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Objective To investigate the effect of CC chemokine receptor 4(CCR4) on sorafenib radiosensitivity and tumorigenesis of hepatocellular carcinoma cells in nude mouse models.Methods Western blot was used to detect the expression of CCR4 in hepatocellular carcinoma cell line.Lentivirus was utilized to construct PLC/PRF/5 and SMMC-7721 cell lines stably overexpressing and silencing CCR4,which were verified by Western blot.The influence of CCR4 on the radiosensitivity of hepatocellular carcinoma cells was assessed by plate clone formation assay.The effect of CCR4 on the tumorigenesis in hepatocellular carcinoma cells in vivo was evaluated by tumorigenesis assay in nude mice.Results CCR4 was highly expressed in highly-metastatic hepatocellular carcinoma cells and lowly expressed in hepatocellular carcinoma cells with low metastases.The PLC/PRF/5 and SMMC-7721 cells,which stably overexpressed and silenced CCR4,were successfully established.Overexpression of CCR4 reversed the inhibitory effect of sorafenib radiotherapy on PLC/PEF/5,whereas knockdown of CCR4 could increase the radiosensitivity of SMMC-7721 to sorafenib.Overexpressing CCR4 could promote the tumorigenicity of PLC/PEF/5,whereas knockdown of CCR4 could inhibit the tumorigenicity of SMMC-7721 in nude mice.Conclusion CCR4 overexpression significantly reduces the radiosensitivity of PLC/PRF/5 and increases the tumorigenicity in nude mice,whereas knockdown of CCR4 considerably increases the chemosensitivity and radiosensitivity of SMMC-7721 and suppresses the tumorigenicity in nude mice.
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Cancer recurrence and severe side effects of currently being used chemotherapeutic agents reduce their clinical efficacy. Thus, there is a constant need to develop alternative anticancer drugs. Sustainable supply is an important challenge facing marine-based drug discovery. Primmorph, a 3D cell culture system, could provide a sustainable source to produce metabolites for anticancer drugs from marine sponges. In the present work, the anticancer activity of primmorph extracts and mesohyls of Negombata magnifica, Hemimycle arabica, Crella spinulata, and Stylissa carteri sponges was evaluated. Anti-proliferative activity was studied in terms of cytotoxicity, colony formation, cell cycle, and apoptosis. Migration was assessed by migration assay and matrix metalloproteinase activity. The expression of proliferation and migration-related genes was analyzed using real time PCR. Migration and proliferation activities of HepG2 cells were inhibited by treatment with primmorph extracts and mesohyls of N. magnifica, H. arabica, and C. spinulata. The mesohyl of S. carteri did not show any anticancer activity although the primmorph extract led to cell cycle arrest. Among the selected sponge species, the prim-morph extract of C. spinulata was the most promising anticancer agent regarding antiproliferative and antimigratory activities. In addition, primmorph extracts have the advantage of working under well-defined and controlled conditions, which allows the easy application as a bioreactor.
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Objective To explore the role and mechanism of miR-20a in the radiosensitivity of hepatocellular carcinoma (HCC). Methods The expression level of miR-20a in HCC cell lines and tissue specimens was detected by real-time fluorescent quantitative PCR.HCC cell line stably over-expressing miR-20a was constructed. The effect of miR-20a on HCC cell radiosensitivity was evaluate by cloning assay. The expression levels of Bcl-2,Caspase-3 and γ-H2AX proteins were quantitatively detected by Western blot. The target gene of the downstream regulation of miR-20a was predicted by bioinformatics analysis,which was further verified by dual luciferase reporter assay,real-time fluorescent quantitative PCR and Western blot. HCC cell line stably overexpressing miR-20a was transfected with pCDNA3. 0-PTEN to investigate the changes in the radiosensitivity of cells and to determine whether PTEN is a functional target gene for miR-20a-induced radioresistance of HCC. Results The expression levels of miR-20a was significantly up-regulated in HCC cell line and tissue specimens ( both P< 0. 05). After identical radiotherapy, the cell survival rate,the radioresistance was strengthened ( P< 0. 05),the expression of Bcl-2 was up-regulated, whereas the expression levels of Caspase-3 and γ-H2AX were down-regulated in the LV-miR-20a group compared with those in the blank and control groups ( WT and LV-con groups). Overexpression of PTEN could reverse the miR-20a-induced radioresistance. Conclusion miR-20a is up-regulated in the HCC cell lines and tissue specimens. Overexpression of miR-20a can promote the radioresistance of HCC cells. PTEN is a functional target gene for miR-20a-induced radioresistance of HCC,indicating that miR-20a/ PTEN site may be an effective molecular target associated with clinical radiotherapy for liver cancer.
