HTPP-MDC inhibits replication and subcellular localization of hepatitis B virus in HepG2.2.15 cells

AIM：To investigate the antiviral effect of hepatocyte-targeting and cell-penetrating peptide and Musca domestica cecropin (HTPP-MDC) fusion polypeptide against hepatitis B virus (HBV) in vitro, and to observe the penetrating ability of HTPP-MDC in hepatocytes. METHODS： HepG2.2.15 cells and Chang liver cells were co-cultured in vitro with HTPP-MDC at different concentrations. MTT assay was used to detect the cytotoxicity of HTPP-MDC in vitro. The levels of HBsAg, HBeAg and HBV DNA in HepG2.2.15 cell culture supernatants were measured by ELISA and real-time fluorescence quantitative PCR. The penetrating ability of HTPP-MDC was detected by laser confocal microscopy. RESULTS：The levels of HBsAg, HBeAg and HBV DNA in the supernatants of HepG2.2.15 cells treated with HTPP-MDC were remarkably reduced. The inhibitory effect of HTPP-MDC depended on the dose and action time of the drug. FITC-labeled HTPP-MDC was observed inside the cells under laser confocal microscope. CONCLUSION：HTPP-MDC strongly inhibits HBV replication in HepG2.2.15 cells. The penetrating ability of HTPP-MDC into hepatocytes indicates that HTPP-MDC is useful in clinic therapy for chronic hepatitis B-related diseases in the future.     

Effect of Paecilomyces cicadae polysaccharide on duplication of hepatitis B virus in HepG2.2.15 cell strain

AIM: To investigative the inhibitory effects of Paecilomyces cicadae polysaccharide (PcPS) against HBV in vitro, and the effects on the Toll like receptor 4 (TLR4) mRNA expression in HepG2.2.15 cell strain. METHODS: HepG2.2.15 cell strain was co-cultured in vitro with PcPS in different concentrations, and lamivudine (LMV) was applied as positive control. MTT assay was employed to detect the cytotoxicities of PcPS in vitro, when the HepG2.2.15 cells was used as target cells. The effects of PcPS on the secretion of HBsAg and HBeAg were assayed by ELISA method. Fluorescence quantitative-PCR (FQ-PCR) was used to detect the inhibitory effects of PcPS on the content of HBV-DNA and TLR4 mRNA in HepG2.2.15 cells. RESULTS: The inhibitory effects of PcPS on the HBsAg and HBeAg were observed and the maximum inhibitory ratio up to 44.8% and 31.0%, respectively. The same inhibitory effects of PcPS on the HBV-DNA replication and TLR4 mRNA expression in HepG2.2.15 cells were also found. CONCLUSION: A certain concentration of PcPS significantly inhibits HBV replication in vitro.     

Effect of proprotein convertases on transforming growth factor β1-induced HBV replication inhibition

AIM: To study the effect of proprotein convertases (PCs) on the transforming growth factor (TGF) β1-induced inhibition of HBV replication.METHODS: HepG2.2.15 cells cultured regularly were exposed to recombinant TGFβ1 at concentration of 2 μg/L or 5 μg/L and/or PC inhibitor at concentration of 20 μmol/L for 18 h. The total RNA and HBV core particle DNA were extracted from these cells, and PC mRNA and core-associated HBV DNA were detected by real-time PCR technique. RESULTS: The mRNA expression levels of 7 PCs in HepG2.2.15 cells were observed with various degrees. Recombinant TGFβ1 significantly up-regulated the mRNA expression of all PCs except for the down-regulation of PC5/6, though PC1/3 and PC2 were up-regulated most obviously. Furin and PACE4 were the predominant PCs before and after TGFβ1 exposure when the basic mRNA expression was taken into account. Further study showed that TGFβ1-induced the inhibition of HBV replication was abrogated by PC inhibitor in HepG2.2.15 cells. CONCLUSION: TGFβ1-induced the inhibition of HBV replication is mediated by the up-regulation of PCs, which might be of many implications in efficient interferences of TGFβ1 on HBV replication.     

