The Function And Mechanism Of RITA In Acute Leukemia Cell

Leukemia is the most common malignant disease which account for 35% in childhood tumor.Above 95% leukemia is acute rather than 3%-5% in chronic.For decades,although significantly improvement obtain in treatment accompany with the application of MDT,there are still 25%³0%of patient relapse,which most probable cause by cell drug resistance so that to search for a new type of chemotherapy targeted drug has become one of the topics in the study of tumor treatment.RITA?Reactivation of P53 and induction of tumor cell apoptosis?is a small molecule compounds which induce tumor cells to apoptosis via prevent the interaction between P53 and MDM2 and that reactive the P53 protein and restore its function in tumor cells,and it has been discovered in recent years and which provides an newly strategy for cancer therapy.It is no allergic reaction,no antibodies existed,and unable transported to extracellular by the resistance protein in RITA which also cause a synergistic effect with others chemotherapy drugs so that to increase drugs sensitivity to tumor cells.As new kind of chemotherapy drugs,RITA suggests a critical role for clinical research.As a molecular drug,RITA play a critical role in proliferation,apoptosis and senescence through the regulation of a variety of signaling pathways in tumor cell.Recently studies have confirmed RITA not only induced tumor cells apoptosis in colon cancer ? neuroblastoma and gastrointestinal tumor.But its function and mechanism still vague in acute leukemia,which neither been reported.Thus,we explore the apoptosis mechanism cause by RITA in acute leukemia.Following are the main points we obtained in this study.?1?RITA inhibits growth of acute leukemia cell.We had detected the growth-inhibition of acute leukemia cell after the RITA treatment via CCK8 cell count text,and the result show that:1?In wild-type P53 NALM6 and Molt4 cell lines,RITA efficiently decreased the count of leukemia cells after treatment in 48 hours,of that time RITA IC₅₀ was calculated to be 0.73 ?mol/L in NALM6 cells and 1.81?mol/L in Molt4 cells,relatively.2?In mutant P53 Reh and CCRF/CEM cell lines,RITA also showed growth inhibition,but the IC₅₀ need for those cell lines were 2³ times compared with wild-type P53 cells.3?HL-60 which lack of P53 were less sensitive to RITA.The IC₅₀ for HL-60 was as 10²7 times as wild-type P53 cells and as 3⁸ times as the mutant P53 cells.From these results suggest that RITA efficiently suppressed the growth of leukemia cells include wild-type,mutant and null P53.Respond to RITA the wild-type P53 leukemia cells made remarkable growth inhibition,while mutant P53 and null P53 were significantly less sensitive.?2?RITA induces apoptosis in acute leukemia cell lines.To examined whether acute leukemia cells were apoptosis through flow cytometry and western blot to examined the expression level of apoptosis protein before and after RITA treatment.The flow cytometry result showed that either wild-type P53 or mutant P53 leukemia cells apoptosis rate increases over time;Western blot results showed cleavage of Caspase-3,and PARP occurred in cells.Taken together,our results suggest RITA induces acute leukemia cells death via apoptosis.?3?RITA induces wild-type P53 leukemia cells apoptosis through target P53.To gain insight into the RITA-induced apoptotic mechanism in cells harboring wild-type P53,we further tested whether RITA influence intracellular P53 expression.Western blot analysis revealed that RITA increased the expression of P53 overtime.In older to identify the differentially expressed genes after RITA treatment,we assessed relevant pathway genes expression profiling by qRT-PCR.The result showed that the expression of MCL-1,C-MYC and 4E-BP1 were decreased,while P21 was increased.Therefore,we speculated that RITA target to P53 concentration and activation,and that regulates protein expression in its downstream pathway hereafter wild-type P53 leukemia cell apoptosis was induced by P53 protein dependent pathway.?4?RITA induces mutant P53 leukemia cells apoptosis through P53-independent pathway.To gain insight into the apoptotic mechanism of RITA-induced apoptosis in leukemia cells harboring mutant P53 also via influence intracellular P53 expression.Western blot analysis result showed that there was no P53 expression before and after RITA treatment,which manifest RITA induces mutant P53 cells apoptosis through P53-independent pathway.Due to DNA damage repair is key mechanism in apoptosis,moreover ATM is the important genes in this pathway so that to we determine whether RITA induces mutant P53 cells apoptosis through DNA damage repair pathway.qRT-PCR result showed that ATM genes expression increased after RITA treatment which suggest that RITA may induce mutant P53 acute leukemia cell apoptosis by DNA damage repair pathway.Above all results demonstrated the function and mechanism of RITA which target to P53 activation in different types of acute leukemia cells,and that regulates mitochondrial apoptosis pathway in wild-type P53 leukemia cell apoptosis.Moreover,we first report that RITA induced the apoptosis not only in leukemia cells harboring wild-type P53 but also in harboring mutant P53.Our study expore the function and mechanism of RITA in acute leukemia cells and which provide an attractive strategy for cancer therapy.