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Objective@#To investigate the effect of hepatitis B core antigen (HBcAg) in promoting the invasion of hepatitis B virus (HBV)-related hepatocellular carcinoma cell line HepG2.2.15 and the role of Toll-like receptor 4 (TLR4) in the mechanism.@*Methods@#TLR4 mRNA and protein expression in HepG2 cells and HepG2.2.15 cells was measured by reverse transcription real-time PCR and Western blot analysis, respectively. HepG2.2.15 cells were transfected with TLR4 specific small interfering RNA (siRNA) to silence TLR4 expression, and stimulated by recombinant HBcAg in culture. The invasion of cells was measured by Transwell invasion assay. The expression of TLR4 signaling pathway-related proteins in the cultured cells and proinflammatory cytokines in the supernatant was also determined. The student’s t-test, one-way ANOVA, and SNK-q test were used for statistical analysis.@*Results@#TLR4 mRNA and protein expression in HepG2.2.15 cells was significantly higher than that in HepG2 cells. TLR4 siRNA transfection remarkably down-regulated TLR4 expression in HepG2.2.15 cells. Inhibiting TLR4 expression and/or HBcAg stimulation did not affect the proliferation of HepG2.2.15 cells. However, HBcAg stimulation significantly increased the invasion ability of HepG2.2.15 cells and the secretion of proinflammatory cytokines [including interferon (IFN)-γ, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α]. Inhibiting TLR4 expression significantly reduced HBcAg-induced cellular invasion. Meanwhile, HBcAg stimulation elevated the expression of MyD88 and TRIF in HepG2.2.15 cells. TLR4 silencing inhibited HBcAg-induced increase in the expression of MyD88, while it showed no significant impact on TRIF expression.@*Conclusion@#HBcAg can promote the invasion of HepG2.2.15 cells. The TLR4/MyD88 signaling pathway may be involved in this process by inducing the expression of proinflammatory cytokines.
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Objective The cancer risk of patients with diabetes mellitus who are treated by metformin declines remarkably in comparison to patients receiving other drug therapies.The article was to investigate the relationship between antineopastic activity and fatty acid synthase (FASN) of metformin in human hepatocellular carcinoma cell(HCC) line HepG2. Methods HepG2 cells were treated with various concentrations of metformin( 0, 1, 5, 10, 15 mmol/L) for 24, 48 and 72 h respectively and cell growth was assessed by CCK-8 assay.Positive control(paclitaxel 10μg/mL) and negative control(metformin 0mmol/L) were set up simultaneously.After being treated with doses of metformin(0, 5, 10,15mmol/L) for 72h, protein expression levels of AMPKα、P-AMPKα、FASN、P-mTOR and P-Akt were measured by western blotting analysis and FASN mRNA expression levels were measured by RT-PCR. Results Being treated with vari-ous doses of metformin(1, 5, 10, 15 mmol/L) for 24, 48 and 72 h, the growth of HepG2 cells were inhibited by metformin in dose-dependent and time-dependent manner( P0.05) .FASN mRNA expression levels decreased significantly in all metformin-treated groups( P<0.05) . Conclusion Met-formin actitiviates AMPK, inhibits mTOR and downregulates FASN, which are implicated in its antineopastic activity on HCC.Although metformin inhibits mTOR activation, it is not involved in Akt upregulation through a negative loop.
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Objective To explore the inhibition effect and the possible mechanism of resveratrol on human hepatocellular carcinoma cell line Bel-7402 both in vitro and vivo.Methods Four Res drugs in the experiment group,the final concentrations were 12.5,25,50,100μmol/L,the control group at the same time set not containing Res drugs,MTT assay was used to measure the inhibition of resveratrol on Bel-7402.The expression of Bcl-2 was detected by RT-PCR and Western Blot.The levels of IL-2,IL-6,IL-12 and TNF-αwere detected by ELISA.Results Resveratrol inhibited Bel-7402 cell proliferation in dose and time manner,and influenced the expression of Bcl-2 mRNA and protein.At the same time,resveratrol inhibited the growth of tumor and improved the levels of IL-2,IL-6,IL-12 and TNF-α.Conclusion Resveratrol could inhibit Bel-7402 cell proliferation both in vitro and vivo, the possible mechanism may be that resveratrol could low down the expression of Bcl-2 and improve the levels of IL-2,IL-6,IL-12 and TNF-α.
