Synergistic Effect Of Argininespecific ADP-ribosyltransferases1and Poly(ADP-ribose)polymerase-1on Apoptosis Induced By Cisplatin In CT26Cells

Objective: To investigate the effect of ART1and PARP-1on CDDPinduced apoptosis in colon carcinoma CT26cells and its possiblemechanism.Methods: Lentivirus vector-mediated ART1-cDNA were transfectedinto murine colon carcinoma CT26cells. The ART1-shRNA CT26cells, theLV-controlCT26cells and the untransfected CT26cells were served ascontrol. All of the cells apopotosis were induced by cisplatin（CDDP）.Expression of ART1mRNA and protein were detected by RT-PCR andWestern Blot. The apoptosis rate induced by CDDP was determined by flowcytometry essay.5-AIQ and Y-27632, inhibitor of PARP-1and ROCK1,were used to clarify the relationship of PARP-1, ROCK1and ART1. Theexpression of PARP-1, PARP1cleavage fragmentation, RhoA, ROCK1,NF-kB, Cox-2and Caspase3were examined by Western Blot.Results:1.Transfection of lentiviral vector-mediated ART1-cDNA in CT26cells was done successfully. The RT-PCR and Western Blot essay showed that: compared with the ART1-shRNA CT26cells, the LV-control CT26cells and the untransfected CT26cells, the expression of ART1inART1-cDNA CT26cells were significantly increased(P<0.05).2.Effect of ART1and PARP-1on CT26cell apoptosis induced byCDDP:①Flow cytometry analysis: results showed that the apoptosis rateincreased in each group of CT26cells induced by CDDP, while theART1-cDNA CT26cells induced by CDDP had a lower apoptosis rate,compared with the ART1-shRNA CT26cells, the un-transfected CT26cellsand the LV-control CT26cells treated in the same manner(P<0.05). Theapoptosis rate of ART1-cDNA CT26cells treated with5-AIQ and CDDPobviously increased, compared to the ART1-cDNA CT26cells treated withCDDP only and the untreated ART1-cDNA CT26cells(P*<0.05).②The results of hoechst33342staining showed that the apoptoticbodies significantly increased in each group of CT26cells treated withCDDP. Moreover, after treatment with CDDP, the apoptosis rate in theART1-cDNA CT26cells declined, compared with the ART1-shRNA CT26cells, the LV-control CT26cells and the un-transfected CT26cells(P<0.05).ART1-cDNA CT26cells treated with5-AIQ and CDDP had a high apoptosisrate, compared with ART1-cDNA CT26cells treated with CDDP only andthe untreated ART1-cDNA CT26cells.(P*<0.05).3.Detection of relevant apoptosis-related proteins: ①Among the groups of cells treated with CDDP, the expression ofPARP1, RhoA, ROCK1, NF-kB and Cox-2and PARP-1was increased in theART1-cDNA CT26cells, when compared to these levels in theART1-shRNA CT26cells, the LV-control CT26cells and the untransfectedCT26cell, while the expression of caspase3and PARP-1cleavagefragment(p89) had an opposite trend(P<0.05). The expression of RhoA,ROCK1, NF-κB, Cox2and PARP-1had the same trend in cells not treatedwith CDDP, except for the PARP-1cleavage fragment (p89) which was thecleavage substrate for activated caspase3which was noted in theCDDP-treated CT26cells only（P<0.05）.②The expression of ROCK1and ART1in the5-AIQ treateduntransfected CT26cells exhibited no obvious changes when compared tothe untreated untransfected CT26cells. In the untransfected CT26cellstreated with Y-27632, the expression of PARP-1was significantly decreasedwhile the expression of ART1exhibited no obvious change (P<0.05).③There was an obvious decreas in NF-κB and Cox-2and an increasein caspase3in the ART1-cDNA CT26cells treated with5-AIQ and CDDP,when compared to the ART1-cDNA CT26cells treated with CDDP only andthe untreated ART1-cDNA CT26cells (P<0.05)Conclusions: ART1-shRNA could increase CT26cell apoptosis,ART1-cDNA could decrease CT26cell apoptosis. ART1and PARP1had asynergistic effect on CDDP induced apoptosis in murine colon carcinoma CT26cells. The mechanism is probably that ART1regulating RhoA/ROCKpathway and affect the expression of NF-κB、PARP-1、Cox-2、caspase3.