Molecular Mechanisms And Signaling Transduction Pathways Of Cell Cycle Arrest And Apoptosis Induced By Tetrandrine In Human Cancer Cells

Tetrandrine(TET),a bisbenylisoquinoline alkaloid isolated from the root of a Chinese herbal Stephenia tetrandra,possesses a broad pharmacological profile including antiheumatic,anti-inflammation,antihypertensive activities.Previous studies have shown that TET had the anticancer action and could enhance the chemosensitivity or radiosensitivity of cancer cell markedly and could induce cell cycle arrest and apoptosis.However,the detailed molecular mechanisms of TET-induced cell cycle arrest and apoptosis have not been defined.In the present study,we elucidate the mechanisms and signaling pathways of TET-induced cell cycle arrest and apoptosis in human cancer cells and provide further evidence to support the use of this drag in clinical cancer therapy.1.TET inhibits cell growth and induces G1 arrest and apoptosis in human colon cancer cells(1) HT-29 cells were treated with various concentrations of TET for various times,. Growth curve and cell viability detection showed that TET inhibited cell growth in a concentration-and time-dependent manner.HCT116 cells were treated with various concentrations of TET for 18h.Growth curve showed that TET also inhibited HCT116 cell growth in a concentration-dependent manner.(2) After 12h incubation with 30μM of TET,the treated HT-29 cells exhibited morphological changes,including that cells became small and round,and some of them fell off from the wall.Ultrastructural feature of apoptosis such as karyopyknosis and apoptotic body were found after 24h.Similar morphological changes were found in TET-treated HCT116 cells.(3) Cell cycle distribution analysis showed that TET(10-30μM) induced G1 arrest in HT-29 and HCT116 cells.Sub-G1 peak(or apoptotic peak) was induced after 30μM TET treatment for 24h. 2.Effect of TET on PI3K/AKT/GSK3βpathway(1).Western blot analysis showed that TET-induced G1 arrest and apoptosis was related with TET-mediated changes of G1 arrest and apoptosis regulatory proteins. TET reduced the levels of AKT,phosphorylated AKT(Ser473),phosphorylated GSK3β(Ser9),cyclin D1,procaspase3 and induced up-regulation of GSK3β, phosphorylated cyclin Dl(Thr286),and cleavage of caspase 3 and PARP, indicating that TET activated PI3K/AKT/GSK3βsignal pathway,which was associated with TET-induced G1 arrest and apoptosis.(2).TET induced up-regulation of p27kip1 in a concentration-and time-dependent manner in HT-29 cells,which might promote G1 arrest and apoptosis induced by TET.(3).Since TET induced down-regulation of cyclin D1,we further explored the mechanisim of down-regulation of cyclin D1.We found the reduction of cyclin D1 protein levels induced by TET was reversed by protease inhibitor MG132. Thus,the down-regulation of cyclinD1 induced by TET may be due to accelerated proteolysis of cyclin D1.(4).It is reported that GSK3βis translocated into the nucleus when activated.To determine whether TET affected the subcellular localization of GSK3β,we performed immunofluorescent staining.In control cells,GSK3βwas expressed predominantly in the cytoplasm.However,GSK3βaccumulated in the nucleus aider 30μM TET treatment for 12 h.These results suggested that TET induced GSK3βnuclear translocation.(5).To explore the relationship between AKT inhibition and GSK3βactivation as well as the effect of AKT inhibition and GSK3βactivation on cell cycle and apoptosis regulatory proteins,we used specific PI3K/AKT inhibitors(wortmannin and LY294002) and wild-type GSK3βplasmid.We observed the changes of cell cycle and apoptosis regulatory proteins after the treatments.The results showed that AKT inhibitors reduced the phosphorylation of GSK3βat Serg,indicating that GSK3βwas a downstream target of AKT.In addition,wortmannin also caused increased phosphorylation of cyclin D1(Thr286),down-regulation of cyclin D1 and cleavage ofcaspase 3 and PARP.Similar to TET,the transfection of HT-29 cells with wild-type GSK3βalso induced enhanced phosphorylation of cyclinD1(Thr286),down-regulation of cyclin D1 and cleavage of PARP, indicating that GSK3βactivation via inhibiton of AKT was involved in TET-mediated changes of G1 arrest and apoptosis regulatory proteins.(6) To determine the contribution of GSK3βactivation to G1 arrest and apoptosis induced by TET,we used GSK3βinhibitors(LiCl and SB216763) and RNAi agent(GSK3βsiRNA).All of LiCl,SB216763 and GSK3βsiRNA attenuated TET-mediated down-regulation of cyclin D1,activation of caspase3 and cleavage of PARP.The role of GSK3βin TET-induced G1 arrest and apoptosis was further confirmed by cell cycle analysis and apoptosis detection.TET significantly increased the G1 phase population and reduced the S phase population. Meanwhile,small population of apoptotic and necrotic cells was accumulated in sub-G1 phase.However,the G1 phase population was significantly decreased in the presence of LiCl,SB216763 and GSK3βsiRNA,respectively.Annexin-V/PI staining and TUNEL fluorescence staining showed that GSK3βsiRNA reversed TET-induced apoptosis.These results suggested that inhibition of GSK3βeffectively attenuated G1 arrest and apoptosis induced by TET.Furthermore,the the percentage of cells in G1 phase or sub-G1 phase was significantly increased in wild-type GSK3βtransfected cells,implicating that GSK3βactivation is sufficient to induce G1 arrest and apoptosis.(7) To assess the role of downstream caspase3 activation mediated by GSK3βin TET-induced apoptosis,we used selective caspase 3 inhibitor Ac-DEVD-CHO. Western blot analysis showed that TET-induced cleavage of caspase 3 and PARP was inhibited by the pretreatment of Ac-DEVD-CHO.Moreover, Ac-DEVD-CHO blocked the TET-induced apoptosis in HT-29 cells,suggesting that activation of caspase 3 is essential for TET-induced apoptosis.3.Effect of TET on MAPK pathwayTo examine the relationship between TET and MAPK pathway,we detected the changes of MAPK after 30μM TET treatment for various times.Western blot showed that the phosphorylations of JNK and p38 were increased,but the phosphorylations of ERK1/2 were decreased in HT-29 cells.Similar results were also observed in TET-treated HCT 116 ceils.These results implicated that TET activated JNK and p38 and inhibited ERK,which might promote G1 arrest and apoptosis.4.Effect of TET on other proteinsWe also investigated the effects of TET on other proteins,such as EGFR,NF-KB, mdm2 and survivin.We found TET induced down-regulation of these proteins.5.Effect of TET on proteasome pathwayTo study the relationship between TET and proteasome pathway,we used proteasome inhibitor MG132.Pretreament of MG132 could inhibit G1 arrest induced by TET treatment alone.RT-PCR showed that TET increased the mRNA levels of E1, E2 and E3.Conclusions:Taken together,our observations highlight the presence of multiple signaling pathways to induce G1 arrest and apoptosis after treatment with TET.(1) PI3K/AKT/ GSK3βpathway is related with TET-induced G1 arrest and apoptosis,and GSK3βactivation play an important role in these processes;(2) TET-induced G1 arrest is associated with increased phosphorylation and ubiquitin-dependent degradation of cyclinD1;(3) TET-induced apoptosis is associated with cleaved activation of caspase3 and its substrate PARP;(4) TET induces nuclear transloeation of GSK3βand up-regulation of p27kipl;(5) TET-induced G1 arrest and apoptosis may be associated with down-regulation of ERK1/2 phosphorylation,NF-κB and mdm2.