| Background & Objective: Esophageal carcinoma is one of the most common gastrointestinal malignancies and the incidence ranks eighth and the cancer-related mortality ranks sixth cancer worldwide. Our country is one of the high incidence of esophageal carcinoma with squamous carcinoma for the more and the fourth leading cause of mortality. The majority of patients with esophageal carcinoma are diagnosed in an advanced stage when symptoms appear and tend to early metastasis. The efficacy and prognosis of esophageal carcinoma is poor. Chemotherapy is one of the primary manners in the treatment of esophageal carcinoma, but the progress made in improving the prognosis is very slowly. Therefore, to discover new therapeutic targets which can enhance sensitivity to chemotherapeutic drugs of esophageal carcinoma is significant. Recent studies showed that RIP1 contains an N-terminal serine / threonine kinase and a C-terminal death domain.RIP1, the first member of the RIP family, plays an important role in regulation of cell death and cell survival.However, the proliferation, apoptosis and sensitivity of RIP1 in esophageal carcinoma cells to drug treatment is unclear currently. The study uses esophageal squamous carcinoma cells KYSE510, KYSE410 as the research object, to observe the roles of RIP1 in DDP-induced apoptosis and explore its mechanism.Methods Cell cultured in vitro conventionally,the viability of human esophageal squamous carcinoma cell lines KYSE510 and KYSE410 exposed to differentconcentrations of DDP were detected by Cell Titer 96® AQueous One Solution Cell Proliferation Assay(MTS) assay.Annexin V/PI staining was used to observe cell apoptosis in KYSE510 and KYSE410 cells. Western blot was used to detect RIP1/caspase-3/PARP/JNK/p-JNK protein expression in KYSE510 cells exposed to DDP.Real-time quantitative PCR was used to detect expression of RIP1 m RNA in esophageal squamous carcinoma cells. The mitochondrial membrane potential of KYSE510 cells with JC-1 were detected by flow cytometry.The apoptosis rate in two cell lines and the change of Reactive oxygen spices(ROS) production in KYSE510 cells treated by DDP and DDP combination with Necrostatin-1(Nec-1) were analyzed by flow cytometry. Small interfering RNA(si RNA) was used to silence RIP1 specificly, using Western blot to detected the expression of RIP1 protein levels in KYSE510 cells after transfection of si RNA. The apoptosis rates in each group treated with si RNA in KYSE510 cells induced by DDP were detected by flow cytometry. The protein expression levels of caspase-3/PARP/JNK/p-JNK in KYSE510 cells after RIP1 silenced were detected by Western blot.Results DDP-induced apoptosis of esophageal squamous carcinoma cells in a dose and time-dependent, KYSE510 and KYSE410 cells exposed to DDP for IC50 were(5.021 ± 0.069) μg / ml and(40.169 ± 0.302) μg / ml. With the latter was eight times than the former, there was a significant difference between the two groups(P <0.01). Apoptotic rate was increased with dose and time, RIP1 protein expression was upregulated in esophageal squamous carcinoma cell lines and m RNA expression levels in KYSE510 cells higher than in KYSE410 cells. Same dose of DDP exposed to the two esophageal squamous carcinoma cell lines, the activation of caspase-3/PARP/p-JNK was significant in KYSE510 cells, but so changesed wasn’t observed in KYSE410 cells. It was significantly reduced after exposuring to DDP association with special inhibitor of RIP1 in KYSE510 cells which were more sensitive to DDP than KYSE410 cells. KYSE510cells exposed to DDP, the mitochondrial membrane potential was decrease than the control group, but ROS produced increasing. After transfection of si RNA silencing RIP1, cell proliferation in KYSE510 cells were increase but the apoptosis rate was significantly lower than the negative control group. The protein expression levels of caspase-3/PARP/Jp-JNK in KYSE510 cells was significantly decreased after RIP1 silenced, while protein expression level of JNK was’ t so change.Conclusion RIP1 maybe participate the apoptosis of human esophageal squamous carcinoma cells to DDP; RIP1 maybe involved in the regulation of caspase-dependent apoptosis and JNK signal transduction pathway in esophageal squamous carcinoma cells. |
