Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis.

Proapoptotic proteins such as Bax, undergo translocation to the mitochondria during apoptosis, where they mediate the release of intermembrane space proteins including cytochrome c. Bax binds to the voltage-dependent anion channel (VDAC). VDAC is a beta-barrel protein located in the outer mitochondrial membrane. In planar lipid bilayers, Bax and VDAC form a channel through which cytochrome c can pass. Hexokinase II (HXK II) also binds to VDAC. HXK II catalyzes the first step of glycolysis and is highly expressed in transformed cells, where over 70% of it is bound to the mitochondria. The present study demonstrates that HXK II interferes with the ability of Bax to bind to mitochondria and release cytochrome c. Detachment of HXK II from the mitochondria-enriched fraction isolated from HeLa cells promoted the binding of recombinant Bax-Delta19 and subsequent cytochrome c release. Similarly, the addition of recombinant HXK II to the mitochondria-enriched fraction isolated from hepatocytes, cells that do not express HXK II endogenously, prevented the ability of recombinant Bax-Delta19 to bind to the mitochondria and promote cytochrome c release. Similar results were found in intact cells, in which the detachment of mitochondrial bound HXK II or its overexpression potentiated and inhibited, respectively, Bax-induced mitochondrial dysfunction and cell death.     

Involvement of NF-κB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A

The plant Artocarpus obtusus is a tropical plant that belongs to the family Moraceae. In the present study a xanthone compound Pyranocycloartobiloxanthone A (PA) was isolated from this plant and the apoptosis mechanism was investigated. PA induced cytotoxicity was observed using MTT assay. High content screening (HCS) was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Reactive oxygen species formation was investigated on treated cells by using fluorescent analysis. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition mRNA levels of Bax and Bcl2 were also checked using RT-PCR. Caspase 3/7, 8 and 9 were measured for their induction while treatment. The involvement of NF-κB was analyzed using HCS assay. The results showed that PA possesses the characteristics of selectively inducing cell death of tumor cells as no inhibition was observed in non-tumorigenic cells even at 30μg/ml. Treatment of MCF7 cells with PA induced apoptosis with cell death-transducing signals, that regulate the MMP by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol. The release of cytochrome c triggered the activation of caspases-9, then activates downstream executioner caspase-3/7 and consequently cleaved specific substrates leading to apoptotic changes. This form of apoptosis was found closely associated with the extrinsic pathway caspase (caspase-8) and inhibition of translocation of NF-κB from cytoplasm to nucleus. The results demonstrated that PA induced apoptosis of MCF7 cells through NF-κB and Bcl2/Bax signaling pathways with the involvement of caspases.     

Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel

Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.     

A role for mitochondrial Bak in apoptotic response to anticancer drugs.

In the present study a clonal Jurkat cell line deficient in expression of Bak was used to analyze the role of Bak in cytochrome c release from mitochondria. The Bak-deficient T leukemic cells were resistant to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. In contrast to wild type Jurkat cells, these Bak-deficient cells did not respond to UV or treatment with these anticancer drugs by membranous phosphatidylserine exposure, DNA breaks, activation of caspases, or release of mitochondrial cytochrome c. The block in the apoptotic cascade was in the mitochondrial mechanism for cytochrome c release because purified mitochondria from Bak-deficient cells failed to release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. The resistance of Bak-deficient cells to VP-16 was reversed by transduction of the Bak gene into these cells. Also, the cytochrome c releasing capability of the Bak-deficient mitochondria was restored by insertion of recombinant Bak protein into purified mitochondria. Following mitochondrial localization, low dose recombinant Bak restored the mitochondrial release of cytochrome c in response to Bax; at increased doses it induced cytochrome c release itself. The function of Bak is independent of Bid and Bax because recombinant Bak induced cytochrome c release from mitochondria purified from Bax(-/-), Bid(-/-), or Bid(-/-) Bax(-/-) mice. Together, our findings suggest that Bak plays a key role in the apoptotic machinery of cytochrome c release and thus in the chemoresistance of human T leukemic cells.     

Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (mPTP) opening

Here we studied the role of mitochondrial permeability transition pore (mPTP) opening in curcumin’s cytotoxicity in melanoma cells. In cultured WM-115 melanoma cells, curcumin induced mitochondrial membrane potential (MPP) decrease, cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) (two mPTP components) mitochondrial association and cytochrome C release, indicating mPTP opening. The mPTP blocker sanglifehrin A (SfA) and ANT-1 siRNA-depletion dramatically inhibited curcumin-induced cytochrome C release and WM-115 cell death. CyPD is required for curcumin-induced melanoma cell death. The CyPD inhibitor cyclosporin A (CsA) or CyPD siRNA-depletion inhibited curcumin-induced WM-115 cell death and apoptosis, while WM-115 cells with CyPD over-expression were hyper-sensitive to curcumin. Finally, we found that C6 ceramide enhanced curcumin-induced cytotoxicity probably through facilitating mPTP opening, while CsA and SfA as well as CyPD and ANT-1 siRNAs alleviated C6 ceramide’s effect on curcumin in WM-115 cells. Together, these results suggest that curcumin-induced melanoma cell death is associated with mPTP opening.     

Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release

Bax mediates cytochrome c release and apoptosis during neurodevelopment. Brain mitochondria that were isolated from 8-day, 17-day, and adult rats displayed decreasing levels of mitochondrial Bax. The amount of cytochrome c released from brain mitochondria by a peptide containing the BH3 cell death domain decreased with increasing age. However, approximately 60% of cytochrome c in adult brain mitochondria could be released by the BH3 peptide in the presence of exogenous human recombinant Bax. Mitochondrial Bax was downregulated in PC12S neural cells differentiated with nerve growth factor, and mitochondria isolated from these cells demonstrated decreased sensitivity to BH3-peptide-induced cytochrome c release. These results demonstrate that immature brain mitochondria and mitochondria from undifferentiated neural cells are particularly sensitive to cytochrome c release mediated by endogenous Bax and a BH3 death domain peptide. Postnatal developmental changes in mitochondrial Bax levels may contribute to the increased susceptibility of neurons to pathological apoptosis in immature animals.     

The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosis

Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (alpha9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.     

Apoptosis of Mo7e leukemia cells is associated with the cleavage of Bcl-2 into a shortened fragment that is not functional for heterodimerization with Bcl-2 and Bax

Bcl-2 is an integral intracellular membrane protein that can protect cells from apoptosis induced by multiple insults in a variety of cell types. During apoptosis, Bcl-2 was cleaved into a shortened fragment (Bcl-2/Delta34) by a caspase-3-like protease in human Mo7e megakaryocytic leukemia cells deprived of exogenous rhGM-CSF. Results from cell fractionation and immunoblot analyses indicated that both Bcl-2 and Bcl-2/Delta34 were located exclusively on the mitochondria of Mo7e cells. Treatment of isolated mitochondria with recombinant caspase-3 induced the same cleavage of Bcl-2 in vitro and caused the release of cytochrome c from the mitochondria into the supernatant. The antiapoptotic effect of Bcl-2/Delta34 was investigated using an in vitro protein translation approach. Both Bcl-2/Delta34 and Bax proteins generated in wheat germ extract were readily relocated to the mitochondria isolated from control Mo7e cells. Insertion of Bax, but not Bcl-2/Delta34, into mitochondria triggered a rapid release of cytochrome c from the mitochondria. Coimmunoprecipitation studies showed that, unlike Bcl-2, the cleaved Bcl-2 fragment was no longer functional for dimerization with either Bcl-2 or Bax. Taken together, these findings showed that the integrity of Bcl-2 is necessary for its function of heterodimerization with Bax, which appears to be one of the mechanisms of antiapoptotic effect of Bcl-2.     

Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis

Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.     

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.     

The mitochondrial permeability transition regulates cytochrome c release for apoptosis during endoplasmic reticulum stress by remodeling the cristae junction

The role of the mitochondrial permeability transition (MPT) in apoptosis and necrosis is controversial. Here we show that the MPT regulates the release of cytochrome c for apoptosis during endoplasmic reticulum (ER) stress by remodeling the cristae junction (CJ). CEM cells, HCT116 colon cancer cells, and murine embryo fibroblast cells were treated with the ER stressor thapsigargin (THG), which led to cyclophilin D-dependent mitochondrial release of the profusion GTPase optic atrophy 1 (OPA1), which controls CJ integrity, and cytochrome c, leading to apoptosis. Interference RNA knockdown of Bax blocked OPA1 and cytochrome c release after THG treatment but did not prevent the MPT, showing that Bax was essential for the release of cytochrome c by MPT. In isolated mitochondria, MPT led to OPA1 and cytochrome c release independently of voltage-dependent anion channel and the outer membrane, indicating that the MPT is an inner membrane phenomenon. Last, the MPT was regulated by the electron transport chain but not mitochondrial reactive oxygen species, since THG-induced cell death was not blocked by antioxidants and did not occur in cells lacking mitochondrial DNA. Our results show that the MPT regulates CJ remodeling for cytochrome c-dependent apoptosis induced by ER stress and that mitochondrial electron transport is indispensable for this process.     

The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.     

