Tunicamycin sensitizes human prostate cells to TRAIL-induced apoptosis by upregulation of TRAIL receptors and downregulation of cIAP2

The addition of tunicamycin to prostate cancer cells enhances cell death mediated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we investigated whether tunicamycin, an endoplasmic reticulum stress inducer, can potentiate TRAIL-induced apoptosis in human prostate cancer cells. We evaluated the combination of tunicamycin and TRAIL and found synergistic promotion of apoptosis in prostate cancer cells. The combined treatment with tunicamycin and TRAIL significantly induced apoptosis, and stimulated caspase-3, -8 and -9 activity, as well as the cleavage of poly (ADP-ribose) polymerase. We found that tunicamycin promoted TRAIL-induced apoptosis by the upregulation of death receptor (DR)4 and DR5 and the downregulation of cellular inhibitor of apoptosis 2 (cIAP2). In addition, downregulation of cIAP2 expression using small interfering RNA significantly attenuated the apoptosis induced by TRAIL. Taken together, our results demonstrate that the combination of tunicamycin and TRAIL may provide a novel strategy for treating prostate cancer by overcoming critical mechanisms of apoptosis resistance.     

Tunicamycin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human prostate cancer cells

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L). In this study, we showed that tunicamycin, a naturally occurring antibiotic, is a potent enhancer of TRAIL-induced apoptosis through up-regulation of DR5 expression. Tunicamycin significantly sensitized PC-3, androgen-independent human prostate cancer cells, to TRAIL-induced apoptosis. The tunicamycin-mediated enhancement of TRAIL-induced apoptosis was markedly blocked by a recombinant human DR5/Fc chimeric protein. Tunicamycin and TRAIL cooperatively activated caspase-8, -10, -9, and -3 and Bid cleavage and this activation was also blocked in the presence of the DR5/Fc chimera. Tunicamycin up-regulated DR5 expression at the mRNA and protein levels in a dose-dependent manner. Furthermore, the tunicamycin-mediated sensitization to TRAIL was efficiently reduced by DR5 small interfering RNA, suggesting that the sensitization was mediated through induction of DR5 expression. Tunicamycin increased DR5 promoter activity and this enhanced activity was diminished by mutation of a CHOP-binding site. In addition, suppression of CHOP expression by small interfering RNA reduced the tunicamycin-mediated induction of DR5. Of note, tunicamycin-mediated induction of CHOP and DR5 protein expression was not observed in normal human peripheral blood mononuclear cells. Moreover, tunicamycin did not sensitize the cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may be a promising candidate for prostate cancer therapy.     

Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels

We have demonstrated that Apo-2 ligand (Apo-2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of human prostate cancer PC-3, DU145, and LNCaP cells in a dose-dependent manner, with PC-3 cells displaying the greatest sensitivity to Apo-2L/TRAIL. Susceptibility of the prostate cancer cell types to Apo-2L/TRAIL-induced apoptosis did not appear to correlate with the levels of the Apo-2L/TRAIL receptors death receptor (DR) 4 (TRAIL receptor 1) or DR5 (TRAIL receptor 2), decoy receptor (DcR) 1 and DcR2, Flame-1, or the inhibitors of apoptosis proteins family of proteins. Apo-2L/TRAIL-induced apoptosis of PC-3 cells was associated with the processing of caspase-8, caspase-10, and the proapoptotic Bid protein, resulting in the cytosolic accumulation of cytochrome c as well as the processing of procaspase-9 and procaspase-3. Cotreatment with the caspase-8 inhibitor z-IETD-fmk or DR4:Fc significantly inhibited Apo-2L/TRAIL-induced apoptosis. Treatment with paclitaxel or taxotere increased DR4 and/or DR5 protein levels (up to 8-fold) without affecting the protein levels of DcR1 and DcR2, Apo-2L/TRAIL, Fas, or Fas ligand. Up-regulation of DR4 and DR5 was not preceded by the induction of their mRNA levels but was inhibited by cotreatment with cycloheximide. Importantly, sequential treatment of PC-3, DU145, and LNCaP cells with paclitaxel followed by Apo-2L/TRAIL induced significantly more apoptosis than Apo-2L/TRAIL treatment alone (P < 0.01). This was also associated with greater processing of procaspase-8 and Bid, as well as greater cytosolic accumulation of cytochrome c and the processing of caspase-3. These findings indicate that up-regulation of DR4 and DR5 protein levels by treatment with paclitaxel enhances subsequent Apo-2L/TRAIL-induced apoptosis of human prostate cancer cells.     