Effects of xeroderma pigmentosum group D and p53 on replication of HBV

AIM:To investigate the effects of xeroderma pigmentosum D (XPD) and p53 on the replication of hepatitis B virus (HBV). METHODS:Recombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transfected into HepG2.2.15 cells by liposome. On the next day, these cells were incubated with pifithrin-α, a p53 inhibitor, at a concentration of 20 μmol/L for 24 h. The cells were divided into 5 groups: blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD+pifithrin-α group and pifithrin-α group. The mRNA expression of XPD, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus X protein (HBx) was detected by RT-PCR. The content of HBsAg and HBeAg in the supernatants of culture medium was measured by ELISA. The content of HBV-DNA in the supernatants of culture medium was examined by fluorescence quantitative PCR. Using the method of bDNA, the content of HBV-DNA in the core particles was assessed. RESULTS:The expression of XPD mRNA was elevated by the transfection of recombinant plasmid pEGFP-N2/XPD. The increase in XPD expression significantly down-regulated the mRNA expression of HBsAg, HBeAg and HBx. The content of HBsAg and HBeAg in the supernatants of culture medium was significantly decreased by the increase in XPD expression. The results of fluorescence quantitative PCR showed that the content of HBV-DNA in the supernatants of culture medium was significantly down-regulated by the increase in XPD expression. bDNA results showed that the content of HBV-DNA in the core particles was significantly decreased by the increase in XPD expression.Pifithrin-α abolished the above-mentioned effects of XPD (all P<0.01). CONCLUSION: XPD inhibits the replication of HBV through p53 pathway. Therefore, XPD and p53 may be the targets for antiviral therapy of hepatitis B.     

Effect of KLF6 gene on nucleotide excision repair capacity in human hepatocellular carcinoma cell line HepG2

AIM: To investigate the roles of KLF6 gene on nucleotide excision repair capacity of human hepatocellular carcinoma cell line HepG2. METHODS: Expression of KLF6 was detected by RT-PCR and Western blotting, followed by HepG2 cells treated with cisplatin (5 mg/L). Host cell reactivation assay was used to assess nucleotide excision capacity of cisplatin-damaged HepG2 cells.RESULTS: Upregulation of KLF6 mRNA or protein in HepG2 cells treated with 5 mg/L concentration of cisplatin was in a time-dependent manner, but KLF6 protein was degraded when exposed to cisplatin at same dose (5 mg/L) for 36 h. HCR assay showed that NER capacity of KLF6 overexpressing HepG2 cells was significantly enhanced, compared to that in control cells. CONCLUSION: DNA damage induced by cisplatin induces the expression of KLF6 gene. KLF6 gene may play an important role in mechanism of nucleotide excision repair in HepG2 cells.     

Effect of gold nanoparticles on reversing adriamycin resistance of human hepatocellular carcinoma HepG2/ADM cells

AIM: To investigate whether gold nanoparticles (GNPs) reverses adriamycin (ADM), resistance of human hepatocellular carcinoma drug-resistant cell line HepG2/ADM and to explore the potential mechanism. METHODS: The sensitivities of HepG2 cells and HepG2/ADM cells to ADM were tested by MTT assay before and after GNPs pretreatment. The apoptotic rate was examined by flow cytometry. The concentration of ADM in HepG2/ADM or HepG2 cells was determined by ultraviolet-visible spectrophotometer. The content of glutathione (GSH) in HepG2/ADM or HepG2 cells by DTNB method. RESULTS: The half maximal inhibitory concentrations (IC₅₀) of ADM for HepG2/ADM cells were(29.46±1.73) mg/L and (15.18±0.85) mg/L before and after GNPs pretreatment,respectively. The IC₅₀ of ADM for HepG2 cells was (9.16±2.03) mg/L before pretreatment. The apoptotic rate in GNPs+ADM group was higher than that in ADM group (P<0.05). The concentration of ADM in HepG2/ADM group was lower than that in HepG2 group (P<0.01). After GNPs pretreatment, the concentration of ADM in HepG2/ADM cells was higher than that before pretreatment. The content of GSH in HepG2/ADM group was higher than that in HepG2 group (P<0.01). After GNPs pretreatment, the content of GSH in the HepG2/ADM cells was lower than that before pretreatment. CONCLUSION: Gold nanoparticles can reverse ADM resistance of human hepatocellular carcinoma drug-resistant cell line HepG2/ADM, reduce the content of GSH and increase the concentration of ADM in HepG2/ADM cells.     

Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the apoptosis and molecular mechanism of human hepatocellular carcinoma HepG2 cells induced by ginsenoside Rh4. METHODS: Human hepatocellular carcinoma HepG2 cells were treated with ginsenoside Rh4 at doses of 10, 20 and 40 μmol/L, and the inhibitory effect of ginsenoside Rh4 on HepG2 cell viability was measured by MTT assay. The apoptotic rate of HepG2 cells was analyzed by flow cytometry. The morphological changes of the HepG2 cells were observed by Hoechst 33258 and TUNEL staining. The expression of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 was determined by Western blot. RESULTS: Ginsenoside Rh4 promoted apoptosis of HepG2 cells in a dose-dependent manner. TUNEL and Hoechst 33258 staining showed that the cells appeared obvious shrinking, swelling and rupture after treated with ginsenoside Rh4 for 24 h. The results of Western blot showed that with the increasing concentrations of ginsenoside Rh4, the expression of pro-apoptotic proteins Bax, cleaved caspase-3 and caspase-9 increased, while anti-apoptotic protein Bcl-2 decreased gradually. CONCLUSION: Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells, and the main mechanism may be related to down-regulation of Bcl-2 and up-regulation of Bax, cleaved caspase-3, and caspase-9.     

