Inhibitory effect of tumor suppressor p33(ING1b) and its synergy with p53 gene in hepatocellular carcinoma

AIM: To investigate the inhibitory effect of tumor suppressor p33ING1b and its synergy with p53 gene in hepatocellular carcinoma (HCC). METHODS: Recombinant sense and antisense p33ING1b plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lines with different endogenous p53 gene expressions, the synergistic effect of p33ING1b with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21WAF1/CIP1. In addition, the expression and mutation rates of p33ING1b in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). RESULTS: Overexpression of p33ING1b inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33ING1b and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21WAF1/CIP1. Immunostaining results showed co-localized P33ING1b with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P<0.01). Among 28 HCC samples, p33ING1b presented a low gene mutation rate (7.1%). CONCLUSION: p33ING1b collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33ING1b normal function may be an important mechanism for the development of HCC retaining wildtype p53.     

Decaprenyl diphosphate synthase subunit 2 as a prognosis factor in hepatocellular carcinoma

AIM:To investigate the involvement of decaprenyl diphosphate synthase subunit 2(PDSS2) in development and progression of human hepatocellular carcinoma(HCC).METHODS:PDSS2 protein expression was examined in well-and poorly differentiated HCC tumor samples.The levels of PDSS2 expression were compared with clinical features and prognosis of HCC patients.The effects of PDSS2 on cell proliferation,cell cycle,apoptosis,cell migration,and invasion in HCC Hep G2 cells were also investigated.RESULTS:PDSS2 was downregulated in poorly differentiated cancer samples compared with welldifferentiated tumor samples,and the expression level was markedly lower in HCC tissues than in histologically normal tissue adjacent to the cancer.Reduced protein expression was negatively associated with the status of HCC progression.In addition,overexpression of PDSS2dramatically suppressed cell proliferation and colony formation,and induced apoptosis in Hep G2 cells by inducing G1-phase cell-cycle arrest.The migration and invasion capabilities of Hep G2 cells were significantly decreased following PDSS2 overexpression.CONCLUSION:Decreased PDSS2 expression is an unfavorable prognostic factor for HCC,and PDSS2 has potent anticancer activity in HCC tissues and Hep G2cells.     

辛伐他汀抑制HepG2细胞增殖作用的机制

目的 体外观察辛伐他汀对人肝癌细胞HepG2增殖、细胞周期及细胞周期蛋白依赖激酶抑制因子p21蛋白表达的影响.方法 采用四甲基偶氮唑盐(MTT)法观察辛伐他汀对HepG2细胞增殖的影响,用流式细胞仪检测辛伐他汀对细胞周期的作用,用免疫细胞化学法观察辛伐他汀对细胞周期蛋白依赖激酶抑制因子p2l蛋白表达的影响.对数据进行析因设计与单因素方差分析.结果 体外辛伐他汀可抑制HepG2细胞的增殖(F浓度=1264,P＜0.001 ;F时间=17.466,P＜0.001;F浓度*时间=35.053,P＜0.001).辛伐他汀处理组G0/G1期细胞增多,但细胞凋亡不明显;体外辛伐他汀可增强HepG2细胞周期蛋白依赖激酶抑制因子p21蛋白的表达(F=512.133,P＜0.001).结论 体外辛伐他汀对HepG2细胞增殖有抑制作用,该作用可能与其使细胞生长阻滞于G0/G1期及增强细胞周期蛋白依赖激酶抑制因子p21蛋白表达有关.     

