Expression level of Bcl-2 determines anti- or proapoptotic function.

Bcl-2 is an oncogene with antiapoptotic function. However, Bcl-2 is converted to a Bax-like death effector by caspases, suggesting that the expression of Bcl-2 may not favor the growth of cancers. We introduced the Bcl-2 gene to gliomas via adenovirus (Adv; Adv-Bcl-2) with the Adv for Fas (Adv-Fas) and the Adv for Fas ligand (Adv-FL) to evaluate the antiapoptotic function of Bcl-2. In U251 glioblastoma cells, Bcl-2 at a low level of expression repressed apoptosis induced by Adv-Fas and Adv-FL, whereas Bcl-2 at a high level of expression did not. On the other hand, Bcl-X(L) showed antiapoptotic function against Fas-mediated apoptosis, irrespective of its expression level. In glioblastoma cells, induction of Bcl-2 alone at a high level induced apoptosis, whereas induction of Bcl-X(L) alone did not. As the multiplicity of infection of Adv-Bcl-2 was increased, the quantity of a cleaved product of Bcl-2 increased. Induction of caspase-inhibitory genes (CrmA and p35) inhibited apoptosis induced by Adv-Bcl-2. Induction of Bcl-2 led to alteration of the membrane potential and structure of the mitochondria. In summary, although Bcl-2 at a low level of expression was antiapoptotic, Bcl-2 at a high level of expression was proapoptotic to Fas-mediated apoptosis. Overexpression of Bcl-X(L) was consistently antiapoptotic to Fas-mediated apoptosis.     

5-Fluorouracil induces apoptosis in human colon cancer cell lines with modulation of Bcl-2 family proteins.

Recently, apoptosis has been implicated as one of the end points of cells exposed to chemotherapeutic agents. The p53 and Bcl-2 family of proteins are involved in chemotherapy-induced apoptosis, but in a cell type-dependent manner. We sought to determine the roles played by the p53 and Bcl-2 family of proteins in 5-fluorouracil (5-FU)-induced apoptosis of human colon cancer cell lines. We first studied the p53 genetic and functional status, and then 5-FU, at inhibitory concentration of 50% (IC50) doses, was used to induce apoptosis, which was confirmed by morphological analysis and enzyme-linked immunosorbent assay (ELISA). Bcl-2, Bcl-X(L), Bax, Bad, Bak and p53 protein expression was analysed by Western blotting. Using five human colon cancer cell lines, we found that equitoxic (IC50) doses of 5-FU induced apoptosis in both wild-type p53 and mutant p53 cells. Analysis of the steady-state levels of Bcl-2 family proteins showed high expression of Bcl-X(L) in all of the cell lines except Colo320. Bcl-2 was expressed in two of them. Bax presented with the lowest basal expression and Bad showed homogeneous expression. On the other hand, Bak expression varied more than fivefold among these cells. In cells containing wild-type p53 (e.g. LoVo), 5-FU-induced apoptosis was accompanied by increased expression of Bax and Bak without consistent modulation of other bcl-2 family proteins. In contrast in cells containing mutant p53 (e.g. DLD1), Bak expression was remarkably increased. There was a significant correlation between chemosensitivity and Bcl-X(L) to Bax ratio, rather than Bcl-2 to Bax. In conclusion, these results suggest that some members of the Bcl-2 family of proteins, in human colon cancer cell lines, are modulated by 5-FU and that the ratio of Bcl-X(L) to Bax may be related to chemosensitivity to 5-FU.     

The prognostic significance of apoptosis-associated proteins BCL-2, BAX and BCL-X in clinical nephroblastoma.

Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers in normal tissues. Various apoptosis-associated regulatory proteins, such as Bcl-2, Bax and Bcl-X, may contribute to the rate of apoptosis in neoplasia. The present study was performed to evaluate the prognostic value of these molecules in a group of 61 Wilms' tumours of chemotherapeutically pre-treated patients using an immunohistochemical approach. Generally, Bcl-2, Bax and for Bcl-X(S/L) were expressed in the blastemal and epithelial components of Wilms' tumour. Immunoreactive blastema cells were found in 53%, 41% and 38% of tumours for Bcl-2, Bax and for Bcl-X(S/L), respectively. An increased expression of Bcl-2 was observed in the blastemal component of increasing pathological stages. In contrast, a gradual decline of Bax expression was observed in the blastemal component of tumours with increasing pathological stages. Also blastemal Bcl-X(S/L) expression decreased with stage. Univariate analysis showed that blastemal Bcl-2 expression and the Bcl-2/Bax ratio were indicative for clinical progression, whereas epithelial staining was of no prognostic value. Multivariate analysis showed that blastemal Bcl-2 expression is an independent prognostic marker for clinical progression besides stage. These findings demonstrate that alterations of the Bcl-2/Bax balance may influence the clinical outcome of Wilms' tumour patients by deregulation of programmed cell death.     

