Phenethyl isothiocyanate-induced apoptosis in PC-3 human prostate cancer cells is mediated by reactive oxygen species-dependent disruption of the mitochondrial membrane potential

The present study was undertaken to gain insights into the molecular mechanism of apoptosis induction by phenethyl isothiocyanate (PEITC), which is a cancer chemopreventive constituent of cruciferous vegetables, using PC-3 human prostate cancer cells as a model. The PEITC-induced cell death in PC-3 cells was associated with disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria to the cytosol and generation of reactive oxygen species (ROS), which were blocked in the presence of a combined mimetic of superoxide dismutase and catalase (Euk134). Ectopic expression of Bcl-xL, whose protein level is reduced markedly on treatment of PC-3 cells with PEITC, conferred partial protection against PEITC-induced apoptosis only at higher drug concentrations (>10 microM). Administration of 12 micromol PEITC/day (Monday through Friday) by oral gavage significantly retarded growth of PC-3 xenografts in athymic mice. For instance, 31 days after the initiation of PEITC administration, the average tumor volume in control mice (721 +/- 153 mm3) was approximately 2-fold higher compared with mice receiving 12 micromol PEITC/day. The PEITC-mediated inhibition of PC-3 xenograft growth was associated with induction of Bax and Bid proteins. In conclusion, the present study indicates that the PEITC-induced apoptosis in PC-3 cells is mediated by ROS-dependent disruption of the mitochondrial membrane potential and regulated by Bax and Bid.     

Bim contributes to phenethyl isothiocyanate-induced apoptosis in breast cancer cells

Phenethyl isothiocyanate (PEITC) is a highly promising cancer chemopreventive constituent of cruciferous vegetables (e.g., watercress) with in vivo efficacy in experimental rodent cancer models. Research thus far implicates apoptosis induction in cancer chemopreventive response to PEITC, but the mechanism of proapoptotic effect is not fully understood. The present study demonstrates that p53 upregulated modulator of apoptosis (PUMA)-independent apoptosis by PEITC is mediated by B-cell lymphoma 2 interacting mediator of cell death (Bim). Exposure of a cell line (BRI-JM04) derived from spontaneously developing mammary tumor of a MMTV-neu transgenic mouse to pharmacological concentrations of PEITC resulted in decreased cell viability coupled with apoptosis induction, characterized by release of histone-associated DNA fragments into the cytosol and cleavage of poly-(ADP-ribose)-polymerase and procaspase-3. The PEITC-induced apoptosis in BRI-JM04 cells was associated with up-regulation of Bak, PUMA, and Bim (long and short forms of Bim), increased S65 phosphorylation of BimEL (extra-long form), and down-regulation of Bcl-xL and Bcl-2. On the other hand, a non-tumorigenic human mammary epithelial cell line (MCF-10A) was significantly more resistant to PEITC-induced apoptosis compared with BRI-JM04 despite induction of Bax and PUMA due to concomitant overexpression of anti-apoptotic proteins, including Bcl-xL, Bcl-2, and Mcl-1. Wild-type HCT-116 cells and its isogenic PUMA knockout variant exhibited comparable sensitivity to PEITC-induced apoptosis. On the other hand, small interfering RNA knockdown of Bim protein imparted partial but statistically significant protection against PEITC-induced apoptosis in BRI-JM04, MCF-7, and MDA-MB-231 cells. In conclusion, the present study provides novel insight into the mechanism of PEITC-induced apoptosis involving Bim.     

Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemopreventive agent

Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.     

Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells

The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in apoptosis induction by phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived cancer chemopreventive agent, with DU145 and LNCaP human prostate cancer cells as a model. The MAPK family of serine/threonine kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-jun N-terminal kinase1/2/3 (JNK1/2/3), and p38 MAPK play an important role in cell proliferation and apoptosis in response to different stimuli. Exposure of DU145 and LNCaP cells to growth suppressive concentrations of PEITC resulted in activation of ERK1/2 and JNKs, but not p38 MAPK, in both cell lines. In DU145 cells, the apoptosis induction by PEITC was statistically significantly attenuated by pharmacological inhibition of JNKs with SP600125. Adenovirus-mediated overexpression of Flag-tagged JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), an inhibitor of JNK, also inhibited PEITC-induced apoptosis in DU145 cells. On the other hand, inhibition of ERK1/2 activation with MEK1 inhibitor PD98059 failed to offer protection against PEITC-induced apoptosis in DU145 cells. In LNCaP cells, the PEITC-induced cell death was not affected by either pretreatment with PD98059 or SP600125 or overexpression of JBD of JIP-1. These results indicate that involvement of MAPKs in apoptosis induction by PEITC in human prostate cancer cells is cell line-specific.     

