Protective effect of RIP and c-FLIP in preventing liver cancer cell apoptosis induced by TRAIL

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the tumor necrosis factor superfamily that can induce tumor selective death by up-regulating death receptor 4 (DR4) and DR5 expression. The study aimed to explore the role of RIP and c-FLIP genes in TRAIL induced liver cancer cell HepG2 and Hep3B apoptosis and related mechanism. RIP and c-FLIP silenced HepG2 and Hep3B cell model were established through siRNA. Western blot was applied to test c-FLIP, RIP, DR4, DR5, FADD, Caspase-3/8/9, ERK1/2, and DFF45 protein expression. Caspase-8 kit was used to detect Caspase-8 expression. Flow cytometry was performed to measure cell apoptosis rate. Acid phosphatase method was applied to determine cell cycle. TRAIL had no significant effect on Caspase-3/8/9, DR4, DR5, ERK1/2, and DFF45 protein expression, but up-regulated c-FLIP and RIP protein expression and reduced FADD expression level. After treated by the chemotherapy drug mitomycin and adriamycin, c-FLIP and RIP expression decreased significantly, while FADD increased. After knockout c-FLIP and RIP gene, HepG2 and Hep3B cell apoptosis rate induced by TRAIL increased obviously. Meanwhile, cell subG1 percentage increased markedly and exhibited G1 phase growth retardation. In addition, after two kinds of gene knockout, Caspase-8 was activated and produce Caspase-3 P20 and P24, leading DFF45 appeared DNA fragment P17 and P25. c-FLIP and RIP can inhibit Caspase-8 activation and prompting HepG2 and Hep3B resistant to cell apoptosis induced by TRAIL.     

肾透明细胞癌中c-FLIP mRNA表达及临床意义

目的探讨细胞型Fas相关死亡域样白介素-1β转换酶抑制蛋白（c-FLIP）在肾透明细胞癌中的表达及其临床意义。方法采用免疫组织化学及RT-PCR方法对56例肾透明细胞癌组织及10例癌旁正常肾组织中c-FLIP蛋白及c-FLIP mRNA表达进行分析,观察c-FLIP mRNA表达与临床分期及预后之间的关系。结果c-FLIP蛋白及c-FLIP mRNA在肾透明细胞癌组织中呈高表达（P〈0.05）,其表达与肾透明细胞癌的临床分期无关,但阳性表达患者的生存率低于阴性表达者（P〈0.05）。结论肾透明细胞癌组织中c-FLIP mRNA表达较高,特异性较强,可作为观察肾透明细胞癌预后的重要指标之一。     

Pentoxifylline induces apoptosis in vitro in cutaneous T cell lymphoma (HuT-78) and enhances FasL mediated killing by upregulating Fas expression

Constitutive nuclear factor-kappaB (NF-kappaB) is known to play an important role in the survival of HuT-78 cells, a cutaneous T cell lymphoma (CTCL) cell line. Here, we have demonstrated that pentoxifylline (PTX), a phosphodiesterase inhibitor, can trigger a series of events leading to apoptosis in HuT-78 cells without affecting NF-kappaB. Apoptosis was ascertained by sub-G1 peak analysis and TUNEL assay. Apoptosis induced by PTX in HuT-78 cells involved mitochondrial hyperpolarization, cytochrome c release, caspase-3 activation and PARP cleavage. Further, it was found that PTX treatment downregulated Bcl-xl and c-FLIP expression without affecting constitutive NF-kappaB but upregulated activator protein-1 (AP-1). Low concentration of PTX upregulated Fas and TRAIL expression in HuT-78 cells. In addition, PTX can act as a scavenger of reactive oxygen intermediate and it could enhance FasL mediated killing in HuT-78 cells. Our results taken together indicated that PTX may be a potential agent for killing CTCL cells.