Imatinib enhances human melanoma cell susceptibility to TRAIL-induced cell death: Relationship to Bcl-2 family and caspase activation

In order to define genetic determinants of primary and metastatic melanoma cell susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we have applied oligonucleotide microarrays to TRAIL-sensitive primary T1 cells and TRAIL-resistant metastatic G1 cells treated or not with TRAIL. T1 and G1 cells are isogenic melanoma cell subclones. We examined 22 000 spots, 4.2% of which displayed differential expression in G1 and T1 cells. Cell susceptibility to TRAIL-mediated apoptosis was found to be correlated with gene expression signatures in this model. Some of the differentially expressed genes were identified as involved in ATP-binding and signaling pathways, based on previously published data. Further analysis provided evidences that c-kit was overexpressed in G1 cells while it was absent in T1 cells. The c-kit inhibitor, imatinib, did not restore TRAIL sensitivity, excluding a role for c-kit in TRAIL resistance in G1 cells. Surprisingly, imatinib inhibited cell proliferation and TRAIL-mediated apoptosis in melanoma cells. We investigated the possible involvement of several molecules, including c-ABL, platelet-derived growth factor receptor (PDGFR), cellular FADD-like interleukin-1 alpha-converting enzyme-like inhibitory protein (c-FLIP)(L/S), Fas-associated DD kinase, p53, p21(WAF1), proteins of B-cell leukemia/lymphoma 2 (Bcl-2) family and cytochrome c. Imatinib did not modulate the expression or activation of its own targets, such as c-ABL, PDGFRalpha and PDGFRbeta, but it did affect the expression of c-FLIP(L), BCL2-associated X protein (Bax) and Bcl-2. Moreover, c-FLIP(L) knockdown sensitized T1 cells to TRAIL-mediated apoptosis, with a sensitivity similar to that of cells previously treated with imatinib. More notably, we found that the resistance to TRAIL in G1 cells was correlated with constitutive c-FLIP(L) recruitment to the DISC and the inhibition of caspase 8, 3 and 9 processing. Moreover, c-FLIP(L) knockdown partly restored TRAIL sensitivity in G1 cells, indicating that the expression level of c-FLIP(L) and its interaction with TRAIL receptor2 play a crucial role in determining TRAIL resistance in metastatic melanoma cells. Our results also show that imatinib enhances TRAIL-induced cell death independently of BH3-interacting domain death agonist translocation, in a process involving the Bax:Bcl-X(L) ratio, Bax:Bcl-X(L)/Bcl-2 translocation, cytochrome c release and caspase activation. Our data indicate that imatinib sensitizes T1 cells by directly downregulating c-FLIP(L), with the use of an alternative pathway for antitumor activity, because PDGFRalpha is not activated in T1 cells and these cells do not express c-kit, c-ABL or PDGFRbeta. Caspase cascade activation and mitochondria also play a key role in the imatinib-mediated sensitization of melanoma cells to the proapoptotic action of TRAIL.     

Bax membrane insertion during Fas(CD95)-induced apoptosis precedes cytochrome c release and is inhibited by Bcl-2

Ligation of the Fas cell surface receptor leads to activation of caspases and subsequent apoptosis. Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that the pro-apoptotic protein, Bax, translocates from the cytosol specifically to the mitochondria following Fas ligation in MCF10A1 breast epithelial cells. Bax translocation was dependent on caspase activation, and preceded the release of cytochrome c and loss of mitochondrial respiratory activity. Bax translocation occurred in concert with activation of downstream caspases as determined by cleavage of a synthetic substrate, proteolysis of poly(ADP-ribose) polymerase, and processing of procaspase-3 and -7. Overexpression of the anti-apoptotic protein, Bcl-2, prevented Bax insertion, cytochrome c release, complete processing of caspase-3 and -7, and full activation of DEVD-specific cleavage activity. These data establish a role for Bax mitochondrial insertion during Fas-mediated apoptosis, and support a model in which Bax insertion amplifies the Fas apoptotic cascade through cytochrome c release and complete processing of caspases-3 and -7. In addition, our findings indicate that prevention of Bax insertion into the mitochondria represents a novel mechanism by which Bcl-2 inhibits Fas-induced apoptosis.     

