Rhein Inhibits Cell Migration And Invasion By Tegulating Rac1/ROS/MMPs Pathway In Human Ovarian Carcinoma Cells

Objective:To study theinhibition of rhein on the migration and invasion in ovarian cancer cells with different lymph node metastasis potentials.To explore the mechanism rhein on the level of NADPH oxidase and ROS and the expression of key proteins of Rac1/ROS/MMPs signaling pathway and MMPs production in SKOV3-PM4 cell.The results can provide a new intervention methods for the study of metastasis and invasion of ovarian cancer.Methods:Ovarian cancer cells with characteristics of highly directional lymphatic metastasis(SKOV3-PM4)was selected as the research objects in this study.MTT assay was used to detect the proliferative effect of SKOV3-PM4 afterrhein treatment.The cells were treated with different concentrations of Rhein combined with PMA(a Racl activator)and NSC23766(Racl inhibitors).In exponential growth phase,SKOV3-PM4 s were divided into nine treatment groups: group I(control);group II(7.7 μM rhein);group III(11.3 μM rhein);group IV(100 n M)PMA alone);group V(PMA plus 7.7 μM rhein)group VI(PMA plus 11.3 μM rhein);group VII(12.5 μM NSC23766 alone);group VIII(NSC23766 plus 7.7 μM rhein);group IX(NSC23766 plus 11.3 μM rhein).The migration ability of cells was detected by Wound healing assay and the invasive ability of cells was measured by Transwell chamber assay.The activity of NADPH oxidase of cellswith different treatment was detected by NBT.The level of reactive oxygen species in cellarlus after different treatment was detected by flow cytometry.The expression level of MMPs protein and the associated protein in Rac1/ROS/MMPs signaling pathway was detected by Western Blot.Results:The proliferation of SKOV3-PM4 cells was inhibited by rhein,and the half maximal inhibitory concentration(IC50)of 48 h was 34.8μM.Non-toxic concentration of rhein was lower than 12 μM.So we selected 7.7 and 11.3μM rhein as the experimental concentration.PMA,a Rac1 activator,can remarkably increase the ability of migration,while inthe cells treated with rhein(11.3 μM)alone,PMA plus rhein(11.3 μM)and NSC23766 plus rhein(11.3 μM),the capacityofmigration signifcantly decreased compared to thecontrol(P<0.05).An NBTassay demonstrated that Rac1 activator PMA remarkably increased the activity of NADPH oxidase(P<0.01),but after the cells treated with rhein(7.7/11.3 μM)alone,PMA plus rhein(11.3 μM)and NSC23766 plus rhein(7.7/11.3μM),the activityof NADPH oxidase signifcantly decreased compared to thecontrol(P<0.01).The generation of intracellular ROS was examinedusing a DCFH-DA probe showed that PMA can remarkably increase the intracellular ROSproduction as compared to control cells(P<0.01).In cellstreated with PMA combined with rhein,the ROS levels weredownregulated clearly compared to cells treated with PMAalone(P<0.05).The results of Western Blot shown that rhein significantly inhibited JNK,AP-1 phosphorylation in the cells treated with PMA.The results obtained from the cells treated with NSC23766 alone or NSC23766 combined with rhein,were consistent with rhein treatment alone.Furthermore,the expression level of MMP-2,-3,-7,-9 and MMP-19 was decreased compared to thecontrol,whereas Nm23-H1,TIMP-2 and TIMP-1 were just the opposite.After PMA treatment,the expression level of MMP-9 protein was increased,whereas MMP-7 and NM23-H1 were just the opposite.In cellstreated with PMA combined with rhein(11.3μM),the expression level of MMP-2-3,-9 and MMP-19 proteinwas significantly reducedcompared to the PMA.After NSC23766 plus rhein treatment,The protein expression level ofMMP-3,-7,-9 and MMP-19 was decreased,whereas NM23-H1,TIMP-2 and TIMP-1 were just the oppositecompared to theNSC23766.The ability of Rhein to inhibit invasion and metastasis of ovarian cancer may be associated with inhibition of phosphorylation of JNK protein in MAPK pathway,further inhibiting MMPs and increased expression of TIMP-1,TIMP-2 and Nm23-H1 protein.Conclution: The results indicated that Rhein can inhibit the migration and invasion of SKOV3-PM4 cells through inhibiting the phosphorylation of JNK and AP-1 proteinin Rac1/ROS/MMPs signaling pathwayand.modulating matrix metalloproteinases.Rac1 played a key role in the activation of matrix Metalloproteinases,Rac1 may be a potential target of lymph node metastasis of ovarian cancer and Rac1 small molecule inhibitor.