RMP Inhibits Cell Apoptosis Induced By Cisplatin Through ATM-mediated NF-κB Activation Pathway

Objective: In this study, we explore the role of RMP in apoptosis induced by cis-platinum and investigate the mechanism of RMP in regulating the apoptosis.Methods: 1. Identified the suitable cisplatin concentration in the induction of apoptosis. 2. The apoptosis of cells treated with cisplatin was measure by flow Cytometry. 3. The protein expression level of Bcl-xl、p65 、AKT、ATM 、PARP and the phosphorylation level of p65、AKT、ATM in different cells after treated with cis-platinum were measure by Western blot. 4. The protein expression level of Bcl-xl、p65 、AKT、ATM、PARP、IκB and the phosphorylation level of p65、IκB、AKT、ATM was detected by western blot followed by transfection with different dose of p CDNA3.1-RMPo and p GPU6/Neo-RMPi. 5. The mitochondrial membrane potential of cells followed treated with cisplatin was detected by flow Cytometry. 6. After treated with the inhibitor of p-p65(BAY-11-7082),the protein level of p-p65 and Bcl-xl were measure by western blot. 7. After treated with the inhibitor of p-ATM(KU55933),the phosphorylation level of p65 and ATM were measure by western blot. 8. Immunoprecipitation was carried out to explore the combination of RMP and PARP、 RMP and p-ATM、PARP and p-ATM. 9. Immunohistochemically staining was applied to detect the expression of p-p65、RMP、p-ATM and Bcl-xl in HCC tissues from patients and the matched non-tumor hepatic tissues.Results:1. The apoptosis was significantly increased in dose-dependent manner of cisplatin. Then 12μg/ml was selected as the working concentration in the following experiment 2. Although apoptosis in all cells increased during the time course after induction with cisplatin, depletion of RMP resulted in more apoptotic cells. However Hep G2 cells with RMP overexpression were resistant to cisplatin with less apoptotic cells compared with untransfected Hep G2 cells. 3. The expression of Bcl-xl and phosphorylation level of p65、ATM was significantly elevated by RMP overexpressed in Hep G2 treated with cisplatin, However PARP cleavage was inhibited in the RMP overexpression cells(RMPo) compared with untransfected cells.There was no significant difference in expression of p65、p-AKT、AKT、ATM. Deleted of RMP has the reverse results. 4. The levels of the phosphorylated form of IκB、p65、ATM and the expression level of Bcl-xl was significantly enhanced by the increasing amount of RMP, although the total expression of IκB、p65、ATM remained unchanged.In opposite, the level of cleaved PARP was reduced by increasing level of RMP. 5. Overexpression of RMP can inhibited mitochondrial membrane potential decline. Deleted RMP can promote mitochondrial membrane depolarization. 6. After treatment with BAY-11-7082, p65 phosphorylation was almost abolished. Meanwhile the expression of Bcl-xl was also decreased in RMP overexpression cell. 7. After KU55933 was applied, KU55933 abolished the increase of ATM phosphorylation meanwhile the phosphorylation of p65 was also decreased in RMP overexpression cell. 8. p-ATM、PARP was immunoprecipitated with RMP in Hep G2 cells treated with cisplatin compared with cells without cisplatin treatment. And more p-ATM、PARP was immunoprecipitated with RMP in Hep G2 cells with overexpression of RMP. 9. Immunohistochemistry showed that RMP expression in HCC was significantly higher than that in the matched non-tumor hepatic tissues. Meanwhile p-ATM 、p-p65 and Bcl-xl also showed elevated expression in HCCs compared with non-tumor hepatic tissues.Conclusion: 1. RMP can inhibit apoptosis of HCC cells induced by cisplatin through up-regulation of Bcl-xl expression.2. RMP up-regulation of Bcl-xl expression via ATM-mediated NF-κB activation.