Overexpression of BCL-X(L) underlies the molecular basis for resistance to staurosporine-induced apoptosis in PC-3 cells

We have reported previously that among human prostate cancer cell lines LNCaP but not PC-3 cells undergo apoptosis after treatment with the protein kinase inhibitor staurosporine (STS). We have now further investigated this model to uncover the molecular mechanism causing resistance to STS-induced apoptosis in PC-3 cells. S-100 lysates of both cell lines showed biochemical changes typical of apoptosis after the addition of cytochrome c and dATP, suggesting that the postmitochondrial phase of apoptosis was intact. Upon addition of STS, the proapoptotic molecules Bax and Bad became predominantly mitochondrial in both cell lines. This, in turn, was followed by loss of mitochondrial transmembrane potential, translocation of cytochrome c to the cytosol, activation of caspase-9, -3, and -7, and cleavage of the apoptotic targets, DNA fragmentation factor and poly(ADP-ribose) polymerase, in LNCaP but not in PC-3 cells. Components of the mitochondrial permeability transition pore, adenine nucleotide transporter and voltage-dependent anion channel, were normally expressed in the correct subcellular fraction of both cell lines. Overexpression of the proapoptotic proteins Bax and Bad, fused to a green fluorescent protein but not of green fluorescent protein alone, induced apoptosis in >80% of PC-3 cells. These experiments suggested that a factor protecting the mitochondria of PC-3 cells mediates resistance to STS-induced apoptosis. A wide search among the antiapoptotic Bcl-2 family members was performed, and Bcl-X(L) was found to be overexpressed in PC-3 cells. Experiments down-regulating Bcl-X(L) expression by using the tyrosine kinase inhibitor genistein, sodium butyrate, or an antisense Bcl-X(L) oligonucleotide restored sensitivity to apoptosis in PC-3 cells. Thus, Bcl-X(L) overexpression is one of the mediators of resistance to STS-induced apoptosis in the prostate cancer cell line PC-3.     

2'-Nitroflavone induces cell cycle arrest and apoptosis in HeLa human cervical carcinoma cells

The mechanism of antitumor action of a synthetic nitroflavone derivative, 2'-nitroflavone, was evaluated in vitro in HeLa human cervix adenocarcinoma cells. We showed that the nitroflavone derivative slowed down the cell cycle at the S phase and increase the population of cells at the G(2)/M phase after 24h of incubation. The treatment with 2'-nitroflavone also induced an apoptotic response, characterized by an increase of the sub-G1 fraction of cells, by cells with chromatin condensation and membrane blebbing, by a typical ladder of DNA fragmentation and by detection of apoptotic cells stained with Annexin V. The observed apoptosis was regulated by caspase-8 and -9, both contributing to the activation of the effector caspase-3. In addition, inhibitors of caspase-8 or -9 partially protected HeLa cells from 2'-nitroflavone-induced cell death. We also found that 2'-nitroflavone did not affect the total amount of Bax and Bcl-2 proteins, although a translocation of Bax from cytosol to mitochondria was evident after 6h of exposure. Furthermore, 2'-nitroflavone decreased the expression of the anti-apoptotic Bcl-X(L) protein, induced the release of cytochrome C to cytosol and increased the levels of Fas and Fas-L. Our results indicated that both death receptor and mitochondria-dependent pathways are involved in the apoptotic cell death triggered by 2'-nitroflavone and suggest that this derivative could be a potentially useful agent for the treatment of certain malignancies.     

The hepatitis B virus X protein abrogates Bcl-2-mediated protection against Fas apoptosis in the liver

