A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation

Although reactive oxygen species (ROS) have been implicated in cadmium (Cd)-induced hepatotoxicity, the role of ROS in this pathway remains unclear. Therefore, we attempted to determine the molecular mechanisms relevant to Cd-induced cell death in HepG2 cells. Cd was found to induce apoptosis in the HepG2 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis and TUNEL staining. In the early stages, both rapid and transient ROS generation triggered apoptosis via Fas activation and subsequent caspase-8-dependent Bid cleavage, as well as by calpain-mediated mitochondrial Bax cleavage. The timing of Bid activation was coincided with the timing at which the mitochondrial transmembrane potential (MMP) collapsed as well as the cytochrome c (Cyt c) released into the cytosol. Furthermore, mitochondrial permeability transition (MPT) pore inhibitors, such as cyclosporin A (CsA) and bongkrekic acid (BA), did not block Cd-induced ROS generation, MMP collapse and Cyt c release. N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of the Cd-induced apoptosis via catalase upregulation and subsequent Fas downregulation. NAC treatment also completely blocked the Cd-induced intracellular ROS generation, MMP collapse and Cyt c release, indicating that Cd-induced mitochondrial dysfunction may be regulated indirectly by ROS-mediated signaling pathway. Taken together, a rapid and transient ROS generation by Cd triggers apoptosis via caspase-dependent pathway and subsequent mitochondrial pathway. NAC inhibits Cd-induced apoptosis through the blocking of ROS generation as well as the catalase upregulation.     

Cholesteryl-hemisuccinate-induced apoptosis of promyelocytic leukemia HL-60 cells through a cyclosporin A-insensitive mechanism

We reported previously that alpha-tocopheryl-succinate (VES) induced apoptosis of cultured human promyelocytic leukemia cells (HL-60) (Free Radic Res 2000;33:407-18). We have now studied the effect of cholesteryl-hemisuccinate (CS) on the fate of HL-60 cells to clarify whether CS has an effect similar to that of VES. CS inhibited the growth of HL-60 cells without differentiation to granulocytes and induced DNA fragmentation and ladder formation. CS inhibited the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt) and initiated the activation of a caspase cascade. CS triggered the reaction leading to the cleavage of Bid and also released cytochrome c (Cyt. c) from mitochondria. In addition, CS induced mitochondrial membrane depolarization and translocation of Bax to mitochondria in HL-60 cells. However, CS did not induce an increase in the concentration of intracellular calcium ions in HL-60 cells. The membrane depolarization, Cyt. c release, and DNA fragmentation were inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor, but not by cyclosporin A, an inhibitor of membrane permeability transition. These results suggested that CS-induced apoptosis of HL-60 cells might be caused by inhibiting Akt phosphorylation following cleavage of Bid through caspase-8 activation and subsequently via an Apaf complex-caspase cascade pathway.     

Critical role of pro-apoptotic Bcl-2 family members in andrographolide-induced apoptosis in human cancer cells

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory activity. In this study, Andro induced apoptosis in human cancer cells via activation of caspase 8 in the extrinsic death receptor pathway and subsequently with the participation of mitochondria. Andro triggered a caspase 8-dependent Bid cleavage, followed by a series of sequential events including Bax conformational change and mitochondrial translocation, cytochrome c release from mitochondria, and activation of caspase 9 and 3. Inhibition of caspase 8 blocked Bid cleavage and Bax conformational change. Consistently, knockdown of Bid protein using small interfering RNA (siRNA) technique suppressed Andro-induced Bax conformational change and apoptosis. In conclusion, the pro-apoptotic Bcl-2 family members (Bid and Bax) are the key mediators in relaying the cell death signaling initiated by Andro from caspase 8 to mitochondria and then to downstream effector caspases, and eventually leading to apoptotic cell death.     

Capsaicin-induced apoptosis is regulated by endoplasmic reticulum stress- and calpain-mediated mitochondrial cell death pathways

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca²⁺, calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.     

Induction of apoptosis in human hepatoblastoma cells by tetrandrine via caspase-dependent Bid cleavage and cytochrome c release

Tetrandrine, a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, induces apoptosis in human T-cell lines, lung carcinoma and hepatoblastoma cells. However, the mechanisms by which tetrandrine inhibits tumor cell growth are poorly understood. The purpose of the present study was to investigate the intracellular signaling mechanism of tetrandrine-induced apoptosis in HepG2 cells. The induction of apoptosis was determined by morphological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Treatment of cells with tetrandrine caused the upregulation of p53, downregulation of Bcl-X(L), cleavage of Bid and Bax, and release of cytochrome c, which were accompanied by activation of caspases 9, 3 and 8. The activation of caspases 9 and 3 preceded that of caspase 8. A broad-spectrum caspase inhibitor and a caspase 8-specific inhibitor completely blocked tetrandrine-induced Bid processing, cytochrome c release, activation of caspase 3, and cell death. These findings and data showing the early release of cytochrome c, cleavage of Bid and downregulation of Bcl-X(L) suggest that the mitochondrial pathway is primarily involved in tetrandrine-induced apoptosis. The activation of caspase 8 after early caspases 9 and 3 activation might act as an amplification loop for activation of upstream signals such as Bid cleavage or cytochrome c release. These data suggest that tetrandrine may constitute a plausible therapeutic for hepatocellular carcinoma.     

