The Mechanism Of Ras-MAPK Pathway Cooperates With P53^N236Sin Anti-apoptosis

Cancer is the first killer of human lives.Moreover,the interaction of inactivated tumor suppressor genes(such as p53)and activated oncogene(such as Ras)is a leading cause of cancer development.Thus,understanding their interaction mechanism will play a key role in tumor therapy.p53 is an important tumor suppressor gene for its average mutation frequency is about 50%in spontaneous human tumors,and most of them are are missense mutation.In addition,mutant p53 will not only loss of function,but also obtain the potential that promote tumorgenesis.In our prelimnary study,we treated p53N236S(the 236th amino acid of p53 is mutated from asparagine to serine,p53S)cells with doxorubicin(Topoisomerase I inhibitors).The genetic background of these five cells are:p53S/S(p53S/S endogenous cells into the Ras-pBabe expression vector),p53S/S,p53J/-+V+Ras(p53 gene knock out of the cells into a p53S expression vector controlled air carrier vector and the Ras-pBabe carrier),p53-’-(p53 gene knocked out of cells),and the wild-type(WT)cells.Western Blot and Flow Cytometry assay have showed that the apoptosis level of p53S cells are significantly lower than that of wild type(WT)cells(p<0.01),which suggest that p53S protein loss the damage stress response function of the wild-type p53 and unable to induce cell apoptosis,thus leading to cell drug resistance.The apoptosis level of p53S/S+Ras cells are lower than p53S/S,and the expression of Multidrug resistance resistance protein 1(MDR1)in p53S/S+Ras cells is increased.It suggesting that the drug resistance ability to doxorubicin could be further enhanced by p53 and Ras cooperation.In addition,our previous work have also found a large number of evidence for the synergistic effect of p53S and Ras,such as promoting tumor formation,cell proliferation and the high expression of oncogene Ras in the context of p53S.These datas suggest that p53S will show gain of function to promote the tumorgenesis when Ras is activated.At present,a large number of studies have shown that activated oncogene Ras can induce its downstream MAPK(ERK1/2,JNK,p38)pathway,by reducing the expression of MDR1,thereby enhancing the drug-resistance of tumor cells.Therefore,this study focous on the Ras induced MAPK pathway to reveal the cooperation mechanism of Ras-MAPK pathway and p53S to resist the doxorubicin induced apoptosis.We detected the MAPK pathways of WT,p53-/-,p53-/-+V+Ras,p53s/s,p53s/s-+Ras cells after doxorubicin treatment,and we found that p38 and ERK1/2 were significantly up-regulated in p53S/S+Ras cells,which suggesting that MAPK was mainly activated by Ras.However,p53 played anauxiliary role in this process.Then,We found that the expression of NF-κB p65 in the nucleus of p53S/S+Ras cells was higher,which suggesting that Ras cooperate with p53S enhances the nuclear localization of NF-κB p65 by activating the p38 and ERK1/2 pathways,thereby enhancing the expression of MDR1,which enhancing the cell’s anti-apoptotic ability.we futher treated cells by ERK1/2 inhibitor(PD0325901)or p38 inhibitor(SB203580)combined with doxorubicin to determine whether ERK 1/2 and p38 were the main pathways for Ras and p53 cooperation.Westem Blot and Flow Cytometry assay results showed that aloughth the apoptosis level of p53S/S+Ras was increased after combination treatment strategy,the increaing degree was not significant when contrast to other cells.This data suggesting that the two MAPK pathways may have a compensatory effect in p53sS/S+Ras cells.Interestingly,the upregulation of p-p38 expression in ERKl/2-inhibited p53S/S+Ras cells may suggest that p38 compensatorily activate.That may be a reason why ERK1/2-inhibited pp53S/S+Ras cells are more resistant to doxorubicin-induced apoptosis than other genotypes.And when p38 was inhibited,p-ERK1/2 also appeared to has slightly upregulation compensatory effect,which suggesting that p38 may be the main pathway for the anti-apoptosis effect of Ras and p53S cooperation.In the study of the mechanism of drug-resistance in cancer cells,it was found that the drug-resistant cells would not only resist to single drug,but also tolerant to different chemotherapeutic drugs that varies in structures and mechanisms.What’s the situation in p53S cells?We futher used cisplatin(DDP,form an adducts with DNA to inhibit the DNA replication process)to deal with p53S/S cells.Intriguingly,not like doxorubicin,p53S/S cells did not show significant anti-apoptotic effect when dealed with cisplatin and the apopotosis level were slightly higher than the WT cells.This discovery aroused our attention.Does the different mechanism of the drug accounts for this phenomenone?In order to confirm our hypothesis,we selected etoposide(ETO)and camptothecin(CAM)as the doxorubiein analogues,and Carmustine(CAR),which has similar effect to cisplatin,to explore the killingability of different drugs on p53S/S cell.The results revealed that p53S/S cells exhibited significant anti-apoptotic effects when treated by topoisomerase inhibitor drugs.However,p53S/S cells showed a slightly higher apoptosis than WT cells after treatment of DNA adduct drugs.Then,we investigated the cisplatin sensitive mechanism of p53S/S cells.Western Blot experiment showed that the expression of p-p53(ser15)was higher in p53S/S+Ras cells,which was associated with time.In addition,the expression of Hsp70 and Hsp90 were slightly increased,so as the expression of Bax,and the expression of Bcl-2 was down-regulated.All together,these data suggesting that cisplatin can induce apoptosis in p53S/S cells.In conclusion,our study further proved that the p53S/S+Ras cells have stronger anti-apoptotic ability than p53S/S and p53-/+V+Ras cells.In addition,when examined the Ras related MAPK pathways,we found high expression of p-ERK1/2,p-p38 in p53S/S+Ras cells after treated by doxorubicin when compared with other genotypes.It suggesting that ERK1/2,p38 activation may be accounts for the tolerance of Ras and p53S to doxorubicin-induced apoptosis.Futhermore,p38 has a significant compensatory effect on p53S/S+Ras cells and may play an important role in the anti-apoptosis effect of the cooperation of Ras and p53S.Moreover,different biological phenomena have been showed when using antitumor drugs to treat p53S/S cells,which suggesting that the drug resistance of p53S cells is related to the pharmacodynamics effect of drugs.The significance of our work is that we revealed the anti-apopotosis mechanism of cooperation between Ras-MAPK pathway and p53S when treated by doxorubicin.Moreover,the results of our study may also benefit clinical drug strategy.