Targeting PGE2/EP4 And Its Downstream Signaling Effectors DNMT1/c-Jun Contribute To Solamargine-inhibited Growth Of Non-Small Lung Cancer Cells

ObjectiveSolamargine is an ingredient of Solanum nigrum which is a Chinese herb consisted FuZheng KangAi decoction.It was reported that Fuzheng KangAi decoction has a good effect to the treatment of lung cancer.Existing studies have shown that solamargine has a significant inhibitory effect to a variety of tumors.And in the previous study,we found that the solamargine has a good inhibitory effect on the cell growth of lung cancer.So we set up this subject to further elucidate the possible molecular mechanisms of solamargine in inhibiting the growth of non-small lung cancer cells through in vivo and in vitro experiments.A549 and H1299 lung cancer cell lines were applied to investigate the inhibition of proliferation and underlying molecular mechanism of solamargine.MethodsThe inhibitory effect of solamargine to the cell viability of lung cancer cells was detected by MTT assay.MTT assay was performed to compare the effects of different concentrations of solamargine to the cell viability of lung cancer cells of A549 and H1299 at 24h,48h and 72h.Flow cytometry(FCM)was used to detect the cell cycle of A549 and H1299 cells.The expression of p-Akt and p-ERK of A549 and H1299 lung cancer cells were detected by Western blot(WB)at different time points.The expression of SP1,EP4,NF-KB,DNMT1 and c-Jun were detected by Western blot assay after 24 h treatment by solamargine to A549 and H1299 lung cancer cells.The expression of EP4 mRNA in A549 and H1299 lung cancer cells was detected by real-time quantitative PCR(qRT-PCR).The activity of EP4 promoter in A549 and H1299 cells was detected by dual-luciferase reporter assay.The effect of p-ERK on the expression of DNMT1 protein was observed by using ERK inhibitor PD98059 in lung cancer cells.The relationship of relevant proteins was studied by exogenous expression(plasmid transfection)experiments in lung cancer cells.Animal experiments were performed by A549-Luc cells inoculated with nude mice to form a transplanted tumor model with solamargine low dose(4mg/kg)and solamargine high dose group(8mg/kg).Intraperitoneal injection was given every other day for 30 days.The fluorescence signals of the low dose and the high dose in the nude mice were compared with that of the control group.The changes of tumor volume,tumor weight to control group were observed before and after treatment.The expression of EP4,DNMT1,c-Jun and p-ERK in the nude mice was detected by Western blot method.ResultsIn this study,both in vitro and in vivo experiments confirmed the solamargine-inhibition to lung cancer cell growth.The results of cell experiments showed that MTT method confirmed that the solamargine had inhibitory effect on the growth of multiple lung cancer cell lines,and the growth inhibition of solamargine on lung cancer A549 and H1299 cells was confirmed,the difference is statistically significant by the increasing of the dosage and the prolongation of the time.Flow cytometry showed that the cell cycle of A549 and H1299 cells could be blocked in G0/G1 phase,the difference is statistically significant(p<0.05).Our results showed that solamargine has an inhibitory effect on the phosphorylation of Akt and inhibited the protein expression of SP1,NF-?B(p65)and EP4 protein.And there is a dose and time dependent manner,the difference is statistically significant(p<0.05).Exogenous expression of Akt could block the effectc of solamargine decreased SPI protein expression,and the difference is statistically significant(p<0.05).Exogenous expression of SP1 showed to have no obvious effect on p-Akt protein expression,the difference is not statistically significant(p>0.05).Overexpression of SP1 can block the effectc of solamargine decreased p65 protein expression,and the difference is statistically significant(p<0.05).Exogenous expression of p65 has no significant effect on the expression of SP1 protein,the difference is not statistically significant(p>0.05).Exogenous expression of p65 can overcome the effect of solamargine decreased EP4 protein expression,and the difference is statistically significant(p<0.05).Exogenous expression of EP4 showed to have no significant effect on the expression of SP1 and p65 protein,and the difference is not statistically significant(p>0.05).Overexpression of EP4 has a positive feedback to the expression of p-Akt protein.At the same time,the qRT-PCR and Dual-luciferase reporter methods demonstrated that solamargine(6.0 ?M)can reduce EP4 mRNA levels and decrease EP4 promoter activity in A549 and H1299 lung cancer cells.The results showed that the expression