The role of the hepatitis B virus protein HBx in liver cell proliferation and apoptosis remains controversial. Using a transgenic mouse model, we have recently shown that HBx stimulates the apoptotic turnover of hepatocytes, independently of p53. In this paper, we tested whether the proapoptotic function of HBx can interfere with Bcl-2 during hepatic apoptosis in vivo. HBx transgenic mice were crossed with PK-hBcl-2 mice that are protected against Fas killing by constitutive overexpression of Bcl-2 in hepatocytes. In a lethal challenge with Fas antibodies, HBx expressed at low levels restored sensitivity to Fas-mediated apoptosis and fulminant hepatic failure in mice overexpressing Bcl-2. Furthermore, cytochrome c release from mitochondria and caspase 3 activation were restored to normal levels in HBx/Bcl-2 mice during transduction of the Fas signal. Thus, the proapoptotic activity of HBx overcomes or bypasses the inhibitory effect of Bcl-2 against Fas cytotoxicity. This effect was not apparently mediated through downregulation of the PK-hBcl-2 transgene or via delocalization of the Bcl-2 protein, and a direct interaction of HBx with Bcl-2, Bcl-X(L) or Bax could not be evidenced in yeast two-hybrid assays. We further show that apoptosis induced by ectopic expression of HBx is associated with mitochondrial membrane alterations and caspase 3 activation. Our data indicate that the dominant function of HBx upon Bcl-2-regulated control of apoptosis might play an important role in the pathogenesis of chronic hepatitis B.     

Co-transduction of p27Kip1 strongly augments Fas ligand- and caspase-8-mediated apoptosis in U-373MG glioma cells

BACKGROUND: p27Kip1 is a potential tumor suppressor gene. As malignant gliomas express Fas at high levels, the relationship between Fas-mediated apoptosis and p27Kip1 expression may improve therapeutic approaches for treating gliomas. MATERIALS AND METHODS: In this study, we transduced U-373MG glioma cells with the Fas ligand or caspase-8 genes using adenovirus vectors after transduction of the p27Kip1 gene to induce cell cycle arrest in U-373MG cells, and evaluated the degree of apoptosis. RESULTS: The results demonstrate that expression of p27Kip1 enhanced Fas ligand- or caspase-8-mediated apoptosis in U-373MG cells. Expression of apoptosis-related genes such as Bax, Bcl-X(L), Bcl-2 or caspase-8 were reduced by p27Kip1 transduction compared with that of beta-actin, whereas p27Kip1 transduction did not affect the expression level of Fas or the Fas ligand. CONCLUSION: Combined transduction of p27Kip1 with Fas ligand or caspase-8 would overide the resistance mechanism to apoptosis in malignant gliomas.     

Apoptosis induced by cryo-injury in human colorectal cancer cells is associated with mitochondrial dysfunction

Cryotherapy, a method of in situ ablation, is used in the treatment of colorectal liver metastases with variable results. During the treatment, the central area of treated tumor undergoes necrotic destruction by lethal cryo-injury; however, the cellular response of tumor exposed to sublethal cryo-injury at the peripheral zone is unclear. In our study, we have identified the induction of apoptosis by cryo-injury at -10 degrees C in 4 colorectal cancer cell lines (HT29, HCT116, KM12C and KM12SM). The apoptosis was characterized by chromatin condensation, transferase-mediated dUTP nick end-labeling (TUNEL) staining, proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and cytokeratin 18, and activation of caspase-3. The occurrence and intensity of cryo-induced apoptosis did not correlate with the functional status of p53 in the cell lines studied. The expression of anti-apoptotic proteins (Bcl-2, Bcl-X(L)) and pro-apoptotic proteins (Bax, Bcl-X(S), Bad, and Bak) in response to cryo-injury varied in this cell line panel. The basal level of Bcl-2/Bax protein ratio correlated inversely to the apoptotic rate. We further demonstrated that Bax level decreased in cytosol and increased in mitochondria, followed by a loss of mitochondrial membrane potential after cryo-injury in HT29 cells. These findings indicate that cryo-injury induces apoptosis in colorectal cancer cells via disruption of mitochondrial integrity. The cryo-induced apoptosis was also identified in a nude mouse tumor xenograft model. Our elucidation of the apoptosis pathway induced by cryo-injury implies that synergistic combination of cryosurgery with pharmacological agents that augment of apoptosis induction may have clinical relevance in treating colorectal liver metastasis.     

Cell permeable BH3-peptides overcome the cytoprotective effect of Bcl-2 and Bcl-X(L)