Deleted in liver cancer 2 suppresses cell growth via the regulation of the Raf‐1–ERK1/2–p70S6K signalling pathway

Background: Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase‐activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation. Aims: In this study, we aimed to investigate the underlying mechanisms of the DLC2 gene in suppressing cell migration and cell growth. HepG2 hepatoma cells were stably transfected with the DLC2γ isoform, which contains the RhoGAP domain. Methods and results: On performing immunofluorescence staining and Western blot analysis, the expression of the focal adhesion protein paxillin was found to be much reduced in DLC2γ‐stable clones. Upon flow cytometric analysis of the cell cycle profiles, the DLC2γ‐stable clones were shown to have a higher population of cells arrested at the G1 phase than the EGFP vector‐stable clone, suggesting that downregulation of RhoA activity in DLC2γ‐stable clones inhibited cell cycle progression. In the DLC2γ‐stable clone, the levels of Raf‐1 and extracellular signal‐regulated kinase (ERK) 1/2 were decreased as compared with those of the parental HepG2, EGFP vector and DLC2γ–GAP defective mutant‐stable clones. Furthermore, the ribosomal kinase p70S6K, a downstream target of ERK1/2, was suppressed in the DLC2‐stable clones. On the contrary, when DLC2 was knocked down by siRNA in HepG2 cells, the expression levels of phospho‐p70S6K and phospho‐ERK1/2 were upregulated. Conclusion: Our data show that DLC2 inhibits the activity of Raf‐1–ERK1/2–p70S6K via its RhoGAP function, resulting in the suppression of cell growth. Further studies on the molecular signalling between DLC2 and p70S6K may provide an insight into its growth suppressor function.     

Retinoic acid modulates the cell‐cycle in fetal rat hepatocytes and HepG2 cells by regulating cyclin‐cdk activities

Abstract: Retinoic acid (RA), the most biologically active metabolite of vitamin A, is known to modulate cell proliferation, apoptosis and differentiation, with different effects depending on the cellular context. Retinoic acid can exert its effects by directly or indirectly influencing the expression of genes involved in the control of cell proliferation. In the present report we investigate the possible correlation between the antiproliferative, differentiative and apoptotic effects previously observed on rat hepatocytes and HepG2 cells, with a possible modulation of cell‐cycle regulators. We demonstrate that RA induces growth arrest and differentiation in HepG2 cells by influencing the activities of cyclin‐cdk complexes involved in the regulation of G1/S transition and S‐phase progression, in particular by modifying the binding of these complexes to p21 and p27 inhibitors. In fetal cells, however, the induction of apoptosis and differentiation by RA was obtained via inhibition of cyclin D1‐cdk4 activity, as result of an increased binding to the p16 inhibitor. Retinoic acid also modulates c‐myc and Bcl‐2 expression. In conclusion, our data suggest that RA could be useful to regulate the reversion of transformed phenotype and could also be utilized as a chemiopreventive agent in cells of hepatic origin.     

MS-275抑制肝癌细胞HepG2增殖及其相关信号传导机制的体外实验研究

目的研究组蛋白去乙酰化酶(HDAC)抑制剂MS-275诱导人肝癌细胞株HepG2细胞周期阻滞作用,并对其信号传导机制进行初步探讨。方法体外培养人肝癌细胞株HepG2;应用MTT比色法观察MS-275对HepG2的生长抑制作用,应用流式细胞仪检测MS-275对细胞周期的影响;Western印迹分析细胞周期蛋白(Cyclin)D1、p21、p-p38MAPK三种蛋白表达的差异。结果HDAC抑制剂MS-275能抑制肝癌细胞生长,具有时间和剂量的依赖性。可以引起细胞周期G0-G1期阻滞。MS-275可以使HepG2细胞p21蛋白表达提高,CyclinD1蛋白表达降低,在SB203580组两蛋白的表达又恢复到对照组水平。p-p38MAPK蛋白在两组表达均提高。结论MS-275能诱导肝癌细胞株的细胞周期阻滞,这种作用可能是通过p38MAPK信号传导途径介导的,与下调CyclinD1的表达、上调p21的表达有关。     

Inhibition of autophagy significantly enhances combination therapy with sorafenib and HDAC inhibitors for human hepatoma cells

