Regualations Of Cytoskeleton And CAMP Signal Pathway In Hela Cell Apoptosis Induced By Trichosanthin

Trichosanthin(TCS)possesses a broad spectrum of biological and pharmacological activities,including abortifacient,anti-HIV and anti-tumor activities through apoptosis pathway in some cells.Previous studies showed that TCS induced the apoptosis of JAR cells via ROS production,internal Ca²⁺elevation and caspase-3 activation.TCS suppressed mitogen-activated protein kinase(MAPK)and Bcl-2 signals leading to the replication inhibition in HSV-1 infected Vero cells.Furthermore, K562 cell apoptosis was induced by TCS via PKC inhibition.Additionally,the data obtained from our laboratory indicated that TCS caused Hela cells with markedly morphological changes,including microvilli disappearance or reduction,cell membrane vesiculation,cell shrinkage,condensation of chromosomes and apoptotic bodies with complete membrane.Further studies on the mechanisms of Hela cell apoptosis induced by TCS,showed that TCS-caused a S-phase arrest in cell cycle associated with a time-dependent increase of caspase-3,8, 9 activities.These preliminary results,therefore,indicate that Hela cells are sensitive to TCS.However,these data are insufficient for elucidating the cellular and molecular mechanisms occurring in Hela cell apoptosis induced by TCS.Objective To investigate the effects of TCS on cytoskeletal remodeling,the quantitative changes of actin and tubulin proteins,and subunit cytoskeletal mRNA levels in apoptotic Hela cell.Furthermore,we determined the specific intracellular signaling transduction pathways to explore the underlying cellular and molecular mechanisms of Hela cell apoptosis induced by TCS. Methods The antiproliferative effect of TCS on HeLa cells was measured with CCK-8.Cytoskeletal remodeling,the quantitative changes of actin and tubulin proteins, and subunit cytoskeletal mRNA levels in apoptotic Hela cell were examined by confocal immunofluorescence microscopy observations,western blot and quantitative real-time PCR assays.Electron microscopy and FCM analysis was employed to observe the apoptosis of Hela cells.Fura-4 fluorescence probe was used to check the cytosolic calcium level.Antisense knockdown of CREB gene expression,and blockade the binding of CREB to the Bcl-2 CRE were carded out to examine whether cAMP response element-binding(CREB)was involved in TCS-mediated Bcl-2 expression.Results(1)TCS induced the execution phase of cell apoptosis was a highly coordinated process of cellular reorganization,depolymerized microfilaments accumulated in the coarsened cytoplasm and apoptotic bodies,accompanied by the formation of a ring microtubule structure beneath the plasma membrane.However,no quantitative changes were observed on actin and tubulin proteins.Furthermore, apoptosis occurred by a suppression of actin and tubulin subunit gene expression.(2) Sequestrating the TCS-increased[Ca²⁺]c with EGTA caused less protective of cell viability,which was related to the absence of apoptotic microtubule structure altered plasma membrane permeability.(3)TCS initiated an influx of extracellular Ca²⁺,and it was required for the suppression of adenyl cyclase(AC)activity and intracellular cAMP formation.Furthermore,this inhibition was abolished by preincubation with an activator of PKC rather than PKA.The expression of p-CREB protein expression inhibited by TCS was reversed by cAMP agonists.Nevertheless,blocking the ERK1/2 and p38 MAPK pathway in TCS/FSK treated cells significantly prevented the increase of cell viability.Furthermore,the phosphorylated ERK1/2 and p38 MAPK protein levels were decreased by the inhibitors of PKC.(4)CREB antisense effectively reduced CREB expression levels,and TCS was able to reduce Bcl-2 level in cells treated with CREB sense.The importance of activating the CREB pathway in Hela cell apoptosis induced by TCS was further demonstrated by a CRE decoy oligonucleotide to block the binding of CREB to the Bcl-2 CRE.As expected,the CRE oligonucleotide treatment resulted in the constant expression of Bcl-2 protein.In contrast,the treatment of CRE oligonucleotide failed to affect the phosphorylated CREB protein level.Conclusion These data suggested that(1)Hela cell apoptosis induced by TCS was accompanied by the cytoskeletal remodeling and suppression of cytoskeletal gene expression,but not by quantitative changes of proteins.(2)TCS-increased intracellular calcium exerted the pivotal role on the formation of apoptotic microtubule ring structure,which participated in the regulating of plasma membrane permeability.(3) TCS mediated Hela cell apoptosis via the regulation of cAMP/PKC/MAPK pathway. (4)TCS suppressed Bcl-2 expression through the inhibition of phosphorylated CREB. Therefore,these results provide deep insights for the understanding of the cellular and molecular mechanisms underlying TCS-caused cell apoptosis.