of p-ERK in solamargine was promoted.The expression of DNMT1 and c-Jun proteins was inhibited.With the increasing of its dosage and treatment time,the effects of solamargine will be showed to be more obviously,and the difference is statistically significant(p<0.05).Exogenous expression of EP4 can block the effect of solamargine decreased the expression of c-Jun protein,and the difference is statistically significant(p<0.05).Exogenous expression of c-Jun showed to have no obviou's effect to solamargine blocked EP4 Protein expression,the difference is not statistically significant(p>0.05).Exogenous expression of EP4 can overcome the effect of solamargine increased the expression of p-ERK protein,and the difference is statistically significant(p<0.05).ERK inhibitor(PD98059)can block the effect of solamargine decreased the expression of DNMT1 protein,and the difference is statistically significant(p<0.05).Exogenous expression of DNMT1 can block the effect of solamargine decreased the expression of c-Jun protein,and the difference is statistically significant(p<0.05).Exogenous expression of c-Jun showed to have no significant effect on solamargine decreasing the expression of DNMT1 protein,,and the difference is not statistically significant(p>0.05).Overexpression of c-Jun has a positive feedback to the expression of p-ERK protein in solamargine.In vivo experiment,the solamargine showed to have a significant inhibitory effect of tumor volume.Compared with the control group,the biofluorescence signal values of the transplanted tumors were significantly lower than those of the low-dose group(4mg/kg)and the high-dose group(8mg/kg),the difference is statistically significant(p<0.05).Compared with the control group,the tumor weight is lower and the tumor volume is smaller than those of the low-dose group and the high-dose group,the difference is statistically significant(p<0.05).The results showed that solamargine has an inhibitory effect on the volume of transplanted tumor,and the difference is much more obvious by the prolongation of administration time.The protein expression of tumor tissue is detected by Western Blot.In the high-dose group,the results showed that solamargine increased p-ERK,decreased the protein expression of EP4,c-Jun and DNMT1 compared with the control group.The difference is statistically significant(p<0.05).The p-ERK increased,compared with the control group,the difference was statistically significant(p<0.05).ConclusionsIn this study,both in vitro and in vivo experiments confirmed the cell growth inhibition effect of solamargine to lung cancer cells.In vitro,the results showed that solamargine can inhibit the cell viability of A549 and H1299 lung cancer cells in a dose-dependent and time-dependent manner.With the increasing dose of solamargine and the prolongated time of administration,the inhibitory effect of solamargine on the cell viability of A549 and H1299 lung cancer cells is more significant.The results showed that solamargine can block the cell cycle in A549 and H1299 cells at G0/G1 phase.Our results show that solamargine inhibits the growth of lung cancer cells through inactivation of p-Akt,followed by the reduction of SP1 and p65.This results in the inhibition of EP4 gene expression.The cross-talk between SP1 and p65,and the positive feedback regulatory loop of PI3K/Akt signaling by EP4 contribute to the overall responses of solamargine in this process.This study unveils the novel mechanism by which solamargine inhibits growth of human lung cancer cells and reemphasizes the potential target of EP4 in lung cancer prevention and treatment.Furthermore,our results show that solamargine inhibits the growth of human lung cancer cells through reduction of PGE2 receptor EP4 protein expression and induction of ERK1/2 signalling.This in turn results in decrease in DNMT1 and c-Jun protein expressions.The inter-correlations between EP4,DNMT1 and c-Jun,and novel feedback regulation of ERK1/2 by c-Jun contribute to the overall responses of solamargine in this process.This study uncovers an additional novel mechanism by which solamargine inhibits growth of NSCLC cells and suggests involvement of additional down-stream signalling and targets of EP4 in lung cancer prevention and treatment.More importantly,our data in vivo were consistent with the findings from that in vitro,confirming the effect of solamargine on lung cancer growth inhibition and regulation of EP4 expression.Moreover,additional studies are needed to further determine the critical role of EP4,DNMT1 and c-Jun in this process using cells stable transfected with shRNAs and exogenous expression vectors of EP4,DNMT1 and c-Jun genes in nude mice model.