AIM:To clarify whether histone deacetylase inhibitors histone deacetylase inhibitors(HDACIs)can sensitize hepatocellular carcinoma(HCC)cells to sorafenib treatment.METHODS:Bax,Bcl-2,ATG5-ATG12,p21,and p27protein levels in Hep3B,HepG2,and PLC/PRF/5 cells were examined by Western blot.CCK8 and a fluorometric caspase-3 assay were used to examine cellular viability and apoptosis levels.The effect of Beclin-1 on sensitization of HCC cells to sorafenib was examined by transfecting Beclin-1 siRNA into Hep3B,HepG2,and PLC/PRF/5 cells.RESULTS:Autophagy inhibition enhances the inhibitory effects of vorinostat and sorafenib alone or in combination on HCC cell growth.Vorinostat and sorafenib synergistically induced apoptosis and cell cycle alterations.Western blot data indicated that HDACIs and Beclin-1 knockdown increased the p53 acetylation level.The knockdown of Beclin-1 enhanced the synergistic effect of the combination of vorinostat with sorafenib.CONCLUSION:HDACIs can sensitize HCC cells to sorafenib treatment by regulating the acetylation level of Beclin-1.     

Effects and mechanisms of silibinin on human hepatoma cell lines

AIM: To investigate in vitro effects and mechanisms of silibinin on hepatocellular carcinoma (HCC) cell growth. METHODS: Human HCC cell lines were treated with different doses of silibinin. The effects of silibinin on HCC cell growth and proliferation, apoptosis, cell cycle progression, histone acetylation, and other related signal transductions were systematically examined. RESULTS: We demonstrated that silibinin significantly reduced the growth of HUH7, HepG2, Hep3B, and PLC/PRF/5 human hepatoma cells. Silibinin-reduced HuH7 cell growth was associated with significantly upregulated p21/CDK4 and p27/CDK4 complexes, downregulated Rb-phosphorylation and E2F1/DP1 complex. Silibinin promoted apoptosis of HuH7 cells that was associated with down-regulated survivin and upregulated activated caspase-3 and -9. Silibinin's anti angiogenic effects were indicated by down-regulated metalloproteinase-2 (MMP2) and CD34. We found that silibinin-reduced growth of HuH7 cells was associated with increased activity of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and decreased p-Akt production, indicating the role of PTEN/ PI3K/Akt pathway in silibinin-mediated anti-HCC effects. We also demonstrated that silibinin increased acetylation of histone H3 and H4 (AC-H3 and AC-H4), indicating a possible role of altered histone acetylation in silibininreduced HCC cell proliferation.CONCLUSION: Our results defined silibinin's in vitro anti-HCC effects and possible mechanisms, and provided a rationale to further test silibinin for HCC chemoprevention.     

Vitamin K2 has growth inhibition effect against hepatocellular carcinoma cell lines but does not enhance anti-tumor effect of combination treatment of interferon-alpha and fluorouracil in vitro.

Several studies have recently reported the efficacy of combination therapy of interferon (IFN) alpha and 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). However, the clinical effect of this treatment was not complete. The new therapeutic modality should be necessary to rise up this clinical response rate. Recently, the anti-tumor effect of Vitamin K2 has been reported in terms of decreased recurrence rate of HCC patients. The aim of this study was to explore the additive or synergistic effect of Vitamin K2 to combined therapy of interferon (IFN) alpha and 5-fluorouracil (5-FU) against hepatocellular carcinoma (HCCs). The study was conducted using three hepatoma cell lines (PLC/PRF/5, Hep3B and HepG2). The 3-(4-5-dimethylthiazol-2-yl)-2, 5-dyphenyl tetrazolium bromide (MTT) assay (48h) revealed anti-tumor effect of IFNalpha and 5-FU. Cell growth assay (3-7 days) showed growth inhibitory effect of Vitamin K2 on three cell lines after day 5. But additional effect of combination treatment of Vitamin K2 and IFNalpha/5-FU was not observed in any time course from 48h to 7 days. Cell cycles were assessed with flowcytometry. Although either Vitamin K2 or IFNalpha/5-FU alone has the influence to the cell cycles, no significant change was shown in the combination of Vitamin K2 and IFNalpha/5-FU. In conclusion, Vitamin K2 itself has potentially growth inhibitory effect for HCC cell lines, but does not enhance the anti-tumor effect of IFNalpha and 5-FU.     