Bax and Bak genes are essential for maximum apoptotic response by curcumin, a polyphenolic compound and cancer chemopreventive agent derived from turmeric, Curcuma longa

Curcumin, an active ingredient of turmeric (Curcuma longa), inhibits proliferation and induces apoptosis in cancer cells, but the sequence of events leading to cell death is poorly defined. The objective of this study was to examine the molecular mechanisms by which multidomain pro-apoptotic Bcl-2 family members Bax and Bak regulate curcumin-induced apoptosis using mouse embryonic fibroblasts (MEFs) deficient in Bax, Bak or both genes. Curcumin treatment resulted an increase in the protein levels of both Bax and Bak, and mitochondrial translocation and activation of Bax in MEFs to trigger drop in mitochondrial membrane potential, cytosolic release of apoptogenic molecules [cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis protein-binding protein with low isoelectric point], activation of caspase-9 and caspase-3 and ultimately apoptosis. Furthermore, MEFs derived from Bax and Bak double-knockout (DKO) mice exhibited even greater protection against curcumin-induced release of cytochrome c and Smac, activation of caspase-3 and caspase-9 and induction of apoptosis compared with wild-type MEFs or single-knockout Bax(-/-) or Bak(-/-) MEFs. Interestingly, curcumin treatment also caused an increase in the protein level of apoptosis protease-activating factor-1 in wild-type MEFs. Smac N7 peptide enhanced curcumin-induced apoptosis, whereas Smac siRNA inhibited the effects of curcumin on apoptosis. Mature form of Smac sensitized Bax and Bak DKO MEFs to undergo apoptosis by acting downstream of mitochondria. The present study demonstrates the role of Bax and Bak as a critical regulator of curcumin-induced apoptosis and over-expression of Smac as interventional approaches to deal with Bax- and/or Bak-deficient chemoresistant cancers for curcumin-based therapy.     

Phenethyl isothiocyanate, a cancer chemopreventive constituent of cruciferous vegetables, inhibits cap-dependent translation by regulating the level and phosphorylation of 4E-BP1

Phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables, exerts significant protection against chemically induced cancer in animal models and inhibits growth of cancer cells in culture and in vivo by causing cell cycle arrest and apoptosis induction. In this study, we report a novel response to PEITC involving the regulation of translation initiation at pharmacologically achievable concentrations. Treatment of human colorectal cancer HCT-116 cells and human prostate cancer PC-3 cells, but not a normal prostate epithelial cell line (PrEC), with PEITC caused an increase in expression of the eukaryotic translation initiation factor 4E (eIF4E) binding protein (4E-BP1) and inhibition of 4E-BP1 phosphorylation. Results from pull-down assay using 7-methyl-GTP Sepharose 4B beads indicated that PEITC treatment reduced cap-bound eIF4E, confirming that increased 4E-BP1 expression and inhibition of 4E-BP1 phosphorylation indeed reduced the availability of eIF4E for translation initiation. Accordingly, results from in vivo translation using luciferase reporter assay indicated that PEITC treatment inhibited cap-dependent translation, in particular the translation of mRNA with secondary structure (stem-loop structure). Ectopic expression of eIF4E prevented PEITC-induced translation inhibition and conferred significant protection against PEITC-induced apoptosis. These results indicate that PEITC modulates availability of eIF4E for translation initiation leading to inhibition of cap-dependent translation. The present study also suggests that inhibition of cap-dependent translation may be an important mechanism in PEITC-induced apoptosis.     

Honokiol, a constituent of oriental medicinal herb magnolia officinalis, inhibits growth of PC-3 xenografts in vivo in association with apoptosis induction.