Protection of betulin against cadmium-induced apoptosis in hepatoma cells

The protective effects of betulin (BT) against cadmium (Cd)-induced cytotoxicity have been previously reported. However, the mechanisms responsible for these protective effects are unclear. Therefore, this study investigated the mechanisms responsible for the protection of BT against Cd-induced cytotoxicity in human hepatoma cell lines. The protection of BT against Cd cytotoxicity was more effective in the HepG2 than in the Hep3B cells. The protection of BT on Cd-induced cytotoxicity in the HepG2 cells appeared to be related to the inhibition of apoptosis, as determined by PI staining and DNA fragmentation analysis. The anti-apoptosis exerted by BT involved the blocking of Cd-induced reactive oxygen species (ROS) generation, the abrogation of the Cd-induced Fas upregulation, the blocking of caspase-8-dependent Bid activation, and subsequent inhibition of mitochondrial pathway. The BT pretreatment did not affect the p21 and p53 expression levels, when compared with those of the treated cells with Cd alone. BT induced the transient S phase arrest at an early stage and the G₀/G₁ arrest at a relatively late stage, but it did not observe the sub-G1 apoptotic peak. In the Hep3B cells, Cd did not induce ROS generation. The BT pretreatment partially inhibited the Cd-induced apoptosis, which was related with the incomplete blockage in caspase-9 or -3 activation, as well as in Bax activation.     

c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine

Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH₂-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIP_L/S and Bcl₂ proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl₂ which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl₂ at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-l-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death.     

The protective effects of selenium on cadmium-induced oxidative stress and apoptosis via mitochondria pathway in mice kidney

Selenium, an essential trace element, showed the significant protective effects against kidney damage induced by some heavy metals. Our previous research have found that the protection effects of selenium on ROS mediated-apoptosis by mitochondria dysfunction in cadmium (Cd)-induced LLC-PK1 cells. The present study as a continuation of our earlier one to investigate the protective effects and mechanism of selenium on Cd-induced apoptosis of kidney in vivo. Cadmium exposure increased the production of reactive oxygen species (ROS) and altered the levels of oxidative stress related biomarkers in kidney tissue. A concomitant by the loss of mitochondrial membrane potential, cytochrome c release and regulation of VDAC, Bcl-2 and Bax were observed. Apoptotic nature of cell death is confirmed by activation of caspase-3, which is also supported by histological examination. During the process, selenium played a beneficial role against Cd-induced renal damage. Pretreatment with selenium partially blocked Cd-induced ROS generation, inhibited Cd induced mitochondrial membrane potential collapse, prevented cytochrome c release, inhibited caspase activation and changed the level of VDAC, Bcl-2 and Bax. Combining all, results suggest that selenium has an ability to inhibit mitochondrial apoptotic pathway in oxidative stress mediated kidney dysfunction caused by cadmium.     

Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (>500 microM) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 microM) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca2+ chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca2+-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.     

Cadmium induces Ca2+-dependent necrotic cell death through calpain-triggered mitochondrial depolarization and reactive oxygen species-mediated inhibition of nuclear factor-kappaB activity

This study investigates the mechanism of cell death induced by cadmium (Cd) in Chinese hamster ovary (CHO) cells. Cells exposed to 4 microM Cd for 24 h did not show signs of apoptosis, such as DNA fragmentation and caspase-3 activation. The pro-apoptotic (Bax) or anti-apoptotic (Bcl-2 and Bcl-xL) protein levels in the Bcl-2 family were not altered. However, an increase in propidium iodide uptake and depletion of ATP, characteristics of necrotic cell death, were observed. Cd treatment increased the intracellular calcium (Ca2+) level. Removal of the Ca2+ by a chelator, BAPTA-AM, efficiently inhibited Cd-induced necrosis. The increased Ca2+ subsequently mediated calpain activation and intracellular ROS production. Calpains then triggered mitochondrial depolarization resulting in cell necrosis. Cyclosporin A, an inhibitor of mitochondrial permeability transition, recovered the membrane potential and reduced the necrotic effect. The generated ROS reduced basal NF-kappaB activity and led cells to necrosis. An increase of NF-kappaB activity by its activator, PMA, attenuated Cd-induced necrosis. Calpains and ROS act cooperatively in this process. The calpain inhibitor and the ROS scavenger synergistically inhibited Cd-induced necrosis. Results in this study suggest that Cd stimulates Ca2+-dependent necrosis in CHO cells through two separate pathways. It reduces mitochondrial membrane potential by activating calpain and inhibits NF-kappaB activity by increasing the ROS level.     