Low-dose 12-O-tetradecanoylphorbol-13-acetate enhances tumor necrosis factor related apoptosis-inducing ligand induced apoptosis in prostate cancer cells.

PURPOSE: Previously, we have shown that c-Fos/activator protein-1 (AP-1) promotes tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by repressing the antiapoptotic molecule c-FLIP(L). In this study, we investigated whether synthetic induction of c-Fos/AP-1 by 12-O-tetradecanoylphorbol-13-acetate (TPA) converts the phenotype of TRAIL-resistant prostate cancer cells to a TRAIL-sensitive phenotype in vitro and in vivo. EXPERIMENTAL DESIGN: Low-dose TPA was used to determine whether LNCaP prostate cancer cells could be converted to a TRAIL-sensitive phenotype in in vitro and in vivo studies. We also assessed whether TPA enhancement of TRAIL-induced apoptosis varies between androgen-sensitive and androgen-insensitive prostate cancer cells and evaluated the role of TRAIL receptors, DR4 and DR5, in TPA-enhanced TRAIL-induced apoptosis. RESULTS: We show that the combination of TRAIL with low-dose TPA has no effect on nonmalignant prostate epithelial cells; however, TPA up-regulates most AP-1 proteins and AP-1 activity, reduces c-FLIP(L), and potentiates TRAIL-induced apoptosis. We show that the combination of TPA + TRAIL is effective in promoting apoptosis in both hormone-sensitive LNCaP and hormone-insensitive LNCaP-C4-2 prostate cancer cells. Although TPA enhances the TRAIL-receptor 1 (DR4) level, sensitization of prostate cancer cells seems to be more dependent on TRAIL-receptor 2 (DR5) than TRAIL-receptor 1 levels. In vivo xenograft experiments suggest that TPA elevates the expression of c-Fos and reduces c-FLIP(L). Combination of TPA with TRAIL-receptor 2 agonist antibody, lexatumumab, effectively increases apoptosis and reduces LNCaP xenograft tumor burden. CONCLUSIONS: TPA, when combined with the proapoptotic agent TRAIL, is effective in changing the phenotype of some TRAIL-resistant prostate cancer cells to a TRAIL-sensitive phenotype.     

Cisplatin and a potent platinum(IV) complex-mediated enhancement of TRAIL-induced cancer cells killing is associated with modulation of upstream events in the extrinsic apoptotic pathway

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediated apoptosis. Combination therapy with platinum complexes may affect TRAIL-induced signaling via modulation of various steps in apoptotic pathways. Here, we show that cisplatin or a more potent platinum(IV) complex LA-12 used in 20-fold lower concentration enhanced killing effects of TRAIL in human colon and prostate cancer cell lines via stimulation of caspase activity and overall apoptosis. Both platinum complexes increased DR5 surface expression in colon cancer cells. Small interfering RNA-mediated DR5 silencing rescued cells from sensitizing effects of platinum drugs on TRAIL-induced caspase-8 activation and apoptosis, showing the functional importance of DR5 in the effects observed. In addition, both cisplatin and LA-12 triggered the relocalization of DR4 and DR5 receptors to lipid rafts and accelerated internalization of TRAIL, which may also affect TRAIL signaling. Collectively, modulations of the initial steps of the extrinsic apoptotic pathway at the level of DR5 and plasma membrane are important for sensitization of colon and prostate cancer cells to TRAIL-induced apoptosis mediated by LA-12 and cisplatin.     