PURPOSE: This study was undertaken to determine the efficacy of honokiol, a constituent of oriental medicinal herb Magnolia officinalis, against human prostate cancer cells in culture and in vivo. EXPERIMENTAL DESIGN: Honokiol-mediated apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation. Knockdown of Bax and Bak proteins was achieved by transient transfection using siRNA. Honokiol was administered by oral gavage to male nude mice s.c. implanted with PC-3 cells. Tumor sections from control and honokiol-treated mice were examined for apoptotic bodies (terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay), proliferation index (proliferating cell nuclear antigen staining), and neovascularization (CD31 staining). Levels of Bcl-2 family proteins in cell lysates and tumor supernatants were determined by immunoblotting. RESULTS: Exposure of human prostate cancer cells (PC-3, LNCaP, and C4-2) to honokiol resulted in apoptotic DNA fragmentation in a concentration- and time-dependent manner irrespective of their androgen responsiveness or p53 status. Honokiol-induced apoptosis correlated with induction of Bax, Bak, and Bad and a decrease in Bcl-xL and Mcl-1 protein levels. Transient transfection of PC-3 cells with Bak- and Bax-targeted siRNAs and Bcl-xL plasmid conferred partial yet significant protection against honokiol-induced apoptosis. Oral gavage of 2 mg honokiol/mouse (thrice a week) significantly retarded growth of PC-3 xenografts without causing weight loss. Tumors from honokiol-treated mice exhibited markedly higher count of apoptotic bodies and reduced proliferation index and neovascularization compared with control tumors. CONCLUSION: Our data suggest that honokiol, which is used in traditional oriental medicine for the treatment of various ailments, may be an attractive agent for treatment and/or prevention of human prostate cancers.     

Mcl-1 down-regulation potentiates ABT-737 lethality by cooperatively inducing Bak activation and Bax translocation

The Bcl-2 antagonist ABT-737 targets Bcl-2/Bcl-xL but not Mcl-1, which may confer resistance to this novel agent. Here, we show that Mcl-1 down-regulation by the cyclin-dependent kinase (CDK) inhibitor roscovitine or Mcl-1-shRNA dramatically increases ABT-737 lethality in human leukemia cells. ABT-737 induces Bax conformational change but fails to activate Bak or trigger Bax translocation. Coadministration of roscovitine and ABT-737 untethers Bak from Mcl-1 and Bcl-xL, respectively, triggering Bak activation and Bax translocation. Studies employing Bax and/or Bak knockout mouse embryonic fibroblasts (MEFs) confirm that Bax is required for ABT-737+/-roscovitine lethality, whereas Bak is primarily involved in potentiation of ABT-737-induced apoptosis by Mcl-1 down-regulation. Ectopic Mcl-1 expression attenuates Bak activation and apoptosis by ABT-737+roscovitine, whereas cells overexpressing Bcl-2 or Bcl-xL remain fully sensitive. Finally, Mcl-1 knockout MEFs are extremely sensitive to Bak conformational change and apoptosis induced by ABT-737, effects that are not potentiated by roscovitine. Collectively, these findings suggest down-regulation of Mcl-1 by either CDK inhibitors or genetic approaches dramatically potentiate ABT-737 lethality through cooperative interactions at two distinct levels: unleashing of Bak from both Bcl-xL and Mcl-1 and simultaneous induction of Bak activation and Bax translocation. These findings provide a mechanistic basis for simultaneously targeting Mcl-1 and Bcl-2/Bcl-xL in leukemia.     

Zerumbone causes Bax- and Bak-mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo

The present study was undertaken to determine the anticancer efficacy of zerumbone (ZER), a sesquiterpene from subtropical ginger, against human breast cancer cells in vitro and in vivo. ZER treatment caused a dose-dependent decrease in viability of MCF-7 and MDA-MB-231 human breast cancer cells in association with G₂/M phase cell cycle arrest and apoptosis induction. ZER-mediated cell cycle arrest was associated with downregulation of cyclin B1, cyclin-dependent kinase 1, Cdc25C, and Cdc25B. Even though ZER treatment caused stabilization of p53 and induction of PUMA, these proteins were dispensable for ZER-induced cell cycle arrest and/or apoptosis. Exposure of MDA-MB-231 and MCF-7 cells to ZER resulted in downregulation of Bcl-2 but its ectopic expression failed to confer protection against ZER-induced apoptosis. On the other hand, the SV40 immortalized mouse embryonic fibroblasts derived from Bax and Bak double knockout mice were significantly more resistant to ZER-induced apoptosis. ZER-treated MDA-MB-231 and MCF-7 cells exhibited a robust activation of both Bax and Bak. In vivo growth of orthotopic MDA-MB-231 xenografts was significantly retarded by ZER administration in association with apoptosis induction and suppression of cell proliferation (Ki-67 expression). These results indicate that ZER causes G₂/M phase cell cycle arrest and Bax/Bak-mediated apoptosis in human breast cancer cells, and retards growth of MDA-MB-231 xenografts in vivo.     

