HSPA9 Inhibits Apoptin Induced Apoptosis In HepG2 Cell Line

Apoptin is a small viral protein that was originally identified in chicken anemia virus.Various studies have shown that Apoptin specifically induces apoptosis in a broad range ofdifferent human cancer cell lines derived from various tumor types but not in their normalcounterpart.Furthermore,apoptosis induced by Apoptin is p53-independent.The molecularmechanism of Apoptin is largely unknown.Previous studies have shown that Apoptininduced tumor cell apoptosis is closely related to tumor-specific phosphorylation andcellular localization.Several Apoptin interacting partners were identified,such as N-mycinteracting protein(NMI),promyelocytic leukemia protein(PML),anaphase promotingcomplex/cyclosome subunit 1(APC/C),death effector domain associated factor(DEDAF),Acid ceramidase,CRM1,imprtin-β1,and Ppil3 et al.These proteins playcrucial roles for Apoptin's selective toxicity.Maddika et al recently reported that,throughits proline-rich sequence(aa 81-86),apoptin interacts with the SH3 domain of the p85regulatory subunit of phosphoinositide 3-kinase(PI3K)and activates PI3K.PI3Kdownstream effector kinase Aktl was also activated and interacted with Apoptin,thenApoptin and activated Akt translocated to the Nucleus.Nuclear Akt activates CDK2 byboth direct and indirect mechanisms,CyclinA-CDK2 complex directly phosphorylatesapoptin at Thr-108 and contributes to the regulation of its subcellular.Identification ofthese Apoptin-interacting proteins extends the knowledge of Apoptin tumor-specifictoxicity,and indicates that searching and identification of Apoptin interacting partners isvery important to further elucidating mechanism of Apoptin tumor-specific toxicity.In an attempt to elucidate the molecular mechanism of apoptosis induced by Apoptin.In this research,prokaryotic native His-Apoptin fusion protein purified with Ni-NTAaffinity chromotography served as a bait for capturing the Apoptin-interacting proteins of inboth tumor cell line(HepG2)and normal cell line(L-02)by employing the pull down assay. The captured proteins were separated by two dimensional gel electrophoresis and analyzedby Imagemaster 2D Platinum 6.0 software.The protein spots were cut from the gel,andthen identified by Mass Spectrum.After that the proteomic information ofApoptin-interacting proteins in both tumor cells and normal cells were obtained.HSPA9 isone of proteins captured from HepG2 cells by pull-down assay.HSPA9(Mortalin/mthsp70/Grp75/PBP74/mot-2)is a heat un-inducible member of hsp70 family ofproteins.It is a 74-kDa protein and has been localized to mitochondria,endoplasmicreticulum,plasma membrane and cytoplasmic vesicles.It is an essential protein thatperforms various functions related to proliferation,functional maintenance and stressresponse.Overexpression of HSPA9 may impart growth or proliferative advantages,prolonging cellular lifespan.Expression study of HSPA9 in tumor cells have revealed thatit is frequently upregulated in tumors.In specially,HSPA9 is the major mitochondrialprotein and it plays a central role in the elaborate translocation system for efficient importand export of proteins.Its role in cell viability and mitochondrial biogenesis wasunderscored by experimental data including the yeast cells knocked out for HSPA9homologue(Sscl)were lethal and the loss of function mutations of HSPA9 causedaggregation of yeast mitochondria..HSPA9 acts as an antiapoptosis protein in tumor cells.So we selected HSPA9 as a target molecule to explore the relationship betweenHSPA9 and Apoptin specific tumor toxicity.The interaction of HSPA9 with Apoptin inHepG2 cells was further verified by co-immunoprecipitation.After that we forcedexpression or knockdown HSPA9 in HepG2 cells to test the effect of HSPA9 on Apoptintumor-specific toxicity.Our data indicated over expression of HSPA9 in HepG2 cellsresulted in partially cytoplasmic distribution of Apoptin and significantly inhibitedapoptosis of HepG2 cells.In contrast,siRNA was applied to suppress of HSPA9 expression,we observed that the apoptosis rate of HepG2 cells induced by Apoptin was elevated.Up to now,the mechanism of Apoptin specific toxicity to tumor is still unclear.MarekLos research group reported that Apoptin is independent of death receptors but involves the loss of mitochondfial membrane potential and the release of mitochondrial cell-deathmediators by a Nur77-dependent pathway.Apoptin nucleus localization and thecytoplasmic translocation of Nur77 are crucial for the toxicity of Apoptin.Our dataindicated,over expression of HSPA9 lead to a part of Apoptin cytoplasmic distribution,meanwhile Apoptin induced apoptosis rate was dropped;whereas down-regulation ofHSPA9 expression in HepG2 cells,Apoptin's toxicity to HepG2 cells was enhanced.Itsuggested that HSPA9 inhibit cancer-specific toxicity of Apoptin through retention Apoptinin cytoplasmic.We speculated that over expression of HSPA9 in HepG2 cells may through two waysinhibit cancer-specific toxicity of Apoptin,on one hand,the more HSPA9 presented incytoplasmic the more Apoptin was stagnated in cytoplasmic,as a result less Nur77 wastanslocated to cytoplasmic.On the other hand,over expression of HSPA9 protectedmitoehondria from the stimulation of proapoptosis factors.Down-regulation of HSPA9levels attenuated mitochondrial stability.It indirectly enhanced activities of Apoptin.Butfurther study is needed.In conclusion,our data suggested that HSPA9 is one of Apoptin-interaction proteins.Over expression of HSPA9 prevented Apoptin from entering the nucleus and carrying outits apoptotic activity,while down-regulation of HSPA9 expression elevated thecancer-specific toxicity of Apoptin significantly.