Protective effect of RIP and c-FLIP in preventing liver cancer cell apoptosis induced by TRAIL
Jichun Sun1, Hongwu Luo1, Wanpin Nie1
1Department of Surgery, The 3rd Xiangya Hospital, Central South University Changsha, Hunan Province, China.
Abstract:
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.
Insights
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in liver cancer cells involves RIP and c-FLIP genes. Silencing these genes enhances TRAIL-induced apoptosis by activating Caspase-8.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Death Signaling
Background:
- TRAIL induces apoptosis in cancer cells by upregulating death receptors DR4 and DR5.
- The roles of RIP and c-FLIP in TRAIL-induced apoptosis in liver cancer cells remain incompletely understood.
- Understanding these mechanisms is crucial for developing effective liver cancer therapies.
Purpose of the Study:
- To investigate the involvement of RIP and c-FLIP genes in TRAIL-induced apoptosis of HepG2 and Hep3B liver cancer cells.
- To elucidate the molecular mechanisms underlying TRAIL resistance mediated by RIP and c-FLIP.
- To assess the potential of targeting RIP and c-FLIP to enhance TRAIL-based cancer therapy.
Main Methods:
- siRNA-mediated gene silencing of RIP and c-FLIP in HepG2 and Hep3B cells.
- Western blot analysis to quantify protein expression levels (c-FLIP, RIP, FADD, Caspases, etc.).
- Caspase-8 activity assay, flow cytometry for apoptosis analysis, and cell cycle determination.
Main Results:
- TRAIL upregulated c-FLIP and RIP, while downregulating FADD in liver cancer cells.
- Chemotherapy drugs reduced c-FLIP and RIP, increasing FADD expression.
- Knockdown of c-FLIP and RIP significantly enhanced TRAIL-induced apoptosis, activated Caspase-8, and induced G1 cell cycle arrest.
- RIP and c-FLIP were identified as inhibitors of Caspase-8 activation, conferring TRAIL resistance.
Conclusions:
- RIP and c-FLIP play critical roles in mediating resistance to TRAIL-induced apoptosis in liver cancer cells.
- Targeting RIP and c-FLIP can overcome TRAIL resistance and potentiate anti-cancer effects.
- These findings highlight RIP and c-FLIP as potential therapeutic targets for enhancing TRAIL-based liver cancer treatments.
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