PKD Sensitizes HepG2 Cells To Radiation Via The Proteasome Pathway

BacgroundBioinformatics analysis showed that a sequence in Hepatitis B virus x protein (HBx) is highly homologous to the "Kunitz domain," a characteristic domain of Kunitz-type serine protease inhibitors which is essential to their inhibitory function.HBx resembles a serine protease inhibitor or its analogue,and bring about transactivation by activating certain transcriptional factors through proteolytic cleavage alteration.Proteasomes are protein complexes inside all eukaryotes and archaea,as well as in some bacteria.In eukaryotes,they are located in the nucleus and the cytoplasm.Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins.The proteasomal degradation pathway is essential for many cellular processes,including the cell cycle,the regulation of gene expression,and responses to oxidative stress.Cell cycle progression is controlled by ordered action of cyclin-dependent kinases(CDKs),activated by specific cyclins that demarcate phases of the cell cycle.Mitotic cyclins have one of the shortest life spans of all intracellular proteins.After a CDK-cyclin complex has performed its function,the associated cyclin is polyubiquitinated and destroyed by the proteasome,which provides directionality for the cell cycle.Proteasome inhibitors have effective anti-tumor activity in cell culture,inducing apoptosis by disrupting the regulated degradation of pro-growth cell cycle proteins.The ability of proteasome inhibitors to induce apoptosis in rapidly dividing cells has been exploited in several recently developed agents for clinical application.In the present study,we investigated whether a synthetic peptide,a peptide from Kunitz domain(PKD),with the same sequence as Kunitz domain of HBx could affect the cell cycle,apoptosis and radiosensitization in HepG2 cells via suppression the activities of proteasome and regulation the expression of valosin-containing protein(VCP),cyclin-dependent kinase,cyclin and associated proteins which involves in the proteasome protein degradation pathway.MethodsProtein analysis software of Vector NTI 6.0 was used to analyze the sequence homogeneity of HBx.The peptide sequence was designed and synthesized by the solid synthetic peptide approach.PKD was identified by high performance liquid chromatography and mass spectrometry.Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys as a fully conserved nine amino acid residues within the carboxyl terminus of all HBx,PKD was chemically synthesized and used to quest its effects on HepG2 cells.MTT assays were used to determine the effects of PKD on HepG2 cell growth.ATPase activity of proteasomes was measured using a colorimetric assay. Peptidase activities of proteasomes such as chymotrypsin-like,trypsin-like,and peptidyl-glutamyl peptide-hydrolyzing-like peptide hydrolase activity were analyzed with various peptidase-specific fluorogenic peptide substrates (LLE-NA,LLVY-AMC and LSTR-AMC).The sequence homogeneity of VCP was analyzed by AlignX software of Vector NTI 6.0.The peptide sequence was designed and synthesized by the solid synthetic peptide approach.The hydrophilicity plot,flexible regions,antigenic index and surface probability plot of VCP were analyzed by DNAStar7 software. According to the homogeneity and antigenicity,a fusion peptide harboring epitopic sequence with 19 amino acid residues of GRLDQLIYIPLEICQRACK from human VCP was designed and chemically synthesized.The VCP fusion peptide was conjugated with KLH.The BALB/c mice were immunized by VCP-KLH to generate VCP polyclonal antibody.The titer was detected by ELISA. Mouse anti-VCP antibody was developed for Western blotting to detect the VCP expression.The expression of P27,Cyclin E,Bax,Bcl-2,Fas and Caspase-8 were detected by immunohistochemical staining.Morphological changes of apoptosis were examinated with FITC-labbled antibody of TFAR19 by fluorescence microscope.Flow cytometry was used to determinate cell cycle phase and apoptosis.Evaluation the radiosensitization of PKD in HepG2 cells was achieved by colony formation assay.ResultsThe purity of PKD was higher more than 97%analysized by high performance liquid chromatography(HPLC).The