Gamma-aminobutyric acid promotes human hepatocellular carcinoma growth through overexpressed gamma-aminobutyric acid A receptor α3 subunit

AIM：To investigate the expression pattern of gamma-aminobutyric acid A（GABAA） receptors in hepatocellular carcinoma（HCC） and indicate the relationship among gamma-aminobutyric acid（GABA）,gamma-aminobutyric acid A receptor α3 subunit（GABRA3） and HCC.METHODS：HCC cell line Chang,HepG2,normal liver cell line L-02 and 8 samples of HCC tissues and paired non-cancerous tissues were analyzed with semiquantitative polymerase chain reaction（PCR） for the expression of GABAA receptors.HepG2 cells were treated with gamma-aminobutyric acid（GABA） at serial concentrations（0,1,10,20,40 and 60 μmol/L）,and their proliferating abilities were analyzed with the 3-（4,5-methylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay,cell doubling time test,colon formation assay,cell cycle analysis and tumor planted in nude mice.Small interfering RNA was used for knocking down the endogenous GABRA3 in HepG2.Proliferating abilities of these cells treated with or without GABA were analyzed.RESULTS：We identified the overexpression of GABRA3 in HCC cells.Knockdown of endogenous GABRA3 expression in HepG2 attenuated HCC cell growth,suggesting its role in HCC cell viability.We determined the in vitro and in vivo effect of GABA in the proliferation of GABRA3-positive cell lines,and found that GABA increased HCC growth in a dose-dependent manner.Notably,the addition of GABA into the cell culture medium promoted the proliferation of GABRA3-expressing HepG2 cells,but not GABRA3-knockdown HepG2 cells.This means that GABA stimulates HepG2 cell growth through GABRA3.CONCLUSION：GABA and GABRA3 play important roles in HCC development and progression and can be a promising molecular target for the development of new diagnostic and therapeutic strategies for HCC.     

GABA stimulates human hepatocellular carcinoma growth through overexpressed GABAA receptor theta subunit

AIM: To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC).METHODS: Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang’s liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed.RESULTS: We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ.CONCLUSION: GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC.     

Smoc2 potentiates proliferation of hepatocellular carcinoma cells via promotion of cell cycle progression

AIM To determine the influence of Smoc2 on hepatocellular carcinoma(HCC) cell proliferation and to find a possible new therapeutic target for preventing HCC progression.METHODS We detected expression of Smoc2 in HCC tissues and corresponding non-tumor liver(CNL) tissues using PCR, western blot, and immunohistochemistry methods. Subsequently, we down-regulated and upregulated Smoc2 expression using siR NA and lentivirus transfection assay, respectively. Then, we identified the effect of Smoc2 on cell proliferation and cell cycle using CCK-8 and flow cytometry, respectively. The common cell growth signaling influenced by Smoc2 was detected by western blot assay. RESULTS The expression of Smoc2 was significantly higher in HCC tissues compared with CNL tissues. Overexpression of Smoc2 promoted HCC cell proliferation and cell cycle progression. Down-regulation of Smoc2 led to inhibition of cell proliferation and cell cycle progression. Smoc2 had positive effect on ERK and AKT signaling.CONCLUSION Smoc2 promotes the proliferation of HCC cells through accelerating cell cycle progression and might act as an anti-cancer therapeutic target in the future.     

