Dysregulation of apoptosis in hepatocellular carcinoma cells

Hepatocellular carcinoma （HCC） is a major health prob- lem, being the sixth most common cancer world-wide. Dysregulation of the balance between proliferation and cell death represents a pro-tumorigenic principle in hu- man hepatocarcinogenesis. This review updates the recent relevant contributions reporting molecular altera- tions for HCC that induce an imbalance in the regulation of apoptosis. Alterations in the expression and/or activation of p53 are frequent in HCC cells, which confer on them resistance to chemotherapeutic drugs. Many HCCs are also insensitive to apoptosis induced either by death receptor ligands, such as FasL or TRAIL, or by transforming growth factor-beta （TGF-β）. Although the expression of some pro-apoptotic genes is decreased, the balance between death and survival is dysregulated in HCC mainly due to overactivation of anti-apoptotic pathways. Indeed, some molecules involved in counter- acting apoptosis, such as Bcl-X1, Mcl-1, c-IAP1, XIAP or survivin are over-expressed in HCC cells. Furthermore, some growth factors that mediate cell survival are upregulated in HCC, as well as the molecules involved in the machinery responsible for cleavage of their pro- forms to an active peptide. The expression and/or activation of the JAK/STAT, PI3K/AKT and RAS/ERKs path- ways are enhanced in many HCC cells, conferring on them resistance to apoptotic stimuli. Finally, recent evi- dence indicates that inflammatory processes, as well as the epithelial-mesenchymal transitions that occur in HCC cells to facilitate their dissemination, are related to cell survival. Therefore, therapeutic strategies to selectively inhibit anti-apoptotic signals in liver tumor cells have the potential to provide powerful tools to treat HCC.

Selective COX-2 inhibitor,NS-398,suppresses cellular proliferation in human hepatocellular carcinoma cell lines via cell cycle arrest

AIM: To investigate the growth inhibitory mechanism of NS-398, a selective cyclooxygenase-2 (COX-2) inhibitor, in two hepatocellular carcinoma (HCC) cell lines (HepG2 and Huh7). METHODS: HepG2 and Huh7 cells were treated with NS-398. Its effects on cell viability, cell proliferation, cell cycles, and gene expression were respectively evaluated by water-soluble tetrazolium salt (WST-1) assay, 4’-6-diamidino-2-phenylindole (DAPI) staining, flow cytometer analysis, and Western blotting, with dimethyl sulfoxide (DMSO) as positive control. RESULTS: NS-398 showed dose- and time-dependent growth-inhibitory effects on the two cell lines. Proliferating cell nuclear antigen (PCNA) expressions in HepG2 and Huh7 cells, particularly in Huh7 cells were inhibited in a time- and dose-independent manner. NS-398 caused cell cycle arrest in the G1 phase with cell accumulation in the sub-G1 phase in HepG2 and Huh7 cell lines. No evidence of apoptosis was observed in two cell lines. CONCLUSION: NS-398 reduces cell proliferation by inducing cell cycle arrest in HepG2 and Huh7 cell lines, and COX-2 inhibitors may have potent chemoprevention effects on human hepatocellular carcinoma.

Molecular mechanisms of apoptosis in hepatocellular carcinoma cells induced by ethanol extracts of Solanum lyratum Thumb through the mitochondrial pathway

AIM To explore the induction effects and mechanism of Solanum lyratum Thumb(ST) on human hepatocellularcarcinoma SMMC-7721 cells through the mitochondrial pathway.METHODS The experiments were conducted on three groups: an experimental group (with ST ethanol extracts' concentration being 2.5, 5 and 10 mg/L), a negative control group (with only nutrient solution, 0 mg/L ST ethanol extracts), and a positive control group (2.5 mg/L DDP). The inhibition rate of cell proliferation was checked by using the methyl thiazolyl tetrazolium method, and cell apoptosis was tested by TUNEL method. Furthermore, RT-PCR was used to examine m RNA expression of Fas, Fas L, caspase-8, caspase-3, p53 and Bcl-2 genes.RESULTS Compared with the negative control group, the inhibition and apoptosis rates of the experimental group with different concentrations of ST extracts on human hepatocellular carcinoma SMMC-7721 cells significantly increased(P<0.05). Besides, the m RNA expression of Fas L and Bcl-2 significantly decreased(P<0.05) while the m RNA expression of Fas, caspase-8, caspase-3 and p53 increased significantly. When compared with the positive control group, the experimental groups with 5 mg/L ST ethanol extracts showed effects similar to the positive control group.CONCLUSION ST ethanol extracts induced the apoptosis of hepatocellular carcinoma SMMC-7721 cells through up-regulated Fas, caspase-8, caspse-3 and p53, and down-regulated Fas L and Bcl-2 in the mitochondrial pathway.