Molecular mechanism of apoptosis induced by schizandrae-derived lignans in human leukemia HL-60 cells.

Schizandrae chinensis, a traditional Chinese medicine herb, has been used to treat hepatitis B disease in Chinese hospital clinic. We have isolated two bioactive compounds, deoxyschizandrin and gamma-schizandrin, from S. chinensis. In the present, we reported that deoxyschizandrin and gamma-schizandrin could induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by DNA fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Further molecular analysis showed that deoxyschizandrin and gamma-schizandrin caused the loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondrion to cytosol, truncation of Bid protein, and activation of caspase-3 and -9. However, they did not increase the intracellular level of reactive oxygen species (ROS). Antioxidants such as N-acetyl cysteine (NAC) and catalase did not block the apoptosis induced by deoxyschizandrin or gamma-schizandrin. These findings suggest that deoxyschizandrin and gamma-schizandrin-induced apoptosis in HL-60 cells involved ROS-independent mitochondrial dysfunction pathway.     

The protection of selenium on ROS mediated-apoptosis by mitochondria dysfunction in cadmium-induced LLC-PK1 cells

Selenium, an essential trace element, showed the significant protective effects against liver and kidney damage induced by some heavy metals. However, the mechanism how selenium suppresses cadmium (Cd)-induced cytotoxicity remains unclear. In this study, we investigated the protective mechanism of selenium on Cd-induced apoptosis in LLC-PK₁ cells via reactive oxygen species (ROS) and mitochondria linked signal pathway. Studies of PI and Annexin V dual staining analysis demonstrated that 20 μM Cd-induced apoptosis as early as 18 h. A concomitant by the generation of ROS, the loss of mitochondrial membrane potential, cytochrome c (cyt c) release, activation of caspase-9, -3 and regulation of Bcl-2 and Bax were observed. N-acetylcysteine (NAC, 500 μM), a free radical scavenger, was used to determine the involvement of ROS in Cd-induced apoptosis. During the process, selenium played the same role as NAC. The anti-apoptosis exerted by selenium involved the blocking of Cd-induced ROS generation, the inhibition of Cd-induced mitochondrial membrane potential collapse, the prevention of cyt c release, subsequent inhibition of caspase activation and the changed level of Bcl-2 and Bax. Taken together, we concluded that Cd-induced apoptosis was mediated by oxidative stress and selenium produced a significant protection against Cd–induced apoptosis in LLC-PK₁ via ameliorating the mitochondrial dysfunction.     

Diallyl disulfide induces apoptosis in human colon cancer cell line (COLO 205) through the induction of reactive oxygen species,endoplasmic reticulum stress,caspases casade and mitochondrial-dependent pathways

In this study, we investigated the effects of DADS on human colon cancer cell line COLO 205 on cell cycle arrest and apoptosis in vitro. After 24 h treatment of COLO 205 cells with DADS, the dose- and time-dependent decreases of viable cells were observed and the IC₅₀ was 22.47 μM. The decreased percentages of viable cells are associated with the production of ROS. Treatment of COLO 205 cells with DADS resulted in G2/M phase arrest and apoptosis occurrence through the mitochondrial-pathway (Bcl-2, Bcl-xL down-regulation and Bak, Bax up-regulation). DADS increased cyclin B, cdc25c-ser-216-9 and Wee1 but did not affect CDK1 protein and gene expression within 24 h of treatment. DADS-induced apoptosis was examined and confirmed by DAPI staining and DNA fragmentation assay. DADS promoted caspase-3, -8 and -9 activity and induced apoptosis were accompanied by increasing the levels of Fas, phospho-Ask1 and -JNK, p53 and decreasing the mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-9 and -3. The COLO 205 cells were pre-treated with JNK inhibitor before leading to decrease the percentage of apoptosis which was induced by DADS. Inhibition of caspase-3 activation blocked DADS-induced apoptosis on COLO 205 cells.     