The roles of JNK and apoptotic signaling pathways in PEITC-mediated responses in human HT-29 colon adenocarcinoma cells

Phenethyl isothiocyanate (PEITC) is a potential chemopreventive agent that is present naturally in widely consumed vegetables, especially in watercress. It has been extensively investigated for its anticancer activities against lung, forestomach and esophageal tumorigenesis. Here we investigated the pro-apoptotic effect of PEITC in HT-29 human colorectal carcinoma cell line, and the mechanism of apoptosis induced by PEITC. PEITC-induced apoptosis was determined by DNA fragmentation assay and diamidino-2-phenylindole (DAPI) staining technique. To understand the mechanisms of apoptosis induced by PEITC, we studied the role of caspases, mitochondria-cytochrome c release, and mitogen-activated protein kinase (MAPK) signaling pathways involved in PEITC-induced apoptosis in HT-29 cells. Both the caspase-3 and -9 activities were stimulated by PEITC. The release of cytochrome c from the mitochondrial inter-space was time- and dose-dependent, with a maximal release at 50 micro M after 10 h treatment. Three MAPKs [JNK (c-Jun N-terminal kinase), extracellular signal-regulated protein kinase (ERK) and p38 kinase] were activated shortly after PEITC treatment in HT-29 cells. Importantly, the SP600125 compound, an anthrapyrazolone inhibitor of JNK, but not the ERK and p38 inhibitor, suppressed apoptosis induced by PEITC. Similarly, this JNK inhibitor attenuated both cytochrome c release and caspase-3 activation induced by PEITC. In summary, this study shows that PEITC can induce apoptosis in HT-29 cells in a time- and dose-dependent manner via the mitochondria caspase cascade, and the activation of JNK is critical for the initiation of the apoptotic processes. This mechanism of PEITC may play an important role in the killing of cancerous cells and offer a potential mechanism for its anticancer action in vivo.     

The proteasome inhibitor PS-341 overcomes TRAIL resistance in Bax and caspase 9-negative or Bcl-xL overexpressing cells

We demonstrate that PS-341, a small molecule inhibitor of the proteasome, markedly sensitizes resistant prostate, colon, and bladder cancer cells to TNF-like apoptosis-inducing ligand (TRAIL)-induced apoptosis irrespective of Bcl-xL overexpression. PS-341 treatment by itself does not affect the levels of Bax, Bak, caspases 3 and 8, c-Flip or FADD, but elevates levels of TRAIL receptors DR4 and DR5. This increase in receptor protein levels is associated with the ubiquitination of the DR5 protein. When PS-341 is combined with TRAIL, the levels of activated caspase 8 and cleaved Bid are substantially increased. In Bax-negative TRAIL-resistant HC-4 colon cancer cells, the combination of PS-341 and TRAIL overcomes the block to activation of the mitochondrial pathway and causes SMAC and cytochrome c release followed by apoptosis. Similarly, murine embryonic fibroblasts lacking Bax undergo apoptosis when exposed to the combination of PS-341 and TRAIL; however, fibroblasts lacking Bak are significantly resistant. Taken together, these findings indicate that PS-341 enhances TRAIL-induced apoptosis by increasing the cleavage of caspase 8, causing Bak-dependent release of mitochondrial proapoptotic proteins.     

Combined inhibitory effects of curcumin and phenethyl isothiocyanate on the growth of human PC-3 prostate xenografts in immunodeficient mice

Earlier studies using prostate cancer cells in culture showed that phenethyl isothiocyanate (PEITC) and curcumin have significant chemopreventive and possibly chemotherapeutic effects. However, their in vivo effects are still lacking. Hence, this study was undertaken to determine the possible in vivo efficacy of prostate cancer-prevention as well as cancer-therapeutic treatment by PEITC and curcumin alone or in combination. We evaluated the effects on tumor growth in vivo, using NCr immunodeficient (nu/nu) mice bearing s.c. xenografts of PC-3 human prostate cancer cells. Molecular biomarkers representing proliferation and apoptosis were determined. Continued i.p. injection of curcumin or PEITC (6 and 5 mumol; thrice a week for 28 days), beginning a day before tumor implantation significantly retarded the growth of PC-3 xenografts. Combination of i.p. administration of PEITC (2.5 mumol) and curcumin (3 mumol) showed stronger growth-inhibitory effects. Next, we evaluated the cancer-therapeutic potential of curcumin and PEITC in mice with well-established tumors, and the results showed that PEITC or curcumin alone had little effect, whereas combination of curcumin and PEITC significantly reduced the growth of PC-3 xenografts. Immunohistochemistry staining and Western blot analysis revealed that the inhibition of Akt and nuclear factor-kappaB signaling pathways could contribute to the inhibition of cell proliferation and induction of apoptosis. Taken together, our results show that PEITC and curcumin alone or in combination possess significant cancer-preventive activities in the PC-3 prostate tumor xenografts. Furthermore, we found that combination of PEITC and curcumin could be effective in the cancer-therapeutic treatment of prostate cancers.     