Effects of luteolin on invasion,migration and adhesion of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the effects of luteolin on invasion, migration and adhesion of human hepatocelluar carcinoma HepG2 cells.METHODS: HepG2 cells were cultured and treated with luteolin at 10, 20 and 40 μmol/L respectively. The invasion capability was examined by cell invasion assay. The migration ability was examined by wound healing assay. The adhesion capability was measured by adhesion assay. The protein levels of E-cadherin, N-cadherin, vimentin and Snai1 were determined by Western blot analysis.RESULTS: Luteolin inhibited the invasion, migration and adhesion ability of HepG2 cells in vitro in a dose-dependent manner. After treatment with luteolin, the expression of E-cadherin was increased significantly and the expression of N-cadherin, vimentin and Snai1 were decreased significantly.CONCLUSION: Luteolin inhibits the invasion, migration and adhesion ability of human hepatocelluar carcinoma HepG2 cells. The mechanism may be related to the regulatory effects of luteolin on epithelial-mesenchymal transition.     

Effects of PAR-2 agonist peptide on proliferation and cytosolic calcium level in hepatoma cells

AIM: To investigate the effects of PAR-2 agonist peptide on the proliferation and cytosolic calcium concentration (［Ca2+］c) in human hepatoma cells HepG2. METHODS: Human hepatoma cell line HepG2 was cultured. The cells were treated with PAR-2 agonist peptide SLIGKV-NH2 and the reverse PAR-2 agonist peptide VKGILS-NH2, respectively. The ［Ca2+］c of hepatoma cells were measured by microfluorimetric techniques based on calcium indicator fura-2/AM. The influences on proliferation of hepatoma cells were determined by MTT method. The changes of cell cycle were evaluated by flow cytometry, and the changes of cyclin D1 mRNA expression were detected by RT-PCR. RESULTS: After treated with 50 μmol/L SLIGKV-NH2, a rapid rise of ［Ca2+］c in HepG2 cells was induced (P<0.01), percent S phase, G2/M phase and proliferation index (PI) of HepG2 cells were elevated (P<0.01), and cyclin D1 mRNA expression was significantly upregulated (P<0.01). The proliferation rates of HepG2 cells treated with 1-50 μmol/L SLIGKV-NH2 were significantly increased, and the effect was in a dose-dependent manner (P<0.01 or P<0.05). No statistical significance of the difference between VKGILS-NH2 and control group was observed (P>0.05). CONCLUSION: PAR-2 agonist peptide induces the rise of ［Ca2+］c in HepG2 cells, upregulates the expression of cyclin D1 mRNA, accelerates the progress of cell cycle, promotes the synthesis of DNA and the proliferation of hepatoma cells via activating PAR-2 in vitro.     

Effects of tanshinone IIA on proliferation,apoptosis and expression of HIF-1α, VEGF and wild-type P53 in human hepatoma HepG2 cells under hypoxia

AIM:To investigate the effects of tanshinone IIA (Tan IIA) on proliferation, apoptosis and its molecular mechanism in human hepatoma HepG2 cells under hypoxic condition. METHODS:Hypoxia model was established by treatment with cobalt chloride (CoCl2). The cells were divided into normoxia control group, hypoxia control group and hypoxia combined at different concentrations of Tan IIA groups. After HepG2 cells were incubated with different concentrations of Tan IIA (0.5, 1.0, 2.0, 5.0 and 10.0 mg/L) for 24 h, 48 h and 72 h under hypoxic condition, the cell proliferation was determined by MTT assay. After Tan IIA was added to the media at different concentrations for 24 h and 48 h, the apoptotic cells were observed by Hoechst 33258 staining. The protein levels of hypoxia-inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and wild-type P53 were detected by Western blotting after cultured with different concentrations of Tan IIA for 48 h. RESULTS:Tan IIA inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner. Tan IIA induced the typical morphology of apoptotic cells and increased the apoptotic rate in a dose- and time-dependent manner after treatment with 1.0 mg/L~5.0 mg/L for 24 h and 48 h under hypoxic condition. The protein levels of HIF-1α and VEGF were weakly expressed in HepG2 cells under normoxia but up-regulated after incubated under hypoxia for 48 h. The protein expression of HIF-1α and VEGF were decreased with the increase in the concentration of Tan IIA under hypoxia. The protein expression of wild-type P53 was increased with the increase in the concentrations of Tan IIA under hypoxia. CONCLUSION: Tan IIA significantly inhibits the proliferation and induces the apoptosis of human hepatoma HepG2 cells under hypoxia, which may be related to the down-regulation of HIF-1α and VEGF and up-regulation of wild-type P53.     

