Curcumin Suppresses Proliferation And Invasion In Human Gastric Cancer Cells By Down-regulation Of PAK1 Activity And Cyclin D1 Expression

IntroductionCancer chemoprevention involves the use of natural or synthetic chemicals to prevent the tumorigenesis of cancer.Curcumin has been widely studied for its anti-inflammatory,anti-angiogenic,antioxidant,wound healing and anti-cancer effects because of its medicinal properties in Indian and Chinese systems of medicine.As a natural anti-cancer agent,curcumin has been paid more attention because of its inhibition effect on tumor.Recently curcumin has been shown to be beneficial in all three stages of carcinogenesis,due to its inhibition of protein kinase C(PKC),EGFR tyrosine kinase,and IkappaB kinase.Subsequently,curcumin inhibits the activation of the transcription factor nuclear factor kappa B(NF-kappaB) and the expression of oncogenes including MAPKs,ERK,PI3K,Akt.Furthermore,the expression of cyclin D1,a proto-oncogene that is overexpressed in many cancers,is also down-regulated by curcumin.It is proposed that curcumin might suppress tumor promotion through blocking signal transduction pathways in the target cells.The p21-activated kinases(PAKs) are important mediators of Rho GTPase signaling,and are implicated in biological processes ranging from cytoskeletal dynamics and motility to tumorigenesis.Abnormal activation of PAK1 and thereafter high expression of cyclin D1 play an important role in tumorigenesis.In addition, PAK1 regulates Cyclin D1 transcription via an NF-kB-dependent pathway.Therefore, PAKs which belong to the protein kinases are important therapeutic targets of tumor and are considered highly druggable owing to their conserved ATP-bingding pocket. Nowadays many small molecule inhibitors were reported targeting PAKs,especially PAK1.However,most of these ATP-competitive inhibitors are not selective.Recently, it was demonstrated that ATP-competitive inhibitors such as imatinib(Gleevec) achieved unusually high kinase selectivity by binding a less conserved region of kinase (inactive conformation).It was also reported that IPA-3,a highly specific and potent non-ATP-competitive inhibitor,targeted the autoregulatory domain of PAK1.In a word,increasing data implicated PAK1 in tumor proliferation and metastasis, thus inhibitors of PAK1 have been suggested as a novel oncologic therapy.Here we report that curcumin targets PAK1 in gastric cancer cells,providing a novel mechanism for curcumin inhibitive effect on proliferation and invasion in human gastric cancer.Materials and Methods1.Curcumin inhibits the proliferation and invasion of gastric cancer cell lines.(1) We detected the protein expression of PAK1 in four cell lines—BGC823, SGC7901,MKN1 and MGC803 by Western Blot.(2) The effects of curcumin on the proliferation of cells were assayed by the MTT method.(3) The effects of curcumin on the invasion of cells were assayed by the Transwell method.2.Curcumin suppresses transition of gastric cancer cells from G1 to S phase and down-regulates the mRNA and protein expression of cyclin D1.(1) Cells were analyzed by flow cytometry(FCM) to detect the effects of curcumin on cell cycle in gastric cancer cells.(2) Detect the protein and RNA levels of cyclin D1 in gastric cancer cells when cells were treated by curcumin.3.Curcumin down-regulated the expression of HER2(Erb2) and the kinase activity of PAK1.(1) The effect of curcumin on the activity of PAK1 in gastric cancer cells was detected by kinase assay.(2) The effect of curcumin on the activity of PAK1 in vitro was detected by kinase assay.(3) The effect of curcumin on the protein expression of HER2 and cyclin D1 in gastric cancer cells was detected by Western Blot.(4) The possibility of curcumin as an ATP-competitive inhibitor was determined by molecular docking studies based on computer. 4.Curcumin inhibits the activity of PAK1 after silencing HER2 transiently in gastric cancer cells.(1) After cells were transfected with siRNA of HER2,the protein expression of HER2 was defined by western blot.(2)The effect of curcumin on PAK1 activity in gastric cancer cells was detected by kinase assay after silencing HER2.Results1.Curcumin inhibits the proliferation and invasion of gastric cancer cell lines.(1) We detected the protein expression of PAK1 in these four cell lines—BGC823, SGC7901,MKN1 and MGC803,and found that all of them had high levels of PAK1 protein expression,especially BGC823 with the highest protein level of PAK1.(2) Assayed by the MTT method,the inhibitory effect of curcumin on the proliferation of gastric cancer cells was dose-dependent.(3) Curcumin could inhibit the invasion of gastric cancer cells.2.Curcumin suppresses transition of gastric cancer cells from G1 to S phase and down-regulates the mRNA and protein expression of cyclin D1.(1) The cell cycle of gastric cancer cells was inhibited by curcumin,and curcumin suppressed transition of cells from G1 to S phase.(2) Curcumin down-regulated the protein and RNA levels in gastric cancer cells.3.Curcumin down-regulates the expression of HER2(Erb2) and the kinase activity of PAK1.(1) Curcumin inhibited the activity of PAK1 induced by EGF in gastric cancer cells,and curcumin could also inhibit the activity of PAK1 as an ATP-competitive inhibitor in vitro.(2) Curcumin down-regulated the protein expression of HER2 and cyclin D1.(3) The binding modes of curcumin in the ATP binding region of PAK1 through molecular docking studies confirmed its effect as an ATP-competitive inhibitor.4.Curcumin can still inhibit the activity of PAK1 induced by EGF after silencing HER2(erbB2/neu) transiently in gastric cancer cells. (1) The 48-hour time point of silencing was employed as the suitable silencing time.(2)After silencing HER2 transiently in gastric cancer cells,curcumin can still inhibit the activity of PAK1.ConclusionCurcumin was highlighted in recent research because of its anti-cancer effect.It suppressed the protein expression of HER2 and also inhibited the kinase activity of PAK1 mediated by EGF.The decrease of PAK1 kinase activity was due to both the inhibition of PAK1 upstream path by curcumin and its effect as an ATP-competitive inhibitor.The down-regulation of cyclin D1 expression by curcumin leaded to the suppression of transition of the cells from G1 to S.The results above contributed to the inhibition of proliferation and invasion of gastric cancer cell lines.In order to discover novel and potent agents for efficient cancer chemotherapy,it is necessary to design and synthesize a number of derivatives around curcumin for further research.