Peptides corresponding to the BH3 domains of Bax (BaxBH3) or Bcl-2 (Bcl2BH3) are potent inducers of apoptosis when fused to the Atennapedia plasma membrane translocation domain (Ant). BaxBH3Ant and Bcl2BH3Ant caused a mitochondrial membrane permeabilization (MMP) and apoptosis, via a mechanism that was not inhibited by overexpressed Bcl-2 or Bcl-X(L), yet partially inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore. When added to isolated mitochondria, BaxBH3 and Bcl2BH3 induced MMP, which was inhibited by CsA. However, Bcl-2 or Bcl-X(L) failed to inhibit MMP induced by BaxBH3 and Bc2BH3 in vitro, while they efficiently suppressed the induction of MMP by the Vpr protein (from human immunodeficiency virus-1), a ligand of the adenine nucleotide translocator (ANT). BaxBH3 but not Bcl2BH3 was found to interact with ANT, and only BaxBH3 (not Bcl2BH3) permeabilized ANT proteoliposomes and induced ANT to form non-specific channels in electrophysiological experiments. In contrast, both BaxBH3 and Bcl2BH3 were able to stimulate channel formation by recombinant Bax protein. Thus, BaxBH3 might induce MMP via an action on at least two targets, ANT and Bax-like proteins. In contrast, Bcl2BH3 would elicit MMP in an ANT-independent fashion. In purified mitochondria, two ligands of ANT, bongkrekic acid and the protein vMIA from cytomegalovirus, failed to prevent MMP induced by BaxBH3 or Bcl2BH3. In conclusion, BaxBH3 and Bcl2BH3 induce MMP and apoptosis through a mechanism which overcomes cytoprotection by Bcl-2 and Bcl-X(L).     

Distinct BAG-1 isoforms have different anti-apoptotic functions in BAG-1-transfected C33A human cervical carcinoma cell line

BAG-1 protein can be expressed as four isoforms of 50, 46, 33 and 29 kDa with different subcellular localizations, which may have different functions in anti-apoptosis, but the exact mechanism remains unclear. We constructed BAG-1 full length and deletion mutated plasmids in a pCR3.1 vector and established stable transfections of BAG-1 isoforms in low BAG-1 expressing C33A cells. Treatment of the transfected cells with cisplatin, staurosporine, paclitaxel and doxorubicine showed that BAG-1 p50, p46 and p33 isoforms enhanced the resistance to apoptosis. BAG-1 p50, p46 and p33 exhibited different degrees of apoptosis inhibition in the transfected cells and BAG-1 p46 isoform had the most pronounced effect on anti-apoptosis. BAG-1 p29 failed to protect the transfected cells from apoptosis. Resistance to apoptosis by BAG-1 isoforms was correlated with decreased caspase-3 activation. We also detected the expression of Bax, Bak, p53, Bcl-2, Bcl-X(L), AIF and MRP1 by Western blots. Bcl-2 protein expression was significantly increased in p50, p46 and p33 transfected cells, while the expression of Bax, Bak, p53, Bcl-X(L) and MRP1 was essentially unchanged. These in vitro results suggest that distinct isoforms of BAG-1 have different anti-apoptotic functions and their functions may be correlated to increased Bcl-2 expression.     

Activation of ERK1/2 protects melanoma cells from TRAIL-induced apoptosis by inhibiting Smac/DIABLO release from mitochondria

We have previously shown that Smac/DIABLO release from mitochondria appears to be the principal pathway by which TRAIL induces apoptosis of human melanoma. We report that TRAIL-induced release of Smac/DIABLO appears to be downregulated by concomitant signaling through the MEK Erk1/2 kinase pathway and that this inhibits TRAIL-induced apoptosis. Inhibition of Erk1/2 signaling by either the MEK inhibitor U0126 or a dominant-negative mutant of MKK1 markedly sensitized melanoma cells to TRAIL-induced apoptosis. The site in the apoptotic pathway acted on by U0126 appeared to be downstream of caspase-8 and Bid but upstream of caspase-3 in that the levels of proteolytic cleavage of caspase-8 and Bid by TRAIL were similar in cells with or without exposure to U0126. Caspase-3 activation and cleavage of its substrates, PARP, ICAD and XIAP, were however increased by cotreatment with U0126. This was associated with a rapid reduction in mitochondrial transmembrane potential (MMP) and increased release of Smac/DIABLO into the cytosol. Exploration of events leading to the changes in MMP revealed an increased translocation of Bax from the cytosol to mitochondria in the presence of U0126. There was also a delayed decrease in the levels of expression of Mcl-1. Bcl-2 and Bcl-X(L). Over expression of Bcl-2 blocked TRAIL-induced apoptosis in the presence of U0126. Cytochrome c appeared not to play a major role in sensitization of melanoma to TRAIL in that caspase-9 activation was not detected in most of the cell lines. These results suggest that Erk1/2 signaling may protect melanoma cells against TRAIL-induced apoptosis by inhibiting the relocation of Bax from the cytosol to mitochondria and that this may reduce TRAIL-mediated release of Smac/DIABLO and induction of apoptosis.     

Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.     

