Transgelin Induces Apoptosis And Senescence Of Prostate Cancer Cell Line LNCaP

Part I Transgelin impacts turnover of actinActin turnover is influenced by various actin-binding proteins (ABPs), and it confers crucial effects on cellular ROS (Reactive oxygen species) and apoptosis. Transgelin is the mammalian homologue of Scpl, which can cause inappropriate actin clumping in yeast. To address transgelin affects turnover of actin and cellular ROS in mammalian cell, human prostate cancer LNCaP cells were transfected with pCDNA3.1-transgelin or vector, followed by detecting turnover of F-actin at 24 h after transfection. Simultaneously, cellular ROS was determined by parallel experiment. We have found that ectopic expression of transgelin leads to aggregation of F-actin and increase of cellular ROS. Conversely, knocking down endogenous transgelin of U2OS cells resulted in increase of F-actin turnover and decrease of cellular ROS. To investigate the effects of transgelin on DNA damage response pathway following the induction of ROS,8-OH-dG, phospho-ATM and yH2AX immunofluorescence stained were performed. We found ectopic expression of transgelin caused oxidative stress, characterized by DNA base damage and double strand break. Western blotting indicates activation of DNA damage response, especially ATM (Ataxia-Telangiectasia Mutated)- Chk2 (checkpoint kinase2)-p53 pathway is caused by introduction of ectopic transgelin through elevation of ROS. Finally, immunofluorescence staing for heterochromatin protein 1(HP1), histochemical staing for P-galactosidase (β-gal) and western blotting were carried out to address the induction of chromatin modeling and cell senescence were caused by ectopic expression of transgelin. Our findings reveal that transgelin can lead to decrease of F-actin turnover, and induce cellular ROS, thereby activating ATM-Chk2-p53 pathway and inducing cell senescence.Part II Transgelin interacts with p53Transgelin, known as an ARA54-associated AR inhibitor, can suppress ARA54-enhaced AR transaction in LNCaP cells. Part I experiment suggests that transgelin activates DNA damage response pathway by induction of cellular ROS. In addition to these effects, we aimed to elucidate the pro-apoptotic effects of the protein on LNCaP and its underlying mechanisms, especially the interaction between transgelin and p53. Using western blotting of p53 and double immunofluorescence staining of p53 with transgelin, we have shown that transfection of transgelin resulted in increasing cytoplasmic translocation of p53 and upregulation of p53 expression. We also found an interaction between transgelin and p53 in vivo by mammalian two-hybrid. To futher confirm the interaction, we found both ectopic and endogenous transgelin coimmunoprecipitated with p53 by coimmunoprecipitation assay.Par tⅢEffects of transgelin on p53 downstream target and intrinsic apoptosis pathway The mitochondrial membrane constitutes the battleground on which pro-and anti-apoptotic factors induce or prevent apoptosis. Cytosolic p53 has function to activate intrinsic apoptosis by induction of mitochondrial outer membrane permeabilization (MOMP). PUMA (p53 upregulated modulator of apoptosis), as p53 downstream target, enhances cytosolic p53 function. Using western blotting, we found that ectopic expression of transgelin resulted in downregulation of Bcl-2, and up-regulation of PUMA and Bax, therefore triggering apoptosis. Additionally, we found the effect was dependent of functional p53 by introduction of transgelin to HCT116 p53-/- and p53+/+ cell lines. Futher, Cell counting, flow cytometric analysis and terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assays were applied to measure the pro-apoptotic effect of transgelin. These results suggest the activation of mitochondria-associated apoptosis pathway of LNCaP cells transfected with transgelin. These results are indicative of p53-mediated mitochondria-associated apoptotic effects of transgelin on LNCaP cells in addition to its known suppressive effects on AR pathway.