Androgens regulate TRAIL-induced cell death in prostate cancer cells via multiple mechanisms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent for prostate cancer because it selectively induces apoptosis in cancer cells but not in normal cells. Previous reports have suggested that androgens regulate TRAIL-induced apoptosis in prostate cancer cells. However, there are discrepancies between these reports of how androgens affect TRAIL-induced cell death. To clarify the role of androgens on TRAIL-induced apoptosis in prostate cancer cells, we investigated the effects of androgen on TRAIL-induced cell death in a dose–response manner. Our results showed that although androgens sensitize LNCaP cells to TRAIL-induced apoptosis, this effect is dose-dependent and biphasic. We found that low levels of androgen are superior to high levels of androgen in term of sensitizing LNCaP cells to TRAIL. We also found that upregulation of DR5 (TRAIL-R2) expression by androgens is critical for sensitizing LNCaP cells to TRAIL. However, low levels of androgen are sufficient to induce DR5 expression and sensitize LNCaP cells to TRAIL-induced cell death. High levels of androgen alter the TRADD/RIP1 ratio, which may contribute to NF-κB activation and sequentially inhibit TRAIL-induced apoptosis.     

Caspase 8-dependent sensitization of cancer cells to TRAIL-induced apoptosis following reovirus-infection.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in susceptible cells by binding to death receptors 4 (DR4) and 5 (DR5). TRAIL preferentially induces apoptosis in transformed cells and the identification of mechanisms by which TRAIL-induced apoptosis can be enhanced may lead to novel cancer chemotherapeutic strategies. Here we show that reovirus infection induces apoptosis in cancer cell lines derived from human breast, lung and cervical cancers. Reovirus-induced apoptosis is mediated by TRAIL and is associated with the release of TRAIL from infected cells. Reovirus infection synergistically and specifically sensitizes cancer cell lines to killing by exogenous TRAIL. This sensitization both enhances the susceptibility of previously resistant cell lines to TRAIL-induced apoptosis and reduces the amount of TRAIL needed to kill already sensitive lines. Sensitization is not associated with a detectable change in the expression of TRAIL receptors in reovirus-infected cells. Sensitization is associated with an increase in the activity of the death receptor-associated initiator caspase, caspase 8, and is inhibited by the peptide IETD-fmk, suggesting that reovirus sensitizes cancer cells to TRAIL-induced apoptosis in a caspase 8-dependent manner. Reovirus-induced sensitization of cells to TRAIL is also associated with increased cleavage of PARP, a substrate of the effector caspases 3 and 7.     

eNOS protects prostate cancer cells from TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anti-cancer agent because it induces apoptosis of most tumor cells with little or no effect on normal cells. In this study, we investigated the effect of TRAIL on human prostate normal and cancer cell lines, and found that the prostate cancer cell lines PC-3, ALVA-31, DU 145 and TSU-Pr1 were sensitive to TRAIL-induced apoptosis, while normal PrEC cells and cancer cell line LNCaP were resistant. No correlation was found between the sensitivity of cells to TRAIL and the expression of TRAIL receptors DR4 and DR5, and pro-apoptotic proteins Bax and Bak. However, LNCaP cells displayed a high Akt activity. Furthermore, we found that endothelial nitric oxide synthase (eNOS), one of the Akt substrates, was highly expressed in LNCaP but not in other cells. Inhibition of eNOS activity by NOS inhibitor sensitized LNCaP cells to TRAIL. Moreover, PC-3 cell clones stably expressing eNOS were resistant to TRAIL-induced apoptosis. Taken together, these results indicate that eNOS can regulate the sensitivity of prostate cancer cells to TRAIL, and down-regulation of eNOS activity may sensitize prostate cancer cells to TRAIL-based therapy.     

