PKD Sensitizes HepG2 Cells to Radiation via the Proteasome Pathway
|Keywords||Hepatitis B virus X protein Kunitz domain Synthetic peptide Proteasome pathway Cell cycle Apoptosis Radiosensitivity HepG2 cells|
Background: bioinformatics analysis showed that the hepatitis B virus X protein (HBx) there are highly homologous to the Kunitz-type serine protease inhibitor \HBx and serine protease inhibitor competitive binding hepatocyte source of serine proteases, disrupt the intracellular transcription factor proteolytic pathways, while exercising their trans-activation functions. The proteasome is ubiquitous in eukaryotes and archaea, a class of protein complexes in prokaryotes. In eukaryotes, the proteasome is located in the nucleus and cytoplasm. Proteasome cell to the main mechanism of regulation of specific proteins and remove misfolded proteins. Proteasome degradation pathway for many cellular processes, including cell cycle regulation of gene expression, oxidative stress, are essential. Cell cycle progression by a series of cell cycle-dependent kinase (CDK) to regulate the CDK is activated by the cell cycle protein (cyclin). Mitotic cell cycle proteins in the protein within the cells of all known life shortest. After its function is exercised in the CDK-cyclin complexes, complexes of cyclin is polyubiquitylation and proteasomal degradation, thus ensuring the normal operation of the cell cycle. Intracellular signals are able to induce apoptosis or programmed cell death, a cell's internal component occurs deconstruction, this process is completed by a specific protease caspase enzyme (caspase), but at the same time the proteasome in the cell exercise a variety of important role in the process of apoptosis. Can affect different types of cell apoptosis induced proteasome inhibition, can promote cell apoptosis in most studies of cells, inhibition of the proteasome. Proteasome inhibitor having effective anti-tumor activity of cultured cells, promoting growth and cell cycle protein to induce apoptosis of tumor cells is regulated by degradation. Therefore, some of the protease inhibitors have been used clinically in cancer therapy has been developed. Objective: In this study, synthesis and HBx Kunitz domain sequence identical peptides (PKD), to explore whether the regulation of proteasome degradation pathway related VCP, cyclins and cell cycle by inhibiting the activity of the proteasome-dependent protein kinase protein expression, and thus HepG2 cell cycle, apoptosis and radiation sensitivity impact. Research methods: analysis, design, synthetic PKD sequence homology of HBx protein analysis software Vector NTI 6.0. The use of solid-phase peptide synthesis technology chemical synthesis and HBx carboxy-terminal nine amino acid sequence exactly peptide Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys, to explore the role of HepG2 cells. By high pressure liquid chromatography (HPLC) and mass spectrometry were identified after purification PKD. PKD HepG2 cells; the detected colorimetric analysis proteasome ATP activity; by analyzing different peptidases specific fluorescent peptide substrate lle-NA LLVY-AMC and LSTR-AMC hydrolysis by MTT method The analysis class proteasome chymotrypsin, tryptase and peptide Valley aminoacyl peptide hydrolase enzyme activity; using AlignX Software analysis in GenBank VCP homology, using the DNAStar 7 software to analyze the characteristics of VCP sequence, based on the hydrophilic structure, flexibility, area, antigenic index and surface probability structure parameters analysis, choose the the human VCP 637 to 647 and 689 to 696 two sequences, designed for the 19 amino acid sequence of the fusion peptide prepared VCP antibodies GRLDQLIYIPLEICQRACK. Using solid phase synthesis of peptide technology VCP fusion peptide with the carrier protein KLH coupled to form VCP-KLH. Routine immunization program with VCP-KLH immunized BALB / c mice, preparation of VCP polyclonal antibody titers detected by ELISA technique. Prepared by the experimental murine anti VCP antibody, Western blotting, and detection of the expression of the VCP. P27, Cyclin E, Bax, Bcl-2, Fas and Caspase-8 expression in HepG2 cells was detected by immunohistochemistry. Apoptosis related protein TFAR19 of using fluorescein (FITC) labeled monoclonal antibody, HepG2 cells marked morphological changes observed PKD induced apoptosis of HepG2 cells. Flow cytometry to detect changes in the cell cycle and apoptosis. The experimental observation PKD radiosensitivity of HepG2 cells formed by cell irradiation and cloning. Results: After synthesis PKD peptide Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys purified by HPLC purity> 97%, the correct amino acid composition and sequence of the MS identification synthesis of PKD. MTT assay 0 mmol / L, 5 mmol / L, 10 mmol / L and 15 mmol / L of PKD HepG2 cell growth impact, results showed that HepG2 cells vitality with the PKD