Effect of blocking IGF-I receptor on growth of human hepatocellular carcinoma cells

AIM： To study the expression level and localization of insulin-like growth factor -Ⅰ receptor （IGF-IR） in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody （αIR3） on the growth of HepG2 cells. METHODS： The expression of IGF-IR in HepG2 cells and Chang liver cells was detected by immunohistochemistry. The influences of αIR3 on proliferation and apoptosis were examined by the 3- （4, 5-dimethylthiazol-2-yl）-2, 5- diphenyltetrazolium bromide （MTT） assay and electron microscopy, respectively. Flow cytometry （FCM） was applied for the analysis of cell cycle and apoptosis was observed under electron microscope. RESULTS： IGF-IR was located in the membranes of both HepG2 and Chang liver cell lines, and the expression level of IGF-IR was higher in HepG2 cells than in Chang liver cells. Treated with 0.1 μg/mL αIR3 for 48 h in vitro, the cell growth index （GI） of HepG2 cells was significantly higher than that of control （103.41% ys 100%, P 〈 0.01）. However, the αIR3 for 24 h at final concentration of 4.0 μg/mL made the GI of HepG2 cells lower than that of control （93.37% vs 100%, P 〈 0.01）. Compared with control, treated with αIR3 for 48 h at final concentrations ranging from 2.0 μg/mL to 4.0 μg/mL markedly reduced the GIs of HepG2 cells （97.63%, 97.16%, 95.13%, 92.53% vs 100%, P 〈 0.05 or P 〈 0.01）, treated with αIR3 for 72 h at final concentrations ranging from 0.2 μg/mL to 4.0 μg/mL decreased the GIs of HepG2 cells obviously （95%, 91.63%, 90.77%, 89.84%, 88.51% vs 100%, P 〈 0.01）, and treated with αIR3 for 96 h at final concentrations ranging from 0.5 μg/mL to 4.0 μg/mL made GIs of HepG2 cells lower significantly （88.86%, 83.97%, 79.81%, 77.24%, 70.51% vs 100%, P 〈 0.05or P 〈 0.01）. Moreover, treated with αIR3 from 24 h to 96 h at final concentrations ranging from 0.2 μg/mL to 4.0 μg/mL reduced the GI of HepG2 cells from 97.63% to 70.51% in a dose- and time-dependent manner. Also, αIR3 treatment for 72 h at final concentration from 0.5 μg/mL to 2.0 μg/mL increased the proportion of G0/G1 phase cells（61.73%, 67.1%, 83.7%,76.87% vs 44.47%, P 〈 0.01） and significantly decreased that of S phase cells（28.63%, 25.13%, 15.63%, 23.13% vs 53.17%, P 〈 0.01）, in contrast to the proportion of G2/M phase cells. The apoptotic rates of HepG2 cells were increased more than that of control （7.83%, 16.13%, 21.1%, 37.73% vs 4.13%, P 〈 0.01）. CONCLUSION： The malignant cell phenotype of human hepatocarcinoma cell is related to overexpression of IGF- IR. The blockage of IGF-IR with αIR3 may contribute to the inhibition of proliferation and induction of apoptosis in HepG2 cells.

The receptor for β2GPⅠon membrane of hepatocellular carcinoma cell line SMMC-7721 is annexin Ⅱ

AIM: To evaluate the receptor protein which can specifically bind to β2GPⅠon the membrane of hepatocellular carcinoma (HCC) cell line SMMC-7721, and to study the biological function of the receptor.METHODS: Through β2GPⅠ-affinity chromatography column, the peptid-polysome-mRNA complex, which can specially bind to β2GPⅠ, stayed with the column and was separated from the whole polysome of liver cells, and then eluted and collected. Using cDNA synthesis kit and cDNA PCR kit, the corresponding cDNA was obtained and sequenced. RT-PCR was used to amplify annexinⅡ, and flow cytometry was used to study the competitive binding of annexinⅡ with β2GPⅠto SMMC-7721.RESULTS: A total of 1.1 kb of the cDNA fragment of the specific binding protein of β2GPⅠon liver cell membrane was obtained. The sequence of cDNA shared high homology with human annexinⅡ (98%). AnnexinⅡ was expressed on the membrane of SMMC-7721, and could compete with β2GPⅠfor combining with SMMC-7721.CONCLUSION: The receptor for β2GPⅠon membrane of SMMC-7721 cells is annexinⅡ, which might bridge HBV to infect hepatocytes.

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.

The Effect of Nano-apatite on the Expression of Telomerase Gene of Human Hepatocellular Carcinoma Cells

To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano-apatite for 4 h at 37 ℃ . The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger- brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity of telomerase gene was both obviously dedined comparing with the control group and the difference had significance （ p 〈 0. 05, p 〈 0.01 ）. The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.

Inhibitory effect of mycophenolate mofetil on human hepatocellular carcinoma cell line HepG-2

To investigate the inhibitory effect of mycophenolate mofetil on human hepatocellular carcinoma cell line HepG-2. Methods： HepG-2 cells were cultured in the presence of the different concentrations of mycophenolate mofetil in vitro. MTT assay was used to analyze the inhibition of cell viability conferred by mycophenolate mofetil. Cell apoptosis was observed using Hoechst33258 staining, and the percentage of HepG-2 cells at different cell cycles was determined through flow cytometry. The ability of cell adhesion was evaluated by in vitro adhesion assay. Gene expressions of factors （ICAM-1 and VCAM-1） were detected by RT-PCR. Results： Mycophenolate mofetil significantly inhibited the growth of HepG-2 cells by inducing the apoptosis of cells and this drug also inhibited the adhesion of HepG-2 cells in a dose-dependent manner. Marked morphological changes characterized in cell apoptosis were demonstrated through Hoechst33258