Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA

AIM To investigate the characteristics of newly established four hepatocellular carcinoma cell lines (SNU 739, SNU 761, SNU 878 and SNU 886) from Korean hepatocellular cancer patients. METHODS Morphologic and genetic studies were done. RESULTS All four lines grew as a monolayer with an adherent pattern, and their doubling times ranged from 20 to 29 hours. The viability rate was relatively high (88%-94%). Neither mycoplasmal nor bacterial contamination was present. The lines showed different patterns in fingerprinting analysis. The hepatitis B virus (HBV) DNA was integrated in the genomes of all four lines, and in all of them HBx, HBc and HBs transcripts were detected by reverse transcriptase PCR methods. Among the three cell lines used as control (Hep 3B, SK Hep1 and Hep G2), only Hep 3B showed HBx expression, and this line was used as a HBV integrated control. The RNA of albumin was detected in three lines (SNU 761, SNU 878 and SNU 886), that of transferrin in two lines (SNU 878, SNU 886), and that of IGF Ⅱ was detected in none of the cell lines. CONCLUSION These well characterized cell lines may be very useful for studying the biology of hepatocellular carcinoma in association with the hepatitis B virus.

Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential

Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of human HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesion molecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis,antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vitro and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.

Potential roles of EZH2, Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma cell line Hep3B

AIM: To investigate the potential roles of enhancer of zeste homolog2(EZH2), Bmi-1 and mi R-203 in cell proliferation and invasion in hepatocellular carcinoma(HCC) cell line Hep3 B.METHODS: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3 B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37?℃. Vectors that containing c DNA of the EZH2 gene or mi R-203 targeted sh RNA plasmid were constructed, and then transfected into Hep3 B cells. The m RNA expression of mi R-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or mi R-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or mi R-203 on tumor cell invasion was detected using Transwell assay.RESULTS: The m RNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3 B cells were significantly higher compared with those in normal samples(P < 0.01), while mi R-203 level was significantly lower in HCC tissues(P < 0.01). Hep3 B cells transfected with EZH2-sh RNA or mi R-203-sh RNA showed lower expression levels of EZH2 and Bmi-1(P < 0.05). Compared with controls, Hep3 B cells transfected with EZH2-sh RNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could inhibit Hep3 B cell proliferation(P < 0.05). The average apoptosis rate of Hep3 B cells transfected with EZH2-sh RNA vector was about 18.631%, while that of Hep3 B cells transfected with sh RNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-sh RNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of mi R-203 could reduce Hep3 B cell invasion(P < 0.05).CONCLUSION: Our study suggests that EZH2 and Bmi-1 are up-regulated while mi R-203 is downregulated in Hep3 B cells. Mi R-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.

Construction of cell lines with CD44 cDNA and its application in hepatocellular carcinoma

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Biological effect of ectopic expression of angiopoietin-1 and -2 in hepatocellular cell line carcinoma cell line

OBJECTIVES: To observe the biological effect of ectopic expression of angiopoietin-l and -2 cDNA onSMMC7721 hepatocellular carcinoma cell line and study the possible role of the angiopoietin gene in thegrowth or metastasis of implantation carcinoma.METHODS: Angiopoietin-1 and -2 cDNA were subcloned into the pcDNA3 vector and subsequentlytransfected into a human SMMC7721 hepatocellular carcinoma (HCC) cell line without detectableangiopoietin gene expression before transfection. Then HCC cells were injected subcutaneously into 30nude mice and the tumor growth speed and amount of newborn vasculature in the HE stained tissue wereobserved every 2 days till 3 weeks or the death of animals.RESULTS: The tumor grew faster with angiopoietin-2 expression; much more blood vessels were seen inthe tumor tissue than that without angiopoietin-2 expression. Angiopoietin-1 gene expression seems to haveno obvious effect on the increase of vasculature and tumor growth.CONCLUSIONS: The angiopoietin gene may play a role in the growth and progression of HCC andangiopoietin-2 seems to promote the angiogenesis of the tumor.