Sulforaphane enhances TRAIL-induced apoptosis through the induction of DR5 expression in human osteosarcoma cells

Sulforaphane (SFN), a naturally occurring isothiocyanate, is an attractive agent because of its potent anticancer effects. SFN suppresses the proliferation of various cancer cells in vitro and in vivo. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also one of the most promising candidates for cancer therapeutics owing to its ability to selectively induce apoptosis in tumor cells. In this study, we report that SFN enhances TRAIL-induced apoptosis in human osteosarcoma cells, Saos2 and MG63. The apoptosis induced by co-treatment with SFN and TRAIL was markedly blocked by a dominant negative form of the TRAIL receptor or caspase inhibitors. The combined use of SFN and TRAIL effectively induced Bid cleavage and the activation of caspases 8, 10, 9 and 3 at ineffective concentrations for each agent. SFN upregulated the expression of death receptor 5 (DR5), a receptor for TRAIL, at mRNA and protein levels in a dose-dependent manner. In addition, the SFN-mediated sensitization to TRAIL was reduced by DR5 siRNA, suggesting that the sensitization was at least partially mediated through the induction of DR5 expression. Furthermore, SFN sensitized TRAIL-induced apoptosis in a p53-independent manner. On the other hand, SFN neither induced DR5 protein expression or enhanced TRAIL-induced apoptosis in normal human peripheral blood mononuclear cells. Thus, combined treatment with SFN and TRAIL might be a promising therapy for osteosarcoma.     

Lipoxygenase inhibitors induce death receptor 5/TRAIL-R2 expression and sensitize malignant tumor cells to TRAIL-induced apoptosis

Lipoxygenases induce malignant tumor progression and lipoxygenase inhibitors have been considered as promising anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. Combined treatment with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, and TRAIL markedly induced apoptosis in Jurkat T-cell leukemia cells at suboptimal concentrations for each agent. The combined treatment efficiently activated caspase-3, -8 and -10, and Bid. The underling mechanism by which NDGA enhanced TRAIL-induced apoptosis was examined. NDGA did not change the expression levels of anti-apoptotic factors, Bcl-x(L), Bcl-2, cIAP-1, XIAP and survivin. The expression of death receptor-related genes was investigated and it was found that NDGA specifically up-regulated the expression of death receptor 5 (DR5) at mRNA and protein levels. Down-regulation of DR5 by small interfering RNA prevented the sensitizing effect of NDGA on TRAIL-induced apoptosis. Furthermore, NDGA sensitized prostate cancer and colorectal cancer cells to TRAIL-induced apoptosis. In contrast, NDGA neither enhanced TRAIL-induced apoptosis nor up-regulated DR5 expression in normal peripheral blood mononuclear cells. Another lipoxygenase inhibitor, AA861, also up-regulated DR5 and sensitized Jurkat and DU145 cells to TRAIL. These results indicate that lipoxygenase inhibitors augment the apoptotic efficiency of TRAIL through DR5 up-regulation in malignant tumor cells, and raise the possibility that the combination of lipoxygenase inhibitor and TRAIL is a promising strategy for malignant tumor treatment.     

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.     

Histone deacetylase inhibitors upregulate death receptor 5/TRAIL-R2 and sensitize apoptosis induced by TRAIL/APO2-L in human malignant tumor cells

Death receptor 5 (DR5) is a receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a promising candidate for cancer therapeutics due to its ability to induce apoptosis selectively in cancer cells. Here, we report that histone deacetylase inhibitors (HDACIs) such as trichostatin A (TSA), sodium butyrate, and suberoylanilide hydroxamic acid (SAHA) upregulated DR5 expression in various human malignant tumor cells. An RNase protection assay demonstrated that HDACIs induced DR5 mRNA markedly but not that of other death receptor family members in Jurkat cells. HDACIs increased DR5 mRNA and protein in a dose- and time-dependent manner. We also show TSA increased DR5 promoter activity using a luciferase promoter assay. Furthermore, we demonstrated that HDACIs strongly sensitized exogenous soluble recombinant human TRAIL-induced apoptosis synergistically in Jurkat and HL-60 cells that were tolerant to TRAIL alone. The combined use of HDACIs and TRAIL in suboptimal concentrations induced Bid cleavage and activation of caspase-8, -10, -3, and -9. Human recombinant DR5/Fc chimera protein, zVAD-fmk pancaspase inhibitor, and caspase-8 and -10 inhibitors efficiently reduced apoptosis induced by cotreatment with HDACIs and TRAIL. Furthermore, TSA did not significantly induce DR5 protein and HDACIs did not enhance TRAIL-induced apoptosis in normal human peripheral blood mononuclear cells. These results suggest that this combined treatment with HDACIs and TRAIL is a promising strategy for new cancer therapeutics.     