Role of renal preS1/S2-Ag and other HBV antigen determination in diagnosis of HBV-associated glomerulonephritis

AIM：To detect the expression of preS1/S2 antigen (preS1/S2-Ag) and other antigens of hepatitis B virus (HBV) in renal tissues of patients with HBV-associated glomerulonephritis (HBV-GN), and to analyze their roles in the diagnosis of HBV-GN.
METHODS：Patients hospitalized in our department from January in 2003 to January in 2013 were retrospectively studied. A total of 49 patients with positive HBV surface antigen (HBsAg) serology, clinical manifestations of hematuria and/or proteinuria, and pathological diagnosis of glomerulonephritis, without systemic lupus erythematosus, anaphylactic purpura, diabetes or hepatitis C, were selected. PreS1/S2-Ag, HBV e antigen (HBeAg), HBsAg and HBV core antigen (HBcAg) in the renal tissues were examined. Five cases of glomerular minimal-change disease (MCD) with negative HBsAg and 5 cases of non-glomerulonephritis with positive HBsAg served as controls. RESULTS：The positive rates of preS1/S2-Ag, HBeAg, HBsAg and HBcAg in the renal tissues from the 49 patients of glomerulonephritis with HBV infection were 32.7% (16 cases), 38.8% (19 cases), 14.3% (7 cases) and 46.9% (23 cases), respectively. Total antigen positive rate was 70.2% (36 cases). The expression of preS1/S2-Ag was located in the cytoplasm of renal tubular epithelial cells, glomerular epithelial cells, endothelial cells and mesangial cells, and positively correlated with the expression of HBcAg (r=0.459, P<001). The 4 antigens were not detected in the 5 cases of HBsAg-negative patients with glomerular MCD. In the 5 cases of HBsAg-positive patients with non-glomerulonephritis, there were 2 cases expressing HBeAg and 1 case expressing HBcAg, but no cases expressing preS1/S2-Ag or HBsAg. CONCLUSION：The expression of preS1/S2-Ag in renal tissues suggests that HBV may invade the cells of renal tissue. Combined detection of the 4 antigens could elevate the rate of diagnosis of HBV-GN.     

In vitro killing effect of adenovirus-mediated herpes simplex virus thymidine kinase gene under regulation of hypoxic response element on hepatoma cell line HepG2

AIM: To investigate the in vitro killing effect of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-TK) driven by hypoxic response element (HRE) on hepatoma cell line HepG2. METHODS: Recombinant adenoviral vector Ad-HRE-TK was constructed with HSV-TK under the control of HRE using AdEasy system. Then Ad-HRE-TK was transfected into hepatoma cell line HepG2 and the cells were cultured under normoxic or hypoxic conditions. After treated with GCV for 3 d, the sensitivity to GCV of HepG2 was measured by MTT method. RESULTS: Over 95% HepG2 cells infected with Ad-HRE-TK cultured under hypoxic condition were killed when the MOI was 100 and the concentration of GCV was 50 mg/L. On the contrary, no killing effect of GCV was observed in cells cultured under normoxic condition. CONCLUSION: HRE promotes the expression of HSV-TK specifically under hypoxic condition and induces the specific killing effect of GCV.     

Inmbition of nodosin on cell proliferation and expression of Bcl-2 and Bax in HepG2 cells

AIM:To investigate the effects of nodosin extracted from Chinese traditional medicine on the proliferation of HepG2 cells cultured in vitroand to detect the protein expression of Bcl-2 and Bax in HepG2 cells. METHODS:HepG2 cells were treated with different concentrations (1.25, 2.5, 5, 10 and 20 μmol/L) of nodosin for 24 h. The morphological changes of HepG2 cells were observed under inverted microscope. The inhibitory rates of HepG2 cell growth were detected by MTT assay. The apoptotic rates and the protein expression of Bcl-2 and Bax were analyzed by flow cytometry. RESULTS:Shrunken and suspended HepG2 cells increased with the increases in the concentrations of nodosin. The apoptotic rates and the expression of Bax increased with the increases in the doses of nodosin, while the expression of Bcl-2 decreased. CONCLUSION: Nodosin inhibits the growth of HepG2 cells in a dose-dependent manner. The inhibition of HepG2 cell growth is induced by decreasing Bcl-2 and increasing Bax, thus promoting cell apoptosis.     