STUDY ON THE EXPRESSION OF CYCLOOXYGENASE-2 IN HEPATOCELLULAR CARCINOMA CELL LINES AND ON THE GROWTH INHIBITION EFFECT OF NS-398

Objective： To investigate the expression of cyclooxygenase -2 （COX-2） in hepatocellular carcinoma cell lines and to explore the effect of NS-398, a selective inhibitor for COX-2, on HepG-2 cell line. Methods： lmmunohistochemistry and RT-PCR were used to investigate COX-2 expression in 6 HCC cell lines. MTT and Flowcytometry were used to evaluate the effect of the selective inhibitor of COX-2, NS-398, on HepG-2 cell lines. Results： All six HCC cell lines showed COX-2 expression at protein level. Five out of 6 cell lines showed COX-2 expression at mRNA level. NS-398 could suppress the growth of HepG-2 cell line, in a time and dose dependant manner. Conclusion： NS-398, a selective inhibitor of COX-2, showed inhibition effect on HepG-2 HCC cell line. The efficacy of inhibition was time and dose dependent, providing a new evidence for chemoprovention of hepatocellular carcinorma with COX-2 selective inhibitors.

Establishment of the human hepatocellular carcinoma cell line HCC－9204 and its characteristics

This study was aimed at providing an experimental model for the research of HCC. Twelve specimens that were pathologically identified as HCC were cultured in vitro . To investigate their biological characteristics, the survived cells were morphologically

Pharmacological Isolation of Experimental Models of Drug-resistant Hepatocellular Carcinoma Cell Line

Drug resistance is one of the major challenges facing the success of chemotherapy against human hepatocarcinoma (HCC) as well as other types of cancer. Studies with cell lines can serve as initial screening for agents that could modulate drug resistance. Development of a good experimental model of drug-resistant cells is a prerequisite for the success of such cellular studies;but could be laborious and generally time-consuming. Additionally, the high mortality rate associated with advanced HCC calls for a probe into the mechanism of resistance by developing experimental model that mimics clinical method of its treatment. Consequently, we have reported a simplified method of selection of drug-resistant hepatocarcinoma cells from human hepatocellular carcinoma (HEPG2) cell line using pharmacologic agents, cisplatin (CDDP) and 5-fluorouracil (5-FU). HEPG2 cell line was incubated for 24 hours with different concentrations of CDDP (0 - 20 μM) or 5-FU (0 - 100 μM). Cell viability was assayed by CCK-8 (Cell Counting Kit) analysis, and the inhibitory concentrations (IC50) for CDDP and 5-FU were established by dose-dependent cytotoxicity curves. The IC50(s) were confirmed by flow cytometric analysis of cell death due to CDDP or 5-FU. Clinical method of treatment was imitated by treating the parental HEPG2 cell line in pulse, at the optimal concentration of either CDDP or 5-FU for 4 to 6 hours. Induction was repeated 6 times, whilst allowing the cells to attain at least 70% confluence between intervals of induction. The resultant drug-resistant sublines, (HEPG2CR) and (HEPG2FR) were found to be stable after over 3 months of drug withdrawal and maintenance in drug-free medium. This was done with the views of establishing a simple, efficient and direct protocol for the development of good cellular models for the study of drug resistance in liver cancer, with possible application in other cancer types.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Effect of cell fusion on metastatic ability of mouse hepatocarcinoma cell lines

AIM To study the effect of cell fusion on metastatic ability of mouse hepatocarcinoma cells and the factors involved in the process of metastasis. METHODS By the method of successively increasing the concentrations, cell fusion and limit dilution, 8 Ag resistant cells were selected, and HGPRT - Hca P cells and eight cloned hybridoma cells were obtained. To observe their metastatic ability, they were inoculated into mice foodtaps and the drainage lymph nodes were examined under microscope. RESULTS The end concentration of 8 Ag which was used to select HGPRT deficient Hca P cells was 30mg/L . All the cells selected died in HAT culture medium in one week. Fused cells appeared approximately 9 days later. They were round, transparent and a little larger than their parental cells. Eight clones of hybridoma cells were obtained and named as PSH1 PSH8. The metastatic rate of HGPRT - Hca P cells and PSH7 cells was 28 6% and 71 4% respectively, the difference being significant ( P <0 05). The metastatic rate of other clones was no more than 20% and there was no significant difference from HGPRT - Hca P cells ( P >0 05). CONCLUSION In normal mice splenic lymphocytes, there are some factors that could inhibit tumor metastasis, however, there are some other factors accelerating tumor cells to metastasize. The establishment of PSH7 provides an experimental model which could be used to study the factors involved in metastasis.