NSC606985, a novel camptothecin analog, induces apoptosis and growth arrest in prostate tumor cells

Purpose  Prostate cancer is a major cause of cancer mortality in American males. Once prostate cancer has metastasized, there is currently no curative therapy available. The development of effective agents is therefore a continuing effort to combat this disease. In the present study, the effects and potential mechanisms of NSC606985 (NSC), a water-soluble camptothecin analog, in prostate cancer cells were investigated. Methods  Prostatic tumor cells, DU-145, LNCaP and PC-3, were used for the study. Cell proliferation, cell cycle, cell apoptosis and caspase 3/7 activity were determined in the presence or absence of NSC. The levels of Bax and Bak, and the release of cytochrome c from mitochondria were analyzed by Western blot. Results  Treatment with NSC at nanomolar concentrations produced a time- and dose-dependent decrease in viable cell numbers of multiple prostate cancer cells. In DU-145 cells, NSC produced a time-and dose-dependent induction of cell apoptosis and cell cycle arrest as evidenced by cell morphological changes, increases in S-phase and sub-G1 cell fractions, an elevation of caspase 3/7 activity, DNA fragmentation and apoptotic cells. NSC increased the levels of apoptotic proteins, Bax and Bak, and induced a release of cytochrome c from mitochondria to cytosol in DU-145 cells. Co-administration of Z-VAD-FMK, a pan-caspase inhibitor, blocked NSC-induced caspase 3/7 activity and cell apoptosis without affecting NSC-induced cell cycle arrest. In contrast, co-administration of a PKCδ inhibitor, rottlerin, had no significant effect on NSC induction of caspase activity, and slightly potentiated NSC-induced cell death. Furthermore, like camptothecin, a mutation of topoisomerase 1 that prevents the binding of camptothecin to the enzyme completely abolished the NSC effect in DU-145 cells. Conclusion  The data obtained suggest that NSC is able to decrease cell growth, induce cell apoptosis and cause growth arrest in prostatic tumor cells, which may involve an interaction with topoisomerase 1 and an activation of mitochondrial apoptotic pathway.     

c-jun/AP-1 activation does not affect the antiproliferative activity of phenethyl isothiocyanate, a cruciferous vegetable-derived cancer chemopreventive agent

Cruciferous vegetable-derived isothiocyanates (ITCs) display potent cancer chemopreventive activity, but also markedly stimulate oncogenic activator protein 1 (AP-1). AP-1 is well known to promote cell survival and proliferation. We examined the impact of AP-1 activation on antiproliferative activity of ITCs, using bladder cancer cells and phenethyl isothiocyanate (PEITC) as models. AP-1 transactivation induced by PEITC was almost completely suppressed by a dominant-negative c-jun (TAM67). However, suppression of AP-1 transactivation did not affect PEITC-induced apoptosis or cell-cycle arrest. Moreover, we previously showed that in response to ITC treatment c-jun was predominantly stimulated among AP-1 family members largely by c-jun N-terminal kinase (JNK) [Food Chem Toxicol 2005; 43: 1373-1380], but neither JNK inhibition nor forced expression of c-jun altered the antiproliferative activity of PEITC. In addition, cyclin D1, which is considered as an AP-1 target gene and promotes cell proliferation, was markedly elevated in PEITC-treated cells. Unexpectedly, neither TAM67 or JNK inhibition, nor forced c-jun expression had a significant impact on cyclin D1 induction by PEITC, indicating that c-jun/AP-1 does not play an important role in cyclin D1 induction by PEITC. In conclusion, despite the known role of c-jun/AP-1 as a stimulator of cell growth and proliferation, our data show that its activation does not diminish the antiproliferative activity of PEITC and is not responsible for cyclin D1 induction by PEITC.     

