced accumulation of sub‐G1 DNA content (apoptotic cell population), apoptotic bodies and chromatin condensation and DNA fragmentation in EJ cells in a dose‐dependent manner. PA also induces activation of caspase‐3, ‐8 and ‐9, and subsequent cleavage of poly (ADP‐ribose) polymerase, and significantly suppressed the inhibitor of apoptosis protein family proteins in a dose‐dependent manner. Furthermore, PA activates Bid and induced the loss of mitochondrial membrane potential (ΔΨ_m) with up‐regulated pro‐apoptotic proteins (Bax and Bad), down‐regulated anti‐apoptotic proteins (Bcl‐2 and Bcl‐xL) and cytochrome c release. In turn, PA increased the generation of reactive oxygen species (ROS); also, the ROS production was blocked by N‐acetyl‐L‐cysteine. The expressions of TNF‐related apoptosis inducing ligand and death receptor 5 were up‐regulated by PA in a dose‐dependent manner, suggesting extrinsic pathway also involved in PA‐induced apoptosis. This study provides evidence that PA might be useful in the treatment of human bladder cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

Harmine Hydrochloride Triggers G2 Phase Arrest and Apoptosis in MGC‐803 Cells and SMMC‐7721 Cells by Upregulating p21, Activating Caspase‐8/Bid,and Downregulating ERK/Bad Pathway

This study aimed to investigate the effects of harmine hydrochloride (HMH) on digestive tumor cells in vitro and its molecular mechanism. MTT assays showed that HMH inhibited the proliferation of some human cancer cell lines and had no obvious inhibitory effects on human LO2 cells. Flow cytometry assays showed that HMH trigged G2 phase arrest in MGC‐803 cells and SMMC‐7721 cells, while the expression of cyclin A, cyclin B, p21, Myt1, and p‐cdc2 (Tyr15) was upregulated. Flow cytometry assays also showed that the percentages of apoptotic cells were increased, the mitochondrial transmembrane potential (ΔΨm) decreased, and the cleavage of caspase‐9, caspase‐3, and poly (Adenosine diphosphate ribose) polymerase (PARP) were observed, the expression of Bad increased, phospho‐Bad (S112) decreased, pro‐caspase‐8 was cleaved, and Bid (22 kDa) was cleaved. The expression of p‐ERK decreased in both cells. In conclusion, these results demonstrated that HMH upregulates the expression of p21, activates Myt1 and inhibits cdc2 by phospho‐cdc2 (Y15), and triggers G2 phase arrest in both MGC‐803 cells and SMMC‐7721 cells. It can also activate the mitochondria‐related cell apoptosis pathway through the caspase‐8/Bid pathway, inhibiting the ERK/Bad pathway and promoting apoptosis in both of these two cell types. Copyright © 2015 John Wiley & Sons, Ltd.     

Epoxyazadiradione Purified from the Azadirachta indica Seed Induced Mitochondrial Apoptosis and Inhibition of NFκB Nuclear Translocation in Human Cervical Cancer Cells

Epoxyazadiradione (EAD) is an important limonoid present in Neem (Azadirachta indica) plant. In the present study, we have purified EAD from Neem seed and studied its anticancer potential in human cervical cancer (HeLa) cells. Cell proliferation inhibition studies indicated that the GI₅₀ value of EAD is 7.5 ± 0.0092 μM in HeLa cells, whereas up to 50 μM concentrations EAD did not affect the growth of normal H9C2 cells. The control drug cisplatin inhibited the growth of both HeLa and H9C2 cells with a GI₅₀ value of 2.92 ± 1.192 and 4.22 ± 1.568 μM, respectively. Nuclear DNA fragmentation, cell membrane blebbing, phosphatidylserine translocation, upregulation of Bax, caspase 3 activity and poly (ADP ribose) polymerase cleavage and downregulation of BCl2 in HeLa cells on treatment with EAD indicated the apoptotic cell death. Increase in caspase 9 activity and release of active cytochrome c to the cytoplasm on treatment with EAD confirmed that the apoptosis was mediated through the mitochondrial pathway. Epoxyazadiradione also inhibited the nuclear translocation of nuclear factor κB in HeLa cells. Thus, our studies demonstrated EAD as a potent and safe chemotherapeutic agent when compared with the standard drug cisplatin that is toxic to both cancer and normal cells equally. Copyright © 2017 John Wiley & Sons, Ltd.