The sequential treatment with ionizing radiation followed by TRAIL/Apo-2L reduces tumor growth and induces apoptosis of breast tumor xenografts in nude mice

TRAIL primarily induces apoptosis in cancer cells but not in normal cells. However, some TRAIL-resistant cancer cell lines have recently been discovered. Ionizing radiation may enhance the apoptosis inducing potential of TRAIL in sensitive cells, and sensitize TRAIL-resistant cancer cells. We assessed the influence of sequential treatment of irradiation followed by TRAIL on intracellular mechanisms of apoptosis of breast tumor cells in vitro and on tumor regression in xenografted athymic nude mice. Irradiation augmented TRAIL-induced apoptosis in breast cancer cells through up-regulation of DR5, and subsequent activation of caspases-3, -8 and -9. Inhibition of p53 by siRNA abrogated irradiation-induced DR5 expression, suggesting the requirement of p53 for DR5 induction. The pretreatment of cells with irradiation followed by TRAIL significantly induced more apoptosis than single agent alone or concurrent treatment with irradiation and TRAIL. The sequential treatment of xenografted mice with irradiation followed by TRAIL-induced apoptosis through caspase-3 activation, completely eradicated the established breast tumors, and enhanced survival of mice without detectable toxicity to normal tissues. The sequential treatment with irradiation followed by TRAIL provides an approach to enhance therapeutic potential of TRAIL. Thus, irradiation can be combined with TRAIL in breast cancer therapy.     

Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways

Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells. The objectives of this study are to investigate the intracellular mechanisms by which TRAIL induces apoptosis. The death receptor Fas, upon ligand binding, trimerizes and recruits the adaptor protein FADD through the cytoplasmic death domain of Fas. FADD then binds and activates procaspase-8. It is unclear whether FADD is required for TRAIL-induced apoptosis. Here we show that the signaling complex of DR4/DR5 is assembled in response to TRAIL binding. FADD and caspase-8, but not caspase-10, are recruited to the receptor, and cells deficient in either FADD or caspase-8 blocked TRAIL-induced apoptosis. In addition, TRAIL initiates the activation of caspases, the loss of mitochondrial transmembrane potential (Deltapsi(m)), the cleavage of BID, and the redistribution of mitochondrial cytochrome c. Treatment of Jurkat cells with cyclosporin A delayed TRAIL-induced Deltapsi(m), caspase-3 activation and apoptosis. Similarly, Overexpression of Bcl-2 or Bcl-X(L) delayed, but did not inhibit, TRAIL-induced Deltapsi(m) and apoptosis. In contrast, XIAP, cowpox virus CrmA and baculovirus p35 inhibited TRAIL-induced apoptosis. These data suggest that death receptors (DR4 and DR5) and Fas receptors induced apoptosis through identical signaling pathway, and TRAIL-induced apoptosis via both mitochondrial-dependent and -independent pathways.     

Doxorubicin pretreatment sensitizes prostate cancer cell lines to TRAIL induced apoptosis which correlates with the loss of c-FLIP expression

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) can induce receptor-mediated apoptosis in prostate cancer cell lines that have been co-treated with the chemotherapeutic agent doxorubicin (Voelkel-Johnson C, et al. Cancer Gene Therapy 2002; 9:164-172). In this study, we report that pretreatment with doxorubicin is sufficient to sensitize cells to TRAIL. To identify possible targets of doxorubicin, we analyzed levels of several Bcl-2 family members, TRAIL receptors and the anti-apoptotic protein c-FLIP. Doxorubicin did not affect steady state levels of Bax, Bcl-2 and Bcl-X(L) in the majority of the prostate cancer cell lines. TRAIL receptor mRNAs (DR4, DR5, and DcR2) were induced by doxorubicin but these changes were not reflected at the protein level. In contrast, in response to doxorubicin, levels of c-FLIP, particularly FLIP(S), decreased in all cell lines tested. The decrease in c-FLIP(S) correlated with onset and magnitude of caspase-8 and PARP cleavage in PC3 cells. In two TRAIL resistant cell lines, DU145 and LNCaP, treatment with TRAIL alone resulted in processing of c-FLIP(L) and initiated abortive caspase-8 proteolysis. TRAIL treatment did not affect levels of c-FLIP(S) in Du145 and LNCaP cells and did not result in PARP cleavage. Therefore, our results suggest that doxorubicin- mediated down regulation of c-FLIP(S) predisposes cells to TRAIL-induced apoptosis.     