Multidomain Bcl-2 homolog Bax but not Bak mediates synergistic induction of apoptosis by TRAIL and 5-FU through the mitochondrial apoptosis pathway

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.     

TRAIL sensitizes for ionizing irradiation-induced apoptosis through an entirely Bax-dependent mitochondrial cell death pathway

The death ligand TRAIL has been suggested as a suitable biological agent for the selective induction of cell death in cancer cells. Moreover, TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation (IR). Here, we show that synergy of TRAIL and IR, that is, crosssensitization between TRAIL and IR for induction of apoptosis, entirely depends on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to activate caspase-3 and -9 when exposed to TRAIL and IR. In contrast, TRAIL sensitized for IR-induced apoptosis and vice versa upon reconstitution of Bax expression. Notably, both DU145 and HCT116 still express significant levels of the multidomain proapoptotic Bcl-2 homolog Bak. This indicates that Bak is not sufficient to mediate crosssensitization and synergism between IR and TRAIL. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade upon DNA damage by IR.     

Versatile prostate cancer treatment with inducible caspase and interleukin-12

To establish optimized conditions for immunity against prostate cancer, we compared the efficacy of multiple approaches in autochthonous and s.c. transgenic adenocarcinoma of the mouse prostate (TRAMP)-based models. Mice immunized with interleukin (IL)-12-containing apoptotic, but not necrotic TRAMP-C2 cell-based, vaccines were resistant to TRAMP-C2 tumor challenge and re-challenge, independently of the route of vaccination (s.c. or i.p.). Administration of gamma-irradiated TRAMP-C2 cells preinfected with adenovirus containing both B7-1 and IL-12 genes, unlike adenovirus containing B7-1 alone, considerably protected C57BL/6 mice from TRAMP-C2 tumor growth and extended the life span of TRAMP mice. Vaccines that included dendritic cells, instead of IL-12, were equally efficient. Whereas injections of ligand-inducible caspase-1- and IL-12-containing adenoviruses cured small s.c. TRAMP-C2 tumors, nanopump-regulated delivery of viruses led to elimination of much larger tumors. The antitumor immune responses involved CD4+-, CD8+-, and natural killer cells and were strengthened by increasing the number of vaccinations. Intraprostatic administration of inducible caspase-1- and IL-12-containing adenoviruses resulted in local cell death and improved survival of adenocarcinoma-bearing TRAMP mice. Thus, tumor cell apoptosis induced by caspase in situ and accompanied by IL-12 is efficient against prostate cancer in a preclinical model.     

Involvement of proapoptotic molecules Bax and Bak in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced mitochondrial disruption and apoptosis: differential regulation of cytochrome c and Smac/DIABLO release

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L induces apoptosis in a wide variety of cancer and transformed cells. Activation of BID, a "BH3-domain-only" Bcl-2 family member, triggers the oligomerization of proapoptotic family members Bak or Bax, resulting in the release of mitochondrial proteins to cytosol. In this study, we have shown the importance of Bax and Bak in TRAIL-induced apoptosis by studying in murine embryonic fibroblasts (MEFs) from Bax(-/-) and Bak(-/-) animals. TRAIL induced cytochrome c release and apoptosis in wild-type, Bid(-/-), Bax(-/-), or Bak(-/-) MEFs, but not in Bax(-/-) Bak(-/-) double knockout (DKO) MEFs. Bid, which functions upstream of cytochrome c release, was cleaved in all of the knockout cells except in Bid(-/-) MEFs. The release of cytochrome c was correlated with caspase-9 activity. TRAIL increased caspase-3 activity in all of the cells except in DKO cells. TRAIL-induced drop in mitochondrial membrane potential was not observed in DKO MEFs. Unlike cytochrome c release, TRAIL-induced Smac/DIABLO release was blocked in Bid(-/-), Bax(-/-), Bak(-/-), or DKO MEFs, suggesting the differential regulation of these mitochondrial proteins during apoptosis. The apoptotic events downstream of mitochondria were intact in DKO MEFs, because microinjection of cytochrome c, or ectopic expression of mature Smac/DIABLO or pretreatment of Smac N7 peptide completely restored TRAIL sensitivity. In conclusion, the data suggest that Bax and Bak differentially regulate the release of cytochrome c and Smac/DIABLO from mitochondria, and Smac/DIABLO can be used to sensitize cells that are deficient in Bax and Bak genes, or resistant to TRAIL.