Redox status of thioredoxin-1 （TRX1） determines the sensitivity of human liver carcinoma cells （HepG2） to arsenic trioxide-induced cell death

lntracellular redox homeostasis plays a critical role in determining tumor cells＇ sensitivity to drug-induced apoptosis. Here we investigated the role of thioredoxin-1 （TRX1）, a key component of redox regulation, in arsenic trioxide （AS2O3）-induced apoptosis. Over-expression of wild-type TRX1 in HepG2 cells led to the inhibition of As2O3-induced cytochrome c （cyto c） release, caspase activation and apoptosis, and down-regulation of TRX1 expression by RNAi sensitized HepG2 cells to As2O3-induced apoptosis. Interestingly, mutation of the active site of TRX1 from Cys^32/35 to Ser^32/35 converted this molecule from an apoptotic protector to an apoptotic promoter. In an effort to understand the mechanisms of this conversion, we used isolated mitochondria from mouse liver and found that recombinant wild-type TRX1 could protect mitochondria from the apoptotic changes. In contrast, the mutant form of TRX1 alone elicited mitochondria-related apoptotic changes, including the mitochondrial permeability transition pore （mPTP） opening, loss of mitochondrial membrane potential, and cyto c release from mitochondria. These apoptotic effects were inhibited by cyclosporine A （CsA）, indicating that mutant TRX1 targeted to mPTP. Alteration of TRX1 from its reduced form to oxidized form in vivo by 2,4-dinitrochlorobenzene （DNCB）, a specific inhibitor ofTRX reductase, also sensitized HepG2 cells to As203-induced apoptosis. These data suggest that TRX1 plays a central role in regulating apoptosis by blocking cyto c release, and inactivation of TRX1 by either mutation or oxidization of the active site cysteines may sensitize tumor cells to As2O3-induced apoptosis.     

Apoptosis-associated mitochondrial outer membrane permeabilization assays

Following most cell death signals, pro-apoptotic Bcl-2 members as Bax and Bak are activated and oligomerize into the mitochondria outer membrane, triggering its permeabilization and release into the cytosol of soluble apoptogenic factors such as cytochrome c involved in caspase activation. Thus, in many studies focused on apoptosis, cytochrome c release within cells is frequently examined to assess Bax/Bak activation and mitochondrial outer membrane permeabilization. In addition, cytochrome c release can also be investigated in vitro in functional mitochondria that have been isolated from cultured cells, offering a number of advantages. Here, protocols for measuring cytochrome c release from intact cells as well as from isolated mitochondria is detailed. Finally, assays to investigate Bax/Bak activation and olimerization are also presented.     

Detailing the role of Bax translocation, cytochrome c release, and perinuclear clustering of the mitochondria in the killing of HeLa cells by TNF

Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.     

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.     

Regulation of intracellular pH mediates Bax activation in HeLa cells treated with staurosporine or tumor necrosis factor-alpha

Induction of apoptosis in HeLa cells with staurosporine produced a rise in the intracellular pH (pH(i)). Intracellular alkalinization was accompanied by translocation of Bax to the mitochondria, cytochrome c release, and cell death. The chloride channel inhibitor furosemide prevented intracellular alkalinization, Bax translocation, cytochrome c release, and cell death. Translocation of full-length Bid to the mitochondria was also prevented by furosemide. The cleavage product of Bid degradation (truncated Bid, tBid) was not detectable in the mitochondria. Its accumulation in the cytosol was prevented by furosemide. Apoptosis induced by tumor necrosis factor-alpha (TNF) lowered pH(i), an effect also accompanied by Bax translocation, cytochrome c release, and cell killing. Furosemide prevented all of these events. TNF induced a depletion of full-length Bid from the mitochondria and the cytosol but induced an accumulation of mitochondrial tBid. Furosemide only delayed full-length Bid depletion and tBid accumulation. The caspase 8 inhibitor IETD did not prevent the translocation of Bax. Although IETD did inhibit the cleavage of Bid and the accumulation of tBid, cell killing was reduced only slightly. It is concluded that with either staurosporine or TNF a furosemide-sensitive change in pH(i) is linked to Bax translocation, cytochrome c release, and cell killing. With TNF Bax translocation occurs as Bid is depleted and can be dissociated from the accumulation of tBid. With staurosporine a role for full-length Bid in Bax translocation cannot be excluded but is not necessary as evidenced by the data with TNF.     

Grifolin, a potential antitumor natural product from the mushroom Albatrellus confluens, inhibits tumor cell growth by inducing apoptosis in vitro

Grifolin is a natural biologically active substance isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens. Here, for the first time, we describe a novel activity of grifolin, namely its ability to inhibit the growth of tumor cells by the induction of apoptosis. Grifolin strongly inhibited the growth of tumor cell lines: CNE1, HeLa, MCF7, SW480, K562, Raji and B95-8. Analysis of acridine orange (AO)/ethidium bromide (EB) staining and flow cytometry showed that grifolin possessed apoptosis induction activity to CNE1, HeLa, MCF7 and SW480. Furthermore, the cytochrome c release from mitochondria was detected by confocal microscopy in CNE1 cells after a 12h treatment with grifolin. The increase of caspase-8, 9, 3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by grifolin, and the underexpression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Owing to the combination of the significant antitumor activity by inducing apoptosis and natural abundance of the compound, grifolin holds the promise of being an interesting antitumor agent that deserves further laboratory and in vivo exploration.     

TanshinoneIIA and Cryptotanshinone Protect against Hypoxia-Induced Mitochondrial Apoptosis in H9c2 Cells

Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA) and cryptotanshinone (CT) on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ) 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak). TIIA and CT (0.3 and 3 μΜ), in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.