dose increase showing decreased, 15mmol / L of PKD have significant cytotoxicity. Analysis of different peptidase specific fluorescent peptide substrate lle-NA LLVY-AMC and LSTR-AMC hydrolysis was found in HepG2 cells, 10mmol / L of PKD can be significantly suppressed the chymotrypsin-like activity of the proteasome (p <0.05), but compared to the trypsin-like peptide - Valley leucyl peptide-like activity was compared with chymotrypsin-like activity of inhibition weak. In addition, further observe changes over time, PKD chymotrypsin-like activity of the proteasome the reaction kinetics change. PKD is not the role of the proteasome chymotrypsin-like activity, the reaction tends to peak in about 12 minutes and getting flattened. Of 10mmol / L PKD is chymotrypsin-like activity of the proteasome has an inhibitory state. Proteasome ATP activity the 2.5h time limit, using anti-proteasome antibody and conjugated to protein G agarose beads for the composite body of the proteasome coimmunoprecipitation. 10mmol / L PKD treated HepG2 cells and compared without PKD processing, PKD significantly inhibit proteasome complex ATP activity. VCP fusion peptide antisera were measured by ELISA titer of 1:10000. 10mmol / L the PKD role HepG2 cells 36h after collection of cell lysis, the extracted proteins by Western blot analysis of the expression of the VCP. For the same protein concentration in the sample, compared to the with PKD processing HepG2 cells, Western blotting of HepG2 cells PKD processing the density of the visible band (97kDa) is significantly reduced, indicating that PKD than significantly inhibited the expression of the VCP. Immunohistochemistry assay HepG2 cells P27, Cyclin E, Bax, Bcl-2, Fas, and Caspase-8 expression by 10mmol / L of PKD role in HepG2 human hepatoma cell line of P27 protein levels were significantly liter , with the blank control group, there were significant sex change. PKD role HepG2 cells p27 to the nucleus and perinuclear brown. PKD is not the role of HepG2 cells staining. The PKD raised P27 expression in HepG2 cells. Most of the PKD synthetic peptide treated HepG2 cells Cyclin E staining was negative, its nucleus is colored blue cytoplasm and cell membrane. Without the PKD intervention of HepG2 Cyclin E protein expression was mainly distributed in the perinuclear cytoplasm and nucleus, were uniformly brownish yellow fine granular. PKD inhibit HepG2 cell cycle protein Cyclin E expression. Of 10mmol / L PKD can significantly promote the expression of Bax in HepG2 cells, Bax protein dark yellow staining, diffuse or localized distributed in the cytoplasm and the nucleus blue dye. Blank control HepG2 cells were negative. PKD to promote Bax protein expression in HepG2 cells. HepG2 cells are the expression of Bcl-2 positive staining cells, Bcl-2 brown granular, mainly localized in the cytoplasm. Of 10mmol / L PKD can completely inhibit the expression of the Bcl-2, no positive staining of the PKD HepG2 cells after treatment. The PKD inhibit HepG2 cells, Bcl-2 protein over-expression. HepG2 cells does not appear obvious positive staining of 10mmol / L the PKD role HepG2 cells after positive expression of Fas reactants brown particles segment in the cytoplasm and membrane. PKD improve the Fas protein expression in HepG2 cells. HepG2 cells compared with the blank control group, 10 mmol / L PKD can significantly increase the expression of Caspase-8 in HepG2 cells, caspase-8 protein dark yellow staining dispersed in the cytoplasm and nucleus. PKD Caspase-8 expression in HepG2 cells. This study demonstrated that the expression of P27, Bax, Fas and Caspase-8 in 10mmol / L PKD HepG2 cells was significantly higher, but cyclin E reduce the expression of Bcl-2. 10mmol / L PKD role HepG2 cells 36 hours which may render the cells remain in the G 0 -G 1 on and produce significant apoptosis in untreated cells entry into S phase. Using fluorescein (FITC) labeled monoclonal antibodies as probes, the tracer a new apoptosis-related molecules of TFAR19 the PKD role of HepG2 cells showed morphological changes typical apoptosis. 5mmol / L PKD cells of the intervention and the control group received different doses of irradiation, clone formation assay by Sigma Plot11 software to multiple target click model SF = 1 - (1-e -D/D0 sup >) n sup> fitting cell survival curves, and calculation of cell radiosensitivity parameters. The 5mmol/LPKD increased radiosensitivity of HepG2 cells 30% (SER D0 = 1.34, SER Dq = 1.24, the SER SF2 = 1.29). Conclusion: The results of this study indicate that PKD is a potential proteasome inhibitor derived from the hepatitis B virus x protein Kunitz domain, by inhibiting the chymotrypsin-like activity of the proteasome and reduce the ATP activity, lowered p97, cyclin E with the expression of Bcl-2, raised the P27, Bax, Fas and Caspase-8 expression caused growth arrest and promote apoptosis of HepG2 cells, increased radiosensitivity of HepG2 cells.