Reversing effect of Tanshinone on malignant phenotypes of human hepatocarcinoma cell line

AIM To study the reversing effect of Chinese drug tanshinone on malignant phenotype of cancer cells.METHODS Human hepatocarcinoma cell line (SMMC-7721) was treated in vitro with 0.5mg/L tanshinone for 4 days, and variation in cell differentiation was detected.RESULTS The morphology of cancer cells was tended toward well differentiation and cell growth was markedly inhibited. BrdU uptake assay and immunohistochemical stain of PCNA showed that the BrdU labeling rate and PCNA positive rate were lower than the controls, but no difference was found statistically as compared with all transretinoic acid. Flow cytometric assay demonstrated that S phase cells decreased and G0/G1 phase cells increased. Expression of c-myc oncogene protein decreased but the c-fos oncogene protein markedly increased.CONCLUSION Tanshinone could reverse the inducing differentiation in human hepatocarcinoma cells (SMMC-7721). It may become a new prospective inducer of cell differentiation to treat cancers.

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth

AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients(who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1(TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1(FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci(12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma(60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36(33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2(100 ng/mL) resulted in more of the cells transitioning into S phase(23.85 ± 2.35 vs 10.94 ± 0.23),and induced a significant migratory(4.0-fold) and invasive(5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION:These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.

Docetaxel shows radiosensitization in human hepatocellular carcinoma cells

AIM: To determine the radiosensitizing potential of docetaxel in human hepatocellular carcinoma SMMC-7721 cells and its mechanisms.METHODS: SMMC-7721 cells were incubated with docetaxel at 0.125, 0.25, and 0.5 nmoL/L for 24 h and at 0.125 and 0.25 nmol/L for 48 h before irradiation. Radiation doses were given from 0 to 10 Gy. Cell survival was measured by a standard clonogenic assay after a 9-d incubation. The reactive oxygen species (ROS) and glutathione (GSH) are detected after being given the same dose of docetaxel for the same time. RESULTS: The sensitization enhancement ratios (SER) for SMMC-7721 cells determined at the 50% survival level were 1.15, 1.21 and 1.49 at 0.125, 0.25, and 0.5 nmol/L for pre-incubation of 24 h, respectively; the SER were 1.42, 1.67 at 0.125 and 0.25 nmol/L, for pre-incubation of 48 h, respectively. The ROS of SMMC-7721 cells increased and GSH decreased after pretreatment with the same doses of docetaxel for 24 or 48 h.CONCLUSION: A radiosensitizing effect of docetaxel could be demonstrated unambiguously in this cell line used. In addition, our data showed that the mechanism of radiopotentiation by docetaxel probably does not involve a G2/M block in SMMC-7721 cells, and ROS generation and GSH deletion may play a key role in the radiosensitizing effect of docetaxel.

Primmorph extracts and mesohyls of marine sponges inhibit proliferation and migration of hepatocellular carcinoma cells in vitro

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. Antiproliferative 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 primmorph 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 welldefined and controlled conditions, which allows the easy application as a bioreactor.

Effect of 5-Aza-2'-deoxycytidine on the expression of p16 in hepatocellular carcinoma cells in vitro

Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.

应用C-to-G碱基编辑器对PCSK9基因靶向敲除的研究

目的:使用C-to-G碱基编辑器(CGBE)Td-CGBE-NG靶向敲除huh-7肝癌细胞系前蛋白转化酶枯草溶菌素9(PCSK9)基因,以CBE(AncBE4max)为参照,评价Td-CGBE-NG对该基因的敲除效果。方法:使用引入终止密码子的策略敲除huh-7肝癌细胞系PCSK9基因。构建碱基编辑器Td-CGBE-NG和AncBE4max,构建重组质粒sg386和sg555,将Td-CGBE-NG和sg386,AncBE4max和sg555分别共转染huh-7细胞,实现c.1447C>G和c.1953C>T的转换,在两位点引入终止密码子S386X(TGA)和Q555X(TAG),使用sanger测序和T载体克隆验证编辑效率;实时荧光定量聚合酶链反应(RT-qPCR)和蛋白免疫印迹法(Western blot)检测PCSK9基因mRNA和蛋白表达水平的变化。结果:sanger测序和T载体克隆结果显示,Td-CGBE-NG和AncBE4max的编辑效率分别为c.1447C>G 33%和c.1953C>T 25%;RT-qPCR结果显示,Td-CGBE-NG使PCSK9 mRNA的表达量降低(t=17.29,P<0.0001);Western blot结果表明,两组huh-7细胞蛋白表达量均明显下降(AncBE4max:t=5.57,P<0.01;Td-CGBE-NG:t=4.912,P<0.01)。结论:Td-CGBE-NG可以有效敲除huh-7肝癌细胞系的PCSK9基因,抑制该基因mRNA和蛋白的表达,为后续Td-CGBE-NG的应用奠定基础。

