Allicin induces apoptosis in gastric cancer cells through activation of both extrinsic and intrinsic pathways

Allicin is an active compound derived from garlic that has been shown to have antitumor properties in vitro. The current study was designed to explore the effects and the underlying mechanism of allicin on gastric cancer cells. The MTT assay was used to detect cell viability. Transmission electron microscopy, Rh123 and propidium iodide staining, annexin V/FITC assay and the mitochondrial membrane potential were used to assess for the presence of apoptosis. Immunocytochemistry, western blot analysis, and Q-RT-PCR were used to detect gene expression. We found that allicin reduced cell viability in a dose- and time-dependent manner, partly through induction of apoptosis in gastric cancer cells. At the molecular level, allicin induced cytochrome c release from the mitochondria and increased caspase-3, -8, and -9 activation, with concomitant upregulation of bax and fas expression in the tumor cells. Allicin treatment inhibited proliferation and induced apoptosis in SGC-7901 cancer cells. Both intrinsic mitochondrial and extrinsic Fas/FasL-mediated pathways of apoptosis occur simultaneously in SGC-7901 cells following allicin treatment. Data from the current study demonstrated that allicin should be further investigated as a novel cancer preventive or therapeutic agent in control of gastric cancer, with potential uses in other tumor types.     

Cantharidin induces apoptosis in human bladder cancer TSGH 8301 cells through mitochondria-dependent signal pathways

Cantharidin has shown potent anticancer activities on many types of human cancer cells. This study was performed to elucidate whether mitochondria and caspases are involved in the modulation of apoptosis and cell cycle arrest by cantharidin in human bladder cancer cells. The effect of cantharidin on cell cycle arrest, apoptosis, caspases, reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ(m)) were measured by flow cytometry, and the levels of apoptosis-associated proteins and its regulatory molecules were studied by Western blotting. Cantharidin-induced apoptosis and DNA damage was determined by flow cytometric analysis, DAPI staining and Comet assay. After cantharidin treatment, the active forms of caspase-3, -8 and -9 were promoted. Cantharidin-induced apoptosis was associated with enhanced ROS and Ca(2+) generations, caused DNA damage, decreased the levels of ΔΨ(m) and promoted Endo G and AIF released from mitochondria. Cantharidin-induced G0/G1 arrest was associated with a marked decrease in the protein expressions of cyclin E and Cdc25c but promoted the levels of p21 and p-p53. Cantharidin-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and PARP, but down-regulation of the protein levels of Bcl-2, resulting in dysfunction of mitochondria then led to Endo G and AIF release for causing induction of apoptosis.     

Inhibition of EGFR signaling augments oridonin-induced apoptosis in human laryngeal cancer cells via enhancing oxidative stress coincident with activation of both the intrinsic and extrinsic apoptotic pathways

Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been reported to have anti-tumor effects, while the epidermal growth factor receptor (EGFR) signal pathway has been reported to play a vital role in the biological progression of several tumors and to be a target for therapeutic intervention. In this work, we show that inhibition of EGFR with tyrphostin AG1478 enhances oridonin-induced cell death in human laryngeal cancer cells HEp-2, a cell line characterized by EGFR gene amplification. The enhanced apoptotic effect correlates with high expression and activation of Bax, FADD, caspase-8 as well as caspase-3 and decreased protein levels of Bcl₂ and SIRT1, suggesting that both the extrinsic and intrinsic apoptosis pathways are involved in the apoptotic processes. However, treatment with oridonin and AG1478 greatly enhances nuclear translocation of apoptosis inducing factor (AIF) without caspase-9 activation, indicating that the apoptosis occurs via a caspase-9-independent mitochondrial pathway. Here, it is the active form of caspase-8 but not caspase-9 that activates downstream effector caspase-3, resulting in the cleavage of critical cellular proteins and apoptosis. Furthermore, the combined use of AG1478 and oridonin augments the production of reactive oxygen species (ROS). Incubation of cells with N-Acetylcysteine (NAC) attenuates the apoptosis and the mitochondrial membrane potential (Δψm) disruption induced by the combination of oridonin and AG1478, which indicates that ROS plays a pivotal role in cell death. In conclusion, targeting EGFR combined with other conventional pro-apoptotic drugs should be a potentially very effective anti-neoplastic therapy for laryngeal cancer.     