Leptomycin B-induced apoptosis is mediated through caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the generation of ROS in U937 leukemia cells

Leptomycin B (LMB), which is originally isolated from Streptomyces, possesses anti-tumor properties in vivo and in vitro. Though it was previously reported that LMB induces cell cycle arrest and p53-mediated apoptosis in certain cancer cells, however, the mechanism by which LMB induces apoptosis remains poorly understood. Here, we investigated the mechanisms of apoptosis induced by LMB in U937 cells. Treatment with LMB concentration-dependently induced cytotoxicity and apoptosis in U937 cells that correlated temporally with activation of caspases and down-regulation of Mcl-1 and XIAP. LMB did not change the expressions of Bcl-2 or Bax. A broad spectrum caspase inhibitor, z-VAD-fmk, blocked caspase-3 activation and elevated the survival in LMB-treated U937 cells, suggesting that caspase-3 activation is critical for LMB-induced apoptosis. Interestingly, Bcl-2 overexpression that blocked cytochrome c release by LMB effectively attenuated the apoptotic response to LMB, suggesting that LMB-induced apoptosis is mediated through the mitochondrial pathway. Antioxidants or antioxidant enzymes had no effects on LMB-induced apoptosis. Data of flow cytometry analysis using 2',7'-dichlorofluorescein-diacetate further revealed no reactive oxygen species (ROS) generation by LMB, indicating that apoptosis induced by LMB is ROS-independent. However, the apoptotic response to LMB was not shown in U937 cells pretreated with the sulfhydryl group-containing antioxidant N-acetylcysteine (NAC). Further analysis suggested that NAC directly binds LMB and abolishes the apoptotic effects of LMB. Collectively, these findings suggest that LMB potently induces apoptosis in U937 cells, and LMB-induced apoptosis in U937 cells is related with cytochrome c release, activation of caspases, and selective down-regulation of Mcl-1 and XIAP.     

Necrotic and apoptotic features of cell death in response to Foscan photosensitization of HT29 monolayer and multicell spheroids

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan. Kinetics of apoptosis and necrosis after Foscan-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Deltapsim) addressed by flow cytometry technique at different time points post-Foscan-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.     

A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 microM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent.     

Mechanisms of thiosulfinates from Allium tuberosum L.-induced apoptosis in HT-29 human colon cancer cells

This study was performed to elucidate the apoptotic pathways by thiosulfinates, major biologically active components of Allium tuberosum L., in HT-29 human colon cancer cells. Thiosulfinates significantly induced cell death in dose- and time-dependent manners in HT-29 cells, which is associated with apoptosis. Thiosulfinates activated the initiator caspase-8, and -9, and the effector caspase-3. In the present study, thiosulfinates were found to stimulate Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates down-regulated the expression of the anti-apoptotic protein Bcl-2, and up-regulated the expression of the pro-apoptotic protein Bax. We also found that thiosulfinates increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, and induced DNA fragmentation and chromatin condensation in HT-29 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibited cell proliferation and activated both the caspase-dependent and caspase-independent apoptotic pathways in HT-29 cells.     

Mechanism of cadmium induced apoptosis in the immunocyte

Cadmium is the major component of polluted environment which can be fatal by mechanisms that are not fully clear. Our study indicates immunosupression may be one of the reason for that. It is well known that cadmium (Cd) has toxic and carcinogenic effects in rhondents and humans, but the effects of cadmium on apoptosis are still not clear. Although some studies have shown that cadmium has apoptotic potential, other studies have shown that cadmium can be anti-apoptotic. In the present study, we aimed to determine the mode of cell death and its mechanism in Swiss albino mice splenocyte by cadmium for its toxic effects. To identify the nature of cell death, our result signifies apoptotic mode of killing. In search of the mechanism behind it we found that cadmium increased cell death and lowered the survival of the host in a dose dependent manner. In search of the reason we found increased expression of the pro-apoptotic proteins p53 in splenic lymphocytes. Here we showed that cadmium induced p53-dependent apoptosis through cooperation between Bcl-xl down regulation without changing the Bcl-2 and Bax expression, the common target of p53. The down regulation of Bcl-xl strongly indicating mitochondrial involvement in apoptosis. It is confirmed by the release of cytochrome c and activation of caspase-3. All of these findings establish an important role of p53 and mitochondrial function in cadmium induced toxic environment in the host.     

Catalpol inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade.

In the present study, using a rat pheochromocytoma (PC12) cell line, the effect of catalpol on H2O2-induced apoptosis was studied. The apoptosis in H2O2-induced PC12 cells was accompanied by down-regulation of Bcl-2, up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol and sequential activation of caspase-1 and caspase-3 then leading to cleavage of poly-ADP-ribose polymerase (PARP). Catalpol not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation, PARP cleavage, and eventually protected against H2O2-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with catalpol can block H2O2-induced apoptosis by the regulation of Bcl-2 family members, as well as suppression of cytochrome c release and caspase cascade activation.