Genistein sensitizes TRAIL-resistant human gastric adenocarcinoma AGS cells through activation of caspase-3

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some cancer cells, including AGS gastric adenocarcinoma cells. However, treatment with TRAIL in combination with subtoxic concentrations of genistein sensitizes TRAIL-resistant AGS cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis are correlated with the activation of death receptors (DR5) and induction of caspase-3 activity, which results in the cleavage of poly(ADP-ribose)polymerase. Both the cytotoxic effect and apoptotic characteristics induced by combined treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role of caspase-3 in the observed cytotoxic effect. These results indicate that caspase-3 is a key regulator of apoptosis in response to combined genistein and TRAIL in human gastric adenocarcinoma AGS cells through the activation of DR5 and mitochondrial dysfunction.     

紫杉醇提高胃癌MGC803细胞对TRAIL的敏感性

目的：研究紫杉醇对TRAIL诱导胃癌细胞凋亡的影响,探讨死亡受体5（DR5）在TRAIL诱导细胞凋亡中的作用。方法：采用MTT法测定细胞活力,流式细胞仪检测细胞凋亡,western blot检测蛋白表达。结果：在MGC803细胞中,100ng/ml的TRAIL导致轻度的增殖抑制和细胞凋亡,TRAIL（100ng/ml）联合紫杉醇（3.41g/ml）引起明显的增殖抑制和细胞凋亡（P〈0.05）。TRAIL单药没有改变死亡受体5（DR5）的表达,而3.41g/ml紫杉醇作用MGC803细胞后明显上调了DR5的表达。结论：紫杉醇通过上调DR5的表达增强TRAIL诱导的胃癌MGC803细胞凋亡。     

Adenovirus E1A reverses the resistance of normal primary human lung fibroblast cells to TRAIL through DR5 upregulation and caspase 8-dependent pathway

Expression of the adenovirus serotype 5 (Ad5) E1A enhances tumor cells to apoptosis by TNF-alpha, Fas-ligand and TNF-related apoptosis-inducing ligand (TRAIL). In this study, we found that E1A expression reversed the resistance of normal primary human lung fibroblast cells (P-HLF) to TRAIL-induced apoptosis. Furthermore, TRAIL dramatically induced apoptosis of P-HLF cells that expressed E1A following either infection with Ad-E1A or transfection with pcDNA3-E1A. Further results demonstrated that E1A specifically upregulated DR5 levels but had nearly no effect on the levels of DR4. E1A dramatically upregulated the exogenous TRAIL, and then increased a substantial amount of TRAIL on the surface of P-HLF cells treated with the expression vectors, both Ad-TRAIL and pIRES-EGFP-TRAIL. The dominant negative FADD mutation (FADD-DN) results revealed that the apoptosis in Ad-E1A and Ad-TRAIL coinfected P-HLF cells was completely blocked following inhibition of the death receptors-associated apoptosis-inducing molecules FADD. Moreover, the caspase 8 inhibitor (Z-IETD-FMK) could efficiently block caspase 8 activation and resulted in inhibition of caspase 3 activation and cleavage. However, The caspase 9 specific inhibitor (Z-LEHD-FMK) could not counteract the synergistic effect of TRAIL-induced apoptosis in combination with E1A, and caspase 3 activation and cleavage were not inhibited by Z-LEHD-FMK. Thus, our results suggest that adenovirus E1A sensitizes P-HLF cells to TRAIL-induced apoptosis involving DR5 upregulation and the caspase 8-dependent pathway. These findings provide the first direct evidence for molecular mechanisms of adenovirus E1A gene products to sensitize normal cells to TRAIL-mediated apoptosis.