synthetic PKD was identified the same sequence with the designed by mass spectrometry.As shown in MTT assay of the effects with different concerntration 0mmol/L, 5mmol/L,10mmol/L and 15mmol/L on HepG2,cell viability decreased in a PKD-dose-dependent manner.While some inhibition was observed in different concerntration of PKD,15mmol/L PKD demonstrated an apparent cytotoxic effect.Chymotrypsin-like,peptidyl-glutamyl peptide-hydrolyzing-like,trypsin-like hydrolase activity of proteasomes were analyzed with fluorogenic peptide substrates of LLE-NA,LLVY-AMC.Chymotryptic activity of proteasomes in HepG2 cells was significantly inhibited by 10mmol/L PKD(p＜0.05).Tryptic activity and peptidylglutamyl peptide hydrolase activity of proteasomes were less inhibited by PKD than chymotryptic activity.ATPase activity of proteasomes was assayed in a time course manner for two and half hours.The proteasome complexes were immunoprecipitated from HepG2 cell lysates using anti-proteasome antibody and protein-G agarose beads.The cells were treated with or without 10mmol/L PKD.The result showed that proteasome activities in the PKD-treated samples were lower than in the sample without treatment.It indicated that the treatment with PKD suppressed the ATPase activities of immunoprecipitated proteasome complexes.The antibody tilter for VCP fusion peptide was more than 1:10000 analysized by ELISA.Western blotting showed that the expression of VCP decreased when cells were treated with 10mmol/L PKD for 36 hours.The intensities of the VCP band(97kDa) were significantly decreased in the cells with PKD treatment compared to the control cells(without treatment).The result indicates that PKD inhibits the expression of VCP in HepG2 cells.The result indicates that PKD inhibits the expression of VCP in HepG2 cells.P27,Cyclin E,Bax,Bcl-2,Fas and Caspase-8 in HepG2 cells were detected by immunohistochemistry.Brown-colored nuclear staining were found in HepG2 cells treated with 10mmol/L PKD,while those have no color without treatment of PKD.Most of HepG2 cells treated with 10mmol/L PKD showed negative immunostaining of Cyclin E.The untreated HepG2 cells revealed yellow-brown color for Cyclin E in the nuclei or cytoplasm.Bax immunostained cells showed a brownish-yellow color in the cytoplasm in HepG2 cells treated with 10mmol/L PKD.The untreated HepG2 cells were negative immunostaining.HepG2 cells without PKD treatment were positive staining for Bcl-2 with dark brown granules in the cytoplasm,but no immunohistochemistry signals appear in HepG2 cells treated with 10mmol/L PKD.PKD could suppress the over expression of Bcl-2.Positive immunostaining was not observed in untreated HepG2 cells.Fas protein was appeared brownish-yellow color in the cytoplasm and membrane of HepG2 cells treated with 10mmol/L PKD.PKD could enhance the expression of Fas.Comparison to the untreated HepG2 cells,10mmol/L PKD increased the expression of Caspase-8.Immunohistochemistry demonstrated dark brown color localized on the nuclei and cytoplasm.Immunohistochemical analys revealed that P27,Bax,Fas and Caspase-8 in HepG2 cells treated with 10mmol/L PKD were expressed higher than in HepG2 cells without PKD treatment.The expression of both cyclin E and Bcl-2 was suppressed by the effect of PKD.Our data of flow cytometry analysis showed that HepG2 cells accumulate largely in the G₀-G₁ phase after being incubated with PKD for 36 hours,while untreated control cells mainly enter into S phase.In addition,the PKD treatment significantly induces cells to proceed to apoptosis. HepG2 cells treated by PKD exhibited characteristic morphology of apoptosis as using FITC-labeled monoclonal antibody to assesse a novel apoptosis-related protein TFAR19.Combined with radiation,5mmol/L PKD could enhance radiosensitivity in HepG2 cells by comparation to the control cells without PKD treatment or a single radiation shot.ConclusionPKD peptide derived from Kunitz domain of HBx protein may apply to be a potential proteasome inhibitor.PKD induces cell growth arrest,apoptosis and enhances radiosensitivity in HepG2 cells via down-regulating the expressions of p97,cyclin E and Bcl-2,and up-regulating the expressions of P27,Bax,Fas and Caspase-8.