Bufalin, a traditional oriental medicine, induces apoptosis in human cancer cells

Bufalin is a traditional oriental medicines which induces apoptosis in some lines of human tumor cells. It constitutes the major digoxin-like immunoreactive component of Chan Su, obtained from the skin and parotid venom glands of toads. Bufalin is cardioactive C-24 steroids that exhibits a variety of biological activities, such as cardiotonic, anaesthetic, blood pressure stimulatory, respiratory and antineoplastic effects. In terms of its anti-tumor activity, bufalin has been demonstrated to inhibit the growth of tumors, such as endometrial and ovarian cancers. This commentary introduces biologic and therapeutic effects of bufalin in treating some cancers. The compound is able to mediate inhibition of cell growth, cell cycle arrest, apoptosis, and expression of genes related to the malignant phenotype in human cancer cells.     

Involvement of both intrinsic and extrinsic pathways in IFN-gamma-induced apoptosis that are enhanced with cisplatin

IFN-gamma has direct anti-proliferative effects on ovarian cancer cell lines and tumour cells isolated from ovarian cancer ascites. The aim of this study was to further elucidate the mechanisms involved. An IFN-gamma-mediated cell cycle blockade was detectable in synchronised cell populations. Apoptosis, which was caspase dependent, was also induced. When caspase activity was blocked, the anti-proliferative effect of IFN-gamma was only partially reduced indicating independent roles for both growth inhibition and apoptosis in its actions. We have demonstrated involvement of the intrinsic apoptotic pathway; IFN-gamma treatment resulted in mitochondrial membrane depolarisation, cytochrome c release into the cytosol and activation of caspase 9. Cytochrome c release was blocked by the presence of a general caspase inhibitor, suggesting a role for caspases upstream of the mitochondria. One candidate is caspase 8, which was also activated in cells treated with IFN-gamma. Levels of Bid, a pro-apoptotic molecule that can mediate mitochondrial membrane permeabilisation when cleaved by caspase 8, were also decreased and indicated a potential link between these two pathways in IFN-gamma-induced apoptosis. Furthermore, together with cisplatin, IFN-gamma exerted a more powerful anti-proliferative effect.     

Pemetrexed induces both intrinsic and extrinsic apoptosis through ataxia telangiectasia mutated/p53‐dependent and ‐independent signaling pathways

Pemetrexed, a new‐generation antifolate, has demonstrated promising single‐agent activity in front‐ and second‐line treatments of non‐small cell lung cancer. However, the molecular mechanism of pemetrexed‐mediated antitumor activity remains unclear. The current study shows that pemetrexed induced DNA damage and caspase‐2, ‐3, ‐8, and ‐9 activation in A549 cells and that treatment with caspase inhibitors significantly abolished cell death, suggesting a caspase‐dependent apoptotic mechanism. The molecular events of pemetrexed‐mediated apoptosis was associated with the activation of ataxia telangiectasia mutated (ATM)/p53‐dependent and ‐independent signaling pathways, which promoted intrinsic and extrinsic apoptosis by upregulating Bax, PUMA, Fas, DR4, and DR5 and activating the caspase signaling cascade. Supplementation with dTTP allowed normal S‐phase progression and rescued apoptotic death in response to pemetrexed. Overall, our findings reveal that the decrease of thymidylate synthase and the increase of Bax, PUMA, Fas, DR4, and DR5 genes may serve as biomarkers for predicting responsiveness to pemetrexed. © 2011 Wiley Periodicals, Inc.     

Apoptin induces apoptosis in human bladder cancer EJ and BIU-87 cells

Objective:To investigate whether apoptin is a apoptosis-inducing protein with a potential for bladder cancertherapy. Methods: We constructed a PCDNA3/Apoptin eukaryotic expression vector, and transfected this vectorinto bladder cancer cell lines BIU-87 and EJ, then observed the results by RT-PCR, transmission electronmicroscopy, MTT assay and the flow cytometry (TUNEL method). Results: PCDNA3/Apoptin successfullyinduced a high level apoptosis in both bladder cancer cell lines, compared with the controls (p<0.05). Conclusions:Apoptin can induce high level apoptosis in human bladder cancer EJ and BIU-87 cells, which suggests a potentialfor human bladder cancer therapy.     

