Resveratrol Combineds With Dihydroartemisinin To Inhibit Cancer Cell Migration Via Regulation Of DLC1/TCTP/Cdc42 Pathway

The migration ability of tumor cells is crucial for the development of tumors,and inhibition its migration ability,as an anti-tumor mechanism,plays an important role in the treatment of tumors.Dihydroartemisinin is the main active metabolite of artemisinin and exerts anti-tumor effects through various ways.Resveratrol is a polyphenolic plant antitoxin with anti-cancer potential.It has been reported that combination therapy with two or more drugs is more effective than treatment with one drug alone.Whether resveratrol combined with dihydroartemisinin can effectively inhibit cancer cell migration through a related mechanism has not been reported.To examine proliferative effect of resveratrol combined with dihydroartemisinin on human cancer cells,two cancer cell lines human hepatoma cell line Hep G2,human breast cancer cell line MDA-MB-231 and one non-tumorigenic human liver cell line HL-7702 were treated without or with different concentrations of dihydroartemisinin and resveratrol alone or in combination for 24 h,and cell proliferation were carried out by MTT assay.The results showed that drugs used alone or in combination inhibited cancer cell proliferation in a dose-dependent manner,and combination drugs showed more effective action than that of either drug used alone,compared to HL-7702 cells.We also calculated the IC₅₀ when drugs used alone or in combination and found that resveratrol combined with dihydroartemisinin had lower IC₅₀than drugs used alone.The drug interaction between resveratrol and dihydroartemisinin was evaluated by CI value,and the results showed that resveratrol combined with dihydroartemisinin generated a synergistic anti-proliferation effect within a certain concentration range in Hep G2 and MDA-MB-231 cells.According to the CI value,we used dihydroartemisinin（25μM）,resveratrol（50μM）and dihydroartemisinin+resveratrol（25+50μM）which had better combined effect for subsequent experiments.In order to examine the effect of drugs on migration ability of cancer cells,we firstly used wound healing assay to detect cancer cell migration rate and then used Rhodamine red fluorescently labeled phalloidin to detect the formation of F-actin.The results showed that the combination drugs more significantly inhibited migration and F-actin formation of cancer cells than drugs used alone.Next,we will explore the molecular mechanisms that drugs inhibit cancer cell migration.Cdc42,a member of Rho family,can regulate JNK/NF-k B signaling pathway to promote the degradation of extracellular matrix mediated by MMP-2/9 and target N-WASP signaling pathway to regulate rearrangement of microfilament cytoskeleton and finally promote cell migration.The tumor suppressor gene DLC1,which down-regulated or deleted in various cancers,primarily localizes to focal adhesions.It can regulate the expression of Cdc42 through Rho GAP domain and inhibit invasion and migration in various cancer cells.The tumor-associated protein TCTP,which localizes in the nucleus and focal adhesions,overexpresses in a variety of cancers and promotes the expression of Cdc42 to enhance the ability of invasion and migration in cancer cells.Our results showed that the combination drugs more significantly up-regulated DLC1 and down-regulated the protein levels of TCTP and Cdc42 than drug used alone in Hep G2 and MDA-MB-231 cell lines.Since DLC1 and TCTP localize in focal adhesions and have a common downstream protein,there may have some correlation between the two proteins.In order to explore the relationship between DLC1 and TCTP,we firstly detected the colocalization between GFP-DLC1-WT and TCTP by immunofluorescence assay and then confirmed there was a mutual interaction between endogenous DLC1 and TCTP by Co-IP experiments in cancer cells.Next,we investigated the regulatory relationship between the two proteins.Silencing DLC1 with si RNA markedly increased the expression of TCTP in MDA-MD-231 cells,implying that DLC1 can negatively regulate TCTP.Subsequently,to further explore whether DLC1 negatively regulate TCTP dependent on its Rho GAP domain,we transfected GFP-DLC1-WT and GFP-DLC1-R718A（GAP-dead mutants）into DLC1-negative A549 lung cancer cell lines.Transfection of GFP-DLC1-WT significantly down-regulated the protein expression of Cdc42compared to transfect GFP-DLC1-R718A or vector,indicating that DLC1 regulated Cdc42 in a Rho GAP-dependent manner.However,cells transfected with GFP-DLC1-WT or GFP-DLC1-R718A showed similar strong decrease of TCTP expression compared to vector,implying that DLC1 regulated TCTP in a Rho GAP-independent manner.In this study,we also explored the effects of drugs on downstream signaling pathway of Cdc42.The results showed that resveratrol combined with dihydroartemisinin significantly inhibited the levels of p-JNK,NF-k B,MMP-2/9 and N-WASP than drugs used alone in Hep G2 and MDA-MB-231 cells.To investigate whether DLC1 inhibited JNK/NF-k B and N-WASP signaling pathways,we knocked down DLC1 that resulted in higher Cdc42,p-JNK,NF-k B and N-WASP levels,suggesting that DLC1 inhibited cancer cell migration through JNK/NF-κB and N-WASP signaling regulated by Cdc42 after drugs treatment.The above results showed that resveratrol combined with dihydroartemisinin significantly up-regulated DLC1 with down-regulation expression of TCTP and Cdc42 and reduced the expression of p-JNK,NF-k B,MMP-2/9 and N-WASP to inhibit migration of cancer cells.Here,we report for the first time that resveratrol synergizes with dihydroartemisinin to inhibit proliferation and migration of cancer cells and explores the molecular mechanisms related cancer cell migration.Furthermore,we explore the interaction and regulation relationship between DLC1and TCTP,which provides new insights for the biological functions of DLC1 and TCTP.