Effects Of Rotundioic Acid(RA)on The Proliferation And Apoptosis Of K562 Cells And Its Effects On The Reversal Of Multi-drug Resistance In K562/ADM Cells

Objective:A derivative of ursolic acid,3β,19β-dihyroxy-urs-12-ene-23,28-dicarboxylic acid(Rotundioic acid,RA)was isolated from Ilex hainanensis Merr.The present study observes the effects of proliferation and apoptosis and investigates the mechanisms of RA in K562 cells;To study the proliferation activity of RA on Adriamycin-tolerance K562 cells(K562/ ADM cells),and the effects of RA promoting antitumor drug sensitivity in K562/ADM cells.To research the effects and mechanisms of RA in K562/ADM cells,to study the relevances among RA and the occurrence,the development,drug resistance of leukemia,RA will be a potential therapeutic drug in the treatments in leukemia.Methods:Use cell culture technique in vitro.Cell counting kit-8(CCK-8)was applied to evaluate the anti-proliferative activity and anticancer drugs sensitivity of RA in K562 and K562/ADM cells.Apoptosis effect of RA in K562 cells was measured by annexin V-FITC/PI double staining with flow cytometry analysis.Real-time quantitative PCR(RT-qPCR)and western blot were used to detecte m RNA and protein expression levels of PARP,Bcl-2,Bax,NF-кB p65,caspase-3 in K562 cells,respectively.RT-qPCR and western blot also were applied to evaluate the levels of transcription and translation of MDR1 both in K562 and K562/ADM cells.The expression levels of MDR1 mRNA and P-gp,and p-JNK,p-p38,p-ERK1/2 were measured in K562/ADM cells following treatments with non-cytotoxic concentrations of RA.Statistic analysis was performed with GraphPad Prism 6 and SPSS 18.0 softwares,Comparisons between two groups were carried out with Student’s t test,comparisons among different groups were carried out with one way analysis of variance.The quantitative data were expressed asx±s,p<0.05 was considered statistically significant.Results:1.RA inhibits the proliferative activity in K562 cells in a concentration-andtime-dependent manner,the proliferative inhibition of 30μg/ml RA was most significantly,the half inhibitory concentration(IC50)of 24 hours was 14.94μg/ml;RA can promote apoptosis effect of K562 cells in a concentration-and time-dependent manner,which was detected by flow cytometry analysis;compared with control group,the total apoptptic rates in high concentration groups show dramatic variation.RT-qPCR and western blot show that RA upregulates the mRNA and protein expression levels of Bax,caspase-3 and PARP in K562 cells,and downregulates the mRNA and protein expression levels of Bcl-2 and NF-кB p65 in the meantime.2.The results of CCK-8 show that RA inhibits proliferative activity in K562/ADM cells in a concentration-and time-dependent manner,the IC50 of 24 hours was 45.14μg/ml;the 10% inhibitory concentration of 24 hours were 6.392μg/ml.The concentration of 4μg/ml would be used in the follow experiments.RA enhances adriamycin sensitivity in K562/ADM cells,reversal Index was 1.61 times,variance exhibits statistically significant(P<0.05),but RA did’t have this effect in K562 cells.The drug index of ADM in K562/ADM cells is 41.76 times.3.After measured by RT-qPCR and western blot,in K562 cells,the mRNA expression of MDR1 is extremely low and its protein product P-glycoprotein(P-gp)is almostly little,but in K562/ADM cells,both the expression levels of MDR1 mRNA and P-gp are really high,and they can be downregulated by RA with a dose-dependent manner.Western blot shows that RA might upregulate the phosphorylation level of p38 and ERK1/2,but it has no effect on the expression of p-JNK,it means that RA reverses the multi-drug resistance of leukemia cells by regulating the transcription and translation levels of MDR1 through regulating MAPK signal pathway.Conclusions:RA may have the proliferative inhibition and pro-apoptotic effects in a concentration-and time-dependent manner in K562 cells by upregulating the expression of pro-apoptotic genes(PARP,caspase-3,Bax)and downregulating the anti-apoptotic gene Bcl-2,besides,may be relative with the reduced expressions of NF-кB p65.So,RA may inhibite proliferative activity and promote apoptosis in K562 cells through regulating the mitochondrial pathway,NF-кB and caspase-3 signalpathways.Further more,we conclude that RA may inhibit the transcription and translation levels of MDR1 by MAPK signal pathway through upregulating the expressions of p-p38,p-ERK1/2,and finally show the reversal effects on multidrug resistance in leukemia cells.