Simultaneous modulation of the intrinsic and extrinsic pathways by simvastatin in mediating prostate cancer cell apoptosis

ABSTRACT: BACKGROUND: Recent studies suggest the potential benefits of statins as anti-cancer agents. Mechanisms by which statins induce apoptosis in cancer cells are not clear. We previously showed that simvastatin inhibit prostate cancer cell functions and tumor growth. Molecular mechanisms by which simvastatin induce apoptosis in prostate cancer cells is not completely understood. METHODS: Effect of simvastatin on PC3 cell apoptosis was compared with docetaxel using apoptosis, TUNEL and trypan blue viability assays. Protein expression of major candidates of the intrinsic pathway downstream of simvastatin-mediated Akt inactivation was analyzed. Gene arrays and western analysis of PC3 cells and tumor lysates were performed to identify the candidate genes mediating extrinsic apoptosis pathway by simvastatin. RESULTS: Data indicated that simvastatin inhibited intrinsic cell survival pathway in PC3 cells by enhancing phosphorylation of Bad, reducing the protein expression of Bcl-2, Bcl-xL and cleaved caspases 9/3. Over-expression of PC3 cells with Bcl-2 or DN-caspase 9 did not rescue the simvastatin-induced apoptosis. Simvastatin treatment resulted in increased mRNA and protein expression of molecules such as TNF, Fas-L, Traf1 and cleaved caspase 8, major mediators of intrinsic apoptosis pathway and reduced protein levels of pro-survival genes Lhx4 and Nme5. CONCLUSIONS: Our study provides the first report that simvastatin simultaneously modulates intrinsic and extrinsic pathways in the regulation of prostate cancer cell apoptosis in vitro and in vivo, and render reasonable optimism that statins could become an attractive anti-cancer agent.     

2-methoxyestradiol up-regulates death receptor 5 and induces apoptosis through activation of the extrinsic pathway

2-Methoxyestradiol (2ME2), a natural metabolite of estradiol, is a potent antitumor and antiangiogenic agent. In vitro, 2ME2 inhibits the proliferation of a wide variety of cell lines and primary cultures, and in numerous models in vivo, it has been shown to be an effective inhibitor of tumor growth and angiogenesis. 2ME2 is currently in several Phase I and Phase II clinical trials under the name Panzem. Although various molecular targets have been proposed for this compound, the mechanism by which 2ME2 exerts its effects is still uncertain. This study shows that 2ME2 uses the extrinsic pathway for induction of apoptosis. 2ME2 treatment results in up-regulation of death receptor 5 (DR5) protein expression in vitro and in vivo and renders cells more sensitive to the cytotoxic activities of the DR5 ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). 2ME2-induced apoptosis requires caspase activation and kinetic studies show the sequential activation of caspase-8, caspase-9, and caspase-3. Blockage of death receptor signaling by expression of dominant-negative Fas-associated death domain severely attenuates the ability of 2ME2 to induce apoptosis. Because 2ME2 administration has not manifested dose-limiting toxicity in the clinic, DR5 expression may serve as a surrogate marker for biological response.     

Bufalin and cinobufagin induce apoptosis of human hepatocellular carcinoma cells via Fas- and mitochondria-mediated pathways

