Effects of sequential treatments with chemotherapeutic drugs followed by TRAIL on prostate cancer in vitro and in vivo

BACKGROUND: Tumor necrosis factor related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo-2L) is a novel anticancer agent, capable of inducing apoptosis preferentially in tumor and transformed cells. TRAIL-R1/death receptor (DR)4 and TRAIL-R2/DR5 are members of the tumor necrosis factor (TNF) receptor family, and can be activated by the TRAIL. We examined the clinical potential of chemotherapeutic drugs and TRAIL for the treatment of prostate cancer. METHODS: Prostate and bladder cancer cells were exposed to chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, doxorubicin, and camptothecin) and TRAIL. Cell viability was measured by sodium 3'[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) assay; expressions of death receptors and Bcl-2 family members were measured by Western blotting, ELISA and ribonuclease protection assay. PC-3 tumor cells xenografted athymic nude mice were exposed to chemotherapeutic drugs and TRAIL, either alone or in combination, to measure tumor growth and survival of mice. Apoptosis was measured by annexin V-FITC/propidium iodide staining, and terminal deoxynucleotidyltransferase-mediated nick end labeling assay. Caspase-3 activity was measured by the Western blotting and immunohistochemistry. RESULTS: TRAIL induced apoptosis with varying sensitivity. Chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, doxorubicin, and camptothecin) significantly augmented TRAIL-induced apoptosis in cancer cells through up-regulation of DR4, DR5, Bax, and Bak, and induction of caspase activation. Mitochondrial pathway enhanced the synergistic interactions between drugs and TRAIL. The sequential treatment of mice with chemotherapeutic drugs followed by TRAIL induced caspase-3 activity, and apoptosis, inhibited angiogenesis, completely eradicated the established tumors, and enhanced survival of mice. CONCLUSIONS: Chemotherapeutic drugs can be used to enhance the therapeutic potential of TRAIL in prostate cancer.     

Synergy is achieved by complementation with Apo2L/TRAIL and actinomycin D in Apo2L/TRAIL-mediated apoptosis of prostate cancer cells: role of XIAP in resistance

BACKGROUND: Tumors have an inherent immunogenicity that can be exploited by immunotherapy. However, often tumors develop mechanisms that render them resistant to most immunologic cytotoxic effector mechanisms. This study examines the underlying mechanism of resistance to Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-mediated apoptosis. METHODS: We studied prostate tumor cell lines for their sensitivity to Apo2L/TRAIL-mediated apoptosis in the presence and absence of the sensitizing agent actinomycin D (Act D). Apoptosis was determined by flow cytometry and signaling for apoptosis by Western blot. RESULTS: Treatment with subtoxic concentrations of Act D significantly sensitizes the tumor cells (CL-1, DU-145, and PC-3 prostate tumor cells) to Apo2L/TRAIL-mediated apoptosis. The cytotoxicity of Act D-sensitized prostate tumor cells was a result of synergistic activation of caspases (caspase-3, -9, and -8), detectable after 6 hr of treatment. Treatment with Apo2L/TRAIL alone, although it was insufficient to induce apoptosis, resulted in the loss of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytoplasm in the absence of significant caspases activation. These findings suggested that a major apoptosis resistance factor blocking the Apo2L/TRAIL apoptotic signaling events is present downstream of the mitochondrial activation. The expression of receptors and anti-apoptotic proteins were examined in Act D-sensitized CL-1 cells. The earliest and the most pronounced change induced by Act D was down-regulation of X-linked inhibitor of apoptosis (XIAP) and up-regulation of Bcl-xL/-xS proteins. The role of XIAP in resistance was demonstrated by overexpression of Smac/DIABLO, which inhibited inhibitors of apoptosis (IAPs) and sensitized the cells to Apo2L/TRAIL. Apo2L/TRAIL receptors (DR4, DR5, DcR1, and DcR2), c-FLIP, Bcl-2, and other IAP members (c-IAP1 and c-IAP2) were marginally affected at later times in the cells sensitized by Act D. CONCLUSION: This study suggests that the combination of Act D-induced down-regulation of XIAP (Signal I) and Apo2L/TRAIL-induced release of cytochrome c (Signal II) leads to the reversal of resistance to Apo2L/TRAIL-mediated apoptosis in the tumor cells. The sensitization of tumor cells to Apo2L/TRAIL by Act D is of potential clinical application in the immunotherapy of drug/Apo2L/TRAIL refractory tumors.     

肾癌中CD133＋细胞和CD133-细胞生物学特性差异的研究

目的：研究人肾癌细胞系786-O和OS-RC-2中CD133＋细胞和CD133-细胞的生物学特性差异。方法：应用流式细胞仪检测肾癌细胞系786-O和OS-RC-2中CD133的膜表达状况;以免疫磁珠法将CD133＋细胞和CD133-细胞分离纯化,比较两者在光镜下的形态、生长特点、体外增殖能力、长期分化能力及体外克隆形成率的差异;流式细胞术分析两者细胞周期分布的变化,采用MTT法分析两者对化疗药物顺铂（DDP）（10、20、50、80／Lg／ml）敏感性的差别。结果：786-O和0S-RC-2均有CD133的少量表达,表达率为（8．12±0．86）％、（6．83±0．20）％,CD133＋细胞和CD133-细胞相比具有较强的体外增殖、克隆形成及长期分化能力;细胞耐药性实验表明CD133＋细胞、CD133-细胞对50bcg／m1DDP敏感性差异有统计学意义（P〈0．05）。结论：肾癌细胞系中CD133＋细胞具有肾癌干细胞的部分特征,能否作为肾癌干细胞的表面标志还需要进一步的实验加以明确。