Sirt1基因稳定低表达的LMH细胞系构建

为构建Sirtl基因低表达的LMH细胞系,根据鸡Sirtl基因的3′UTR序列设计gSmiR30、gSmiR70和gSmiR913个miRNAs,以质粒pLNCX-gSmiRn(n为30,70或91)分别过表达这3个miRNAs48h后,依次检测其对CHO-K1细胞中报告载体psiCHECH2-gSirtl-UTR672所表达的荧光素酶活性的影响以及对LMH细胞中Sirtl基因表达的干扰情况;然后选用干扰效果较好的pLNCX-gSmiR30包装的反转录病毒感染LMH细胞,以终浓度1μg·mL^-1的G418筛选稳定过表达gSmiR30的LMH-gSmiR30细胞系,荧光定量PCR和Westernblot检测Sirtl基因的表达。结果表明:人工设计的3个miRNAs可抑制含靶位点的海肾荧光素酶活性在97%以上;且LMH细胞中Sirtl的mRNA表达水平均能被这3个miRNAs下调40%以上;筛选得到的LMH-gSmiR30细胞中绿色荧光蛋白的表达率在99%以上,细胞内Sirt1基因的mRNA水平下降75%,SIRT1蛋白也明显减少。这一研究成功获得了Sirt1基因低表达的LMH-gSmiR30细胞系,为进一步研究Sirt1基因在鸡肝脏中的功能提供了一种很好的细胞模型。

Differential Expression of Genes in HepG2 Cells Caused by UC001kfo RNAi as Shown by RNA-seq

The differential expression of genes in HepG2 cells caused by UC001 kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-sh UC001 kfo lentivirus particles. The expression of UC001 kfo m RNA in the HepG2-sh UC001 kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lnc RNA UC001 kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different m RNAs. The results showed that m RNAs were differentially expressed between the HepG2-sh UC001 kfo cell line and the HepG2 cell line. The UC001 kfo m RNA was significantly down-regulated in the stable cell line HepG2-sh UC001kfo（P〈0.001）. Additionally, we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics, cell adhesion, invasion and migration. The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lnc RNA UC001 kfo. Lnc RNA UC001 kfo may play a role in regulating cancer cell invasion and metastasis. It was suggested that m RNAs were differentially expressed in the HepG2 cell line after the down-regulation of lnc RNA-UC001 kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed m RNAs may participate in cell invasion and metastasis.

In vitro antitumor efficacy of Kochia indica extract on human hepatocellular carcinoma cell line with or without 5-fluorouracil

Aim: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the sixth most common cancer worldwide. The resistance to chemotherapy is a major obstacle in the treatment of HCC, necessitating the discovery of additional agents. There is a growing use of anticancer complementary and alternative medicine worldwide. Therefore, the aim of present study was focused on the confirmation of the suitability and validity of the new markers which would be achieved by demonstrating their significant change and reproducible expression during disease and disease management. Methods: HepG2 cell line was used to provide a source for HCC cells. The cell cultures were divided into 4 groups: control untreated group, 5-fluorouracil (5-FU) treated group as a standard chemotherapy for HCC (positive control) with the following doses (15.625, 31.2, 62.5, 125, 250 μg/mL), Kochia indica extract treated group with the following concentration (12.5, 25, 50, 100, 200 μg/mL) and the group treated with a combination of 5-FU and Kochia indica in different ratios. Results: Treatment with Kochia indica extract, 5-FU and the combined treatment showed a significant cytotoxicity to HepG2 cells, with different IC50 values, when compared to the control. Regarding toxic effect, 5-FU showed IC50 = 237.56 μg/mL which is lower cytotoxic in compared to Kochia indica with IC50 =120.5 μg/mL. The results also revealed that tumor cells were more resistant to 5-FU. Alternatively, the co-treatment with Kochia indica extract ameliorated the toxicity induced by 5-FU and enhanced its therapeutic potency, either by synergistic effect of both agents and/or due to its flavonoid components that may enhance the physiological properties of the cell membranes, facilitating 5-FU entrance into tumor cells. This decreased its therapeutic dose to less than 250 μg/mL by combination therapy. Conclusion: Present findings assume that Kochia indica extract co-therapy can ameliorate the side effects of 5-FU on HepG2 by enhancing its cellular uptake.

Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.