Role of heat shock protein 90 and tubulin in oxidative stress preconditioning

AIM:To find the role of heat shock protein 90 （HSP90） and tubulin in oxidative stress preconditioning in HepG2 cells. METHODS:The different doses of H2O2 were used to induce cell injury in HepG2 cells. MTT assay, Western blotting and confocal laser microscopy were also used. RESULTS:MTT colorimetry showed that preconditioning (50 mmo1/L H2O2) provided a temporary resistance against subsequent oxidative stress (500 mmol/L H2O2). Western blotting demonstrated that preconditioning increased the levels of HSP90 and tubulin in HepG2 cells, and lessen the declining of HSP90 and tubulin after stress. Tubulin and HSP90's colocalizations in cells with different doses of H2O2 were also observed under laser scanning confocal microscope. CONCLUSION:Tubulin might play important role in oxidative stress preconditioning in HepG2 cells by combining with HSP90.     

Role of preconditioning to oxidative stress in HepG2 cells

AIM:To induce preconditioning and oxidativ e stress by H₂O₂ in HepG2 cells.METHODS:The different doses of H₂O₂ were used to induce apo ptosis in HepG2 cells,which was estimated by AO/EB staining,MTT assay and flow cytometry.RESULTS:The different group of HepG2 cells stained with AO/EB s howed different staining state.The high dose of H₂O₂ resulted in the increa se in apoptosis rate of HepG2 cells and made MTT activity decreased.However,af ter pretreated with low dose of H₂O₂,the apoptosis rate was decreased and M TT activity was increased.CONCLUSION:The high dose H₂O₂ induced apoptosis in HepG2 ce lls and the low dose H₂O₂ protected HepG2 cells against the oxidative stress .     

Ginsenoside Rh2 enhances the apoptotic effect ofcisplatin on PC-3M prostatic cancer cells

AIM:To study the inducing apoptotic effect of Cisplatin (DDP) combined with Ginsenoside Rh₂ on PC-3M prostatic cancer cells in vitro.METHODS:Cell viability was examined by MTT test and the apoptosis of PC-3M by electrophoresis of NP-40 extracting fragmental DNA and flowcytometry.RESULTS:Pc-3M cells were exposed to 15.0 mg/L Rh₂ combined with 0.4 mg/L DDP: ①Cell viabililty inhibitory rate was increased by 1.44 times from 25.0% (with 0.4 mg/L DDP) to 61.03%. ②Characteristic apoptotic DNA laddering was found more obviously than using either agent alone. ③Apoptotic rate of PC-3M was increased from 0.32% (with 0.4 mg/L DDP) to 52.2%, which showed similar effect with 4.0 mg/L DDP, it was much higher than that of 15.0 mg/L Rh₂ alone (13.6%) (P＜0.01).CONCLUSION:Rh₂ greatly enhances the apoptotic effect of DDP on PC-3M prostatic cancer cells.     

Expression,purification and activity detemination of recombinant human keratinocyte growth factor in Pichia pastoris

AIM:To determine the conditions of recombinant human keratinocyte growth factor(hKGF) expression for large-scale production using Pichia pastoris(P.pastoris). METHODS:The DNA fragment encoding hKGF mature peptide was artificial synthesized. This artificial gene, which consisted of 20 codons of preference in P. pastoris, was cloned into secretory expression vector pPICZαA and transformed into P. pastoris X-33 strain. Under the optimal conditions in a 5 L fermentor, the target protein was purified using the combination of ultrafiltration and chromatography. The pro-proliferative activity of this protein for rhesus monkey lung epitelial cell line 4MBr-5 was detected by MTT assay. RESULTS:After 60 h of fermentation induced by methanol at 20℃, the expression level of recombinant hKGF was up to 14.1% of the total supernatant proteins. Subsequent filtration through heparin affinity chromatography and Sephadex G-25 yielded 12 mg/L target protein with purity > 95.0%. The glycosylated hKGF stimulated the proliferation of 4MBr-5 cells in vitro and its ED₅₀ was 57μg/L. CONCLUSION:The glycosylated hKGF was successfully expressed using P. pastoris and was able to improve the proliferation of 4MBr-5 cells in vitro.