Potential role of vitamin K2 as a chemopreventive agent against hepatocellular carcinoma

Vitamin K, a cofactor necessary for the production of several antihemorrhagic factors, can inhibit the growth of various types of cells derived from neoplasms. In hepatoma cells, vitamin K₂ causes cell-cycle arrest and apoptosis. Vitamin K₂ is widely used in Japan to treat osteoporosis. The safety, relatively low cost and ease of use of vitamin K₂ have led to good compliance with treatment. The result of preliminary clinical trials in patients with chronic liver diseases are intriguing and suggest that vitamin K₂ might reduce the risk of hepatocellular carcinoma (HCC) in patients with liver cirrhosis as well as prevent disease recurrence after curative therapy in patients with HCC. This article reviews the potential role of vitamin K₂ as a chemopreventive agent against HCC and discusses future directions for clinical trials.     

Changes in the expression of melatonin receptors induced by melatonin treatment in hepatocarcinoma HepG2 cells

Abstract:  Hepatocellular carcinoma (HCC) is one of the most common cancers and its incidence is increasing worldwide. Melatonin, an indoleamine hormone, exerts anti-oxidant, immunomodulatory, anti-aging, and antitumor effects. Previous studies have shown that melatonin can act through specific receptors, including MT₁, MT₂, MT₃ receptors as well as a nuclear receptor belonging to the orphan nuclear receptor family. Recently, we have described their role in the oncostatic and pro-apoptotic effects of melatonin on HepG2 human HCC cells. However, the potential role of the different melatonin cellular receptors on its antiproliferative effects remains unknown. In the present study, we examined the effect of melatonin treatment on HepG2 human HCC cells, analyzing cell cycle arrest and melatonin receptor expression. Melatonin was administered for 2, 4, and 6 days at 1000 or 2500 μ m . Melatonin induced a dose- and time-dependent inhibition on cell proliferation. This treatment caused an alteration in the cell cycle, with an increase in the number of cells in G₂/M phase at both 1000 and 2500 μ m melatonin concentrations, and a significant increase on S phase cell percentage by the highest dose. Furthermore, increases in protein expression of MT₁, MT₃, and retinoic acid-related orphan receptor-α were found after melatonin treatments. These increases were coincident with a significant induction in the expression of p21 protein, which negatively regulates cell cycle progression. Our results confirm the antitumor effect of melatonin in HCC cells, suggesting that its oncostatic properties are related, at least in part, to changes on the expression of their different subtypes of receptors.     

Specific COX-2 inhibitor, meloxicam, suppresses proliferation and induces apoptosis in human HepG2 hepatocellular carcinoma cells

BACKGROUND AND AIMS: Cyclooxygenase-2 (COX-2) is associated with carcinogenesis. The aim of this study was to investigate the expression of COX-2 in four hepatocellular carcinoma (HCC) cell lines, and evaluate the effect of a selective COX-2 inhibitor, meloxicam, in HepG2, a high COX-2 expressing cell line. METHODS: Expression of COX-2 was detected using RT-PCR, Western blotting and immunohistochemical analysis. Cell proliferation was measured using MTT assay. Cell cycle distribution was determined by flow cytometry. Apoptosis was detected with TUNEL method. Expression of proliferating cell nuclear antigen (PCNA), cell cycle regulatory proteins including cyclins A, B1, D1 and E, and apoptosis-related proteins including Fas, Fas ligand and Bcl-2 were examined using Western blotting. RESULTS: Cyclooxygenase-2 was intensely expressed in HepG2, HLE and BEL7402 cells, but weakly expressed in SMMC-7402 cells. Meloxicam suppressed proliferation of HepG2 cells in a dose- and time-dependent manner, resulting in cell cycle arrest in S phase and cell accumulation in G0/G1 phase. Expression of PCNA, cyclin A but not cyclin B1, cyclin D1 or cyclin E was down-regulated by meloxicam. Meloxicam also induced apoptosis of HepG2 cells, with increased expression of Fas ligand, but the expression of Fas and Bcl-2 was not affected by meloxicam treatment. CONCLUSIONS: The present study demonstrates that the specific COX-2 inhibitor meloxicam suppresses proliferation and induces apoptosis in HCC cells that express COX-2, suggesting that COX-2 inhibition may offer a novel chemopreventive and therapeutic approach for HCC.