Bufadienolides bufalin and cinobufagin are cardiotonic steroids isolated from the skin and parotid venom glands of the toad Bufo bufo gargarizans Cantor. They have been shown to induce a wide spectrum of cancer cell apoptosis. However, the detailed molecular mechanisms of inducing apoptosis in hepatocellular carcinoma (HCC) are still unclear. In the present study, the apoptosis-inducing effect of bufalin and cinobufagin on HCC cell line HepG(2) was investigated. We found bufalin and cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells. This apoptotic induction was associated with an increase in Fas, Bax and Bid expression, a decrease in Bcl-2 expression, disruption of the mitochondrial membrane potential, release of cytochrome c, activation of caspase-3, -8, -9 and -10, and the cleavage of poly(ADP-ribose)polymerase (PARP), which indicated that bufalin and cinobufagin induced apoptosis through both Fas- and mitochondria-mediated pathways. In addition, caspase activation during bufalin- and cinobufagin-induced apoptosis was further confirmed by caspase-3 inhibitor Z-DEVD-FMK, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK and caspase-10 inhibitor Z-AEVD-FMK. The results showed that bufalin- and cinobufagin-induced apoptosis was blocked by these inhibitors and particularly by caspase-10 inhibitor. Taken together, bufalin and cinobufagin induce apoptosis of HepG(2) cells via both Fas- and mitochondria-mediated pathways, and a Fas-mediated caspase-10-dependent pathway might play a crucial role.     

Induction of apoptosis by the synthetic retinoid MX3350-1 through extrinsic and intrinsic pathways in head and neck squamous carcinoma cells

Retinoids have shown promise in cancer prevention and therapy. As some retinoids exhibit undesirable side effects, new retinoid analogs have been synthesized. In this study, we examined the effects of the retinoid MX3350-1 on human head and neck squamous cell carcinoma (HNSCC) cell lines. MX3350-1 suppressed the growth of 7/8 HNSCC cell lines by >65%. This inhibition appeared to be due to induction of apoptosis as revealed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Studies with cell line UMSCC17B indicated that apoptosis was induced within 1-2 days and involved activation of caspase-8, -9, and -3. Inhibitors of these caspases suppressed MX3350-1-induced apoptosis. MX3350-1 decreased the levels of antiapoptotic Bcl-2 and Bcl-XL, increased proapoptotic Bax, induced mitochondrial membrane permeabilization (MMP), and cytochrome c release from mitochondria to cytosol. The antioxidant butylated hydroxyanisol and the MMP inhibitor cyclosporin A (Cs A) blocked apoptosis induced by MX3350-1. In contrast, retinoid receptor antagonists failed to inhibit apoptosis. MX3350-1 increased the levels of Fas-ligand, Fas, and Fas-associated death domain, and enhanced activation of procaspase-8 and cleavage of its substrate Bid. Soluble Fas rescued the cells from MX3350-1-induced apoptosis. These results demonstrate that MX3350-1 induces apoptosis by activating both extrinsic and intrinsic apoptosis pathways and suggest that further studies on the potential of this retinoid for prevention and therapy of HNSCCs are warranted.     

Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells

Intact mycobacteria and mycobacterial cell wall extracts have been shown to inhibit the growth of human and murine bladder cancer. Their mechanism of action is, however, poorly understood. Mycobacterium phlei mycobacterial cell complex (MCC) is a cell wall preparation that has mycobacterial DNA in the form of short oligonucleotides complexed on the cell wall surface. In this study, we have investigated the possibility that MCC has anti-cancer activity that is mediated by two different mechanisms – a direct effect on cancer cell proliferation and viability and an indirect effect mediated by the production of interleukin 12 (IL-12), a cytokine known to possess anti-cancer activity. We have found that, although MCC is a potent inducer of IL-12 and IL-6 synthesis in monocytes and macrophages either in vitro or in vivo, it is unable to induce the synthesis of either IL-12, IL-6 or granulocyte–macrophage colony-stimulating factor (GM-CSF) by the human transitional bladder cancer cell lines HT-1197 and HT-1376. MCC is not directly cytotoxic towards these cancer cells, but induces apoptosis as determined by nuclear DNA fragmentation and by the release of nuclear mitotic apparatus protein. Mycobacterium phlei DNA associated with MCC is responsible for the induction of apoptosis. Our results indicate that MCC directly effects bladder cancer cells by inhibiting cellular proliferation through the induction of apoptosis, and has the potential for an indirect anti-cancer activity by stimulating cancer-infiltrating monocytes/macrophages to synthesize IL-12. © 1999 Cancer Research Campaign     

Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells

Over the coming years, skin cancer could become a significant public health problem. Previous results indicate that ursolic acid (UA), a pentacyclic triterpene acid, has pleiotropic biologic activities such as antiinflammatory and antiproliferative activities on cancer cells. As UA represents a promising chemical entity for the protection of human skin, in agreement with tests done by the cosmetic industry, we investigated its effects on the M4Beu human melanoma cell line. In this report, we demonstrated for the first time that UA had a significant antiproliferative effect on M4Beu, associated with the induction of an apoptotic process, characterized by caspase-3 activation, the downstream central effector of apoptosis. We demonstrated that UA-induced apoptosis was dependent on the mitochondrial intrinsic pathway, as shown by transmembrane potential collapse (DeltaPsim) and by alteration of the Bax-Bcl-2 balance, with a concomitant increase in Bax expression and decrease in Bcl-2 expression. We also showed that UA-induced DeltaPsim was associated with apoptosis-inducing factor leakage from mitochondria. Taken together, our results suggest that UA-induced apoptosis on M4Beu cells is accomplished via triggering of mitochondrial pathway. In conclusion, UA could be an encouraging compound in the treatment or prevention of skin cancer and may represent a new promising anticancer agent in the treatment of melanoma.     

Arsenic trioxide induces apoptosis in human gastric cancer cells through up-regulation of p53 and activation of caspase-3

Arsenic trioxide (As(2)O(3)) can induce clinical remission in patients suffering from acute promyelocytic leukemia, through induction of apoptosis and activation of caspases. We investigated the potential use of As(2)O(3) in human gastric cancer and its possible mechanisms. Human gastric cancer cell lines AGS and MKN-28 were treated with various concentrations (0.1 to 100 microM) of As(2)O(3) for 24 to 72 hr. Apoptosis was determined by acridine orange staining, flow cytometry and DNA fragmentation. Protein levels of p53, p21(waf1/cip1), c-myc, bcl-2 and bax were detected by Western blotting. Effects of As(2)O(3) on caspase-3 protease activity, its protein concentration and cleavage of poly(ADP)-ribose polymerase (PARP) were also studied. As(2)O(3) inhibited cell growth and induced apoptosis in both cell lines, though AGS cells were more sensitive. As(2)O(3) induced apoptosis in AGS cells in a concentration- and time-dependent manner. Treatment resulted in a marked increase in p53 protein levels as early as 4 hr. Co-incubation with p53 anti-sense oligo-nucleotide suppressed As(2)O(3)-induced intracellular p53 over-expression and apoptosis. As(2)O(3) increased the activity of caspase-3, with appearance of its 17 kDa peptide fragment, and cleavage of PARP, with appearance of the 85 kDa cleavage product, both in parallel with the induction of apoptosis. Both the tripeptide caspase inhibitor zVAD-fmk and the specific caspase-3 inhibitor DEVD-fmk partially suppressed As(2)O(3)-induced caspase-3 activation and apoptosis. As(2)O(3) inhibits cell growth and induces apoptosis in gastric cancer cells, involving p53 over-expression and activation of caspase-3. The potential use of this compound in the treatment of gastric cancer is worth further investigation.     

Artemisinin induces apoptosis in human cancer cells

BACKGROUND: Artemisinin is a chemical compound extracted from the wormwood plant, Artemisia annua L. It has been shown to selectively kill cancer cells in vitro and retard the growth of implanted fibrosarcoma tumors in rats. In the present research, we investigated its mechanism of cytotoxicity to cancer cells. MATERIALS AND METHODS: Molt-4 cells, in complete RPMI-1640 medium, were first incubated with 12 microM of human holotransferrin at 37 degrees C in a humid atmosphere of 5% CO2 for one hour. This enhanced the iron supply to the cells. The cells were then pelleted and transferred to a complete RPMI-1640 containing 200 microM of an analog dihydroartemisinin (DHA) and incubation was started (0 h). In addition, some culture samples were treated with holotransferrin alone and some (controls) were assayed without neither holotransferrin nor DHA treatment. Cells were counted and DNA diffusion assay was used to evaluate apoptosis and necrosis in each sample at 0 h and at 1, 2, 4 and 8 h of incubation. RESULTS: DHA treatment significantly decreased cell counts and increased the proportion of apoptosis in cancer cells compared to controls (chi2=4.5, df=1, p<0.035). Addition of holotransferrin significantly further decreased cell counts (chi2=4.5, df=1, p<0.035) and increased apoptosis (chi2=4.5, df=1, p<0.035). No necrotic cells were observed. CONCLUSION: This rapid induction of apoptosis in cancer cells after treatment with DHA indicates that artemisinin and its analogs may be inexpensive and effective cancer agents.     