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Objective:To establish proteomic technique system of human hepatocellular carcinoma cell line QCY-7701. Method:Two different lysis buffer were taken to extract cell proteins. After two-dimensional gel electrophuresis (2-DE) and PDQuest analysis, 10 good-matched protein spots were chosen to be identified by MALDI-TOF-MS. Results:A steady 2-DE electrophregram of hepatocellular carcinoma cell line QGY-7701 was established. Compared with lysis bufferⅠ,protein quantities extracted from lysis bufferⅡincreased by 25%;protein spots in 2-DE gel increased by 32%. 9 out of 10 candidate protein spots were successfully identified (P
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Objective: To study the influence of emodin on fluorouracil(5-FU) induced apoptosis by elevating reactive oxygen species(ROS)of human hepatocellular carcinoma cell lines HepG2. Methods: The intracellular ROS levels were detected when exposed to various concentrations of emodin for the indicated periods by flow cytometry(FCM).MTT assay and FCM were used to assess cell viability,apoptosis and cell cycle distribution after treatment with 5-FU alone or combined with various concentrations of emodin.Transmission electron microscope was applied to observe the ultrastructural change of cells. Results: Emodin elevated intracellular ROS level in a dose-and time-dependent manner,and intracellular ROS level of HepG2 cell incubated in 4 ?g/ml emodin reached peak in 2-4 h.Emodin combined with 5-FU sensitized HepG2 cell to 5-FU induced apoptosis via generation of ROS in a time-and dose-dependent manner. Conclusion: Enhancement of ROS generation by emodin may be a novel strategy to improve the curative effect of chemotherapeutic drugs.
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In the present study, the influence of essential free fatty acids on AFP secretion and cell growth of BEL-7402 human hepatocellular cacinoma cell line was investigated by radiommunoassay. The results demonstrated that 40 - 50?g/ ml concentration of linolenic acid could inhibit the AFP secretion obviously( P 0.05) . All of these studies about anti-cancer effect of linolenic acid will provide the principle for the patient for health care and therapy.
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We used retroviral vector pLXSN to construct recombinant retroviral vectors with the human apoptosis gene, interleukin-l? converting enzyme (ICE). The vectors were introduced into packaging cell line PA317 by electroporation method. The G418 resistant colonies were selected, and the supematants of the colonies were used to infect the human hepatocellular carcinoma cell line SMMC7721. G418 resistant colonies of SMMC7721 were named SMMC7721-MCE and SMMC7721-neo. The results of RT-PCR analysis showed that exogenous hICE gene had successfully integrated into the genome of SMMC7721-hICE cells. The proliferation rate and tumorigenicity of cells in nude mice were examined. Our data showed that the growth rate and the tumorigenicity of SMMC7721-hICE cells in nude mice were considerablely decreased comparing with parent SMMC7721 and SMMC7721-neo. These results suggested that the retroviral vector expressing hICE gene was successfully constructed and could suppress the growth ability and tumorigenicity of human hepatocellular carcinoma cells, which provided a basis for further investigation of hICE gene therapy.
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Objective: To investigate the cytotoxicity of ICE gene transfection in Combination with Antitumor Chemicals killing Hepatocellular Carcinoma Cells in vitro.Methods: The recombinant plasmid pLXSN-hICE was transferred to virus packing cell PA317 by electroporation method. And then the retrovirus containing human ICE cDNA generated by these PA317 cells were used to transfect human hepatocellular carcinoma cell line HepG2. The apoptosis of transferred cells were examined by gel electrophoresis. The influence of chemotherapeutic drug Carbo-platin to the proliferation of hepatocellular carcinoma cell line SMMC7721 and its derivative cells(SMMC7721-hICE,SMMC7721-antisence hICE,SMMC7721-neo)was observed by incorporation of 3H-TDR. Results: Electrophoresis of DNA displayed the apoptosis ladder of HepG2 transfected by ICE gene. The proliferation of SMMC7721-hICE was significantly suppressed in vitro induced by Carbo-alatin compared to the other three cell lines. Conclusion: ICE gene transfection could greatly increase the susceptibility of SMMC7721 cells to apoptotic cell death following chemotherapy. These findings suggest that combining ICE gene transfection with utilizing antitumor drugs would represent a novel approach for the effective treatment of hepatocellular carcinoma.