Functional screening of genes suppressing TRAIL-induced apoptosis: distinct inhibitory activities of Bcl-XL and Bcl-2

TNF-related apoptosis-inducing ligand (TRAIL) is known to selectively induce apoptosis in various tumour cells. However, downstream-signalling of TRAIL-receptor is not well defined. A functional genetic screening was performed to isolate genes interfering with TRAIL-induced apoptosis using cDNA retroviral library. Bcl-X(L) and FLIP were identified after DNA sequencing analysis of cDNA rescued from TRAIL-resistant clones. We found that increased expression of Bcl-X(L), but not Bcl-2, suppressed TRAIL-induced apoptosis in tumour cells. Western blot and immunohistochemical analyses showed that expression of Bcl-X(L), but not Bcl-2, was highly increased in human breast cancer tissues. Exposure of MDA-MB-231 breast tumour cells to TRAIL induced apoptosis accompanied by dissipation of mitochondrial membrane potential and enzymatic activation of caspase-3, -8, and -9. However, SK-BR-3 breast tumour cells exhibiting increased expression level of Bcl-X(L) were resistant to TRAIL, though upon exposure to TRAIL, caspase-8 and Bid were activated. Forced expression of Bcl-X(L), but not Bcl-2, desensitised TRAIL-sensitive MDA-MB-231 cells to TRAIL. Similar inhibitory effects were also observed in other tumour cells such as HeLa and Jurkat cells stably expressing Bcl-X(L), but not Bcl-2. These results are indicative of the crucial and distinct function of Bcl-X(L) and Bcl-2 in the modulation of TRAIL-induced apoptosis.     

Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2.

Proapoptotic members of the Bcl-2 family, including Bax, Bak, and Bid, directly trigger the mitochondrial release of apoptogenic cytochrome c and apoptosis-inducing factor into the cytoplasm. One of the crucial steps before Bax can exert its proapoptotic activity is translocation from the cytoplasm to the mitochondria, but the molecular mechanism of this translocation is not understood. To investigate the mechanism of apoptosis-associated Bax translocation, we used an in vitro system comprising isolated mitochondria and cytosol. We found that both endogenous and exogenous added recombinant Bax translocated to the mitochondria more efficiently in the presence of cytosol from cells with VP16-induced apoptosis than with cytoplasm from normal cells. This apoptosis-dependent promotion of Bax translocation was not seen with cytosol that was prepared from VP16-treated cells expressing Bcl-2. Cytosol from cells with VP16-induced apoptosis, but not that from normal cells or Bcl-2-expressing cells, induced cytochrome c release from isolated mitochondria, which, as assessed by immunodepletion experiments, was mainly mediated by Bax. These results suggest that Bcl-2 exerts its antiapoptotic activity partly by inhibiting the translocation of Bax through the modification of cytosolic factors that are involved in such translocation during apoptosis.     

Pinocembrin triggers Bax-dependent mitochondrial apoptosis in colon cancer cells

Bioflavanoids are the major pigments in plants with multitude of biological activities including inhibition of proliferation or induction of apoptosis in tumor cells. Even though the safety records of most flavanoids are exceptional, its therapeutic use is still in its infancy. We have isolated pinocembrin (5,7-dihydroxyflavanone) from Alpinia galanga that showed cytotoxicity against a variety of cancer cells including normal lung fibroblasts with relative nontoxicity to human umbilical cord endothelial cells. The compound induced loss of mitochondrial membrane potential with subsequent release of cytochrome c and processing of caspase-9 and -3 in colon cancer cell line HCT 116. Processing of caspase-8 was minimal. The initial trigger for mitochondrial apoptosis appears to be by the translocation of cytosolic Bax protein to mitochondria. Overexpression of proapoptotic Bax protein sensitized the colon cancer cells to pinocembrin-induced apoptosis and Bax knockout cells were resistant to pinocembrin-induced apoptosis. Antiapoptotic protein Bcl-X(L) only partially prevented apoptosis induced by this compound. The Bax-dependent cell death involving classical cytochrome c release and processing of caspase-9 and -3 suggests that pinocembrin is a classical mitochondrial apoptosis inducer. But the failure of Bcl-X(L) overexpression to completely prevent apoptosis induced by this compound suggests that pinocembrin is capable of triggering mitochondrial-independent cell death that needs to be clarified. The existence of cell death upon Bcl-X(L) overexpression is a promising feature of this compound that can be exploited against drug resistant forms of cancer cells either alone or in combination with other drugs.