Valproic acid induces apoptosis in human leukemia cells by stimulating both caspase-dependent and -independent apoptotic signaling pathways

We investigated the effects of valproic acid (VPA) on the growth and survival of human leukemia cell lines. VPA induced cell death in all of the nine cell lines tested in a dose dependent manner. VPA-treatment induced apoptotic changes in MV411 cells including DNA fragmentation, phosphatidylserine externalization, cytochrome c release from mitochondria, and activation of caspases-3, -8, and -9. A caspase inhibitor, zVAD-FMK, inhibited the DNA fragmentation induced by VPA but not cell death. These findings suggest that VPA exerts an anti-leukemic effect by both caspase-dependent and -independent apoptotic signaling pathways.     

Leukemia inhibitory factor induces apoptosis and proliferation of human carcinoma cells through different oncogene pathways

Leukemia inhibitory factor (LIF) affects the growth of carcinoma cells, and we thus analyzed its underlying mechanisms. Carcinoma cells constitutively express LIF mRNA, and 23 lines (92.0%) and all (100%) of 25 lines express LIF receptor mRNAs of LIFRβ and gp130, respectively. Exogenous addition of LIF promoted significant cell proliferation in 4 lines (MCF-7, ZR-75-1, Hs-700T and Panc-1) and suppressed cell growth in 3 lines (AZ-521, GBK-1 and HT-29). LIF significantly induced an immediate early response of genes c-fos and junB 3 hr after stimulation, but not of c-jun during the process of proliferation of MCF-7 and Hs-700T cells, with maximum levels at 30–60 min. The cell-cycle-related gene cyclin E was also induced in MCF-7 and Hs-700T cells, whereas cyclinA, cdk2, c-myc, c-myb and p53 mRNAs were not induced. On the other hand, LIF inhibited growth and increased the rate of cell death of AZ-521 and GBK-1 cells. LIF increased the number of TUNEL-positive cells in AZ-521 cells and DNA fragmentation in AZ-521 and GBK-1 cells. LIF induced apoptosis related genes c-myc and ICE during suppression of cell growth, but p53, p21, c-fos, cyclin A and cyclin E were not induced. Our results suggest that LIF is linked to cell proliferation and apoptosis in some human carcinoma cell lines. It is considered that this is related to differences in signal transduction and induction of oncogenes. Int. J. Cancer 72:687–695, 1997. © 1997 Wiley-Liss, Inc.     

Se-methylselenocysteine induces apoptosis through caspase activation in HL-60 cells

Apoptosis, a programmed process of cell suicide, has been proposed as the most plausible mechanism for the chemopreventive activities of selenocompounds. In our study, we found that Se-methylselenocysteine (MSC) induced apoptosis through caspase activation in human promyelocytic leukemia (HL-60) cells. Measurements of cytotoxicity, DNA fragmentation and apoptotic morphology revealed that MSC was more efficient at inducing apoptosis than selenite, but was less toxic. Moreover, MSC increased both the apoptotic cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 activity, whereas selenite did not. We next examined whether caspases and serine proteases are required for the apoptotic induction by MSC. A general caspase inhibitor, z-VAD-fmk, dramatically decreased cytotoxicity in MSC-treated HL-60 cells and several other apoptotic features, such as, caspase-3 activation, the apoptotic DNA ladder, TUNEL-positive staining and the DNA double-strand break. Interestingly, a general serine protease inhibitor, AAPV-cmk, also effectively inhibited MSC-mediated cytotoxicity and apoptosis. These results demonstrate that MSC is a selenocompound that efficiently induces apoptosis in leukemia cells and that proteolytic machinery, in particular caspase-3, is necessary for MSC-induced apoptosis. On the other hand, selenite-induced cell death could be derived from necrosis rather than apoptosis, since selenite did not significantly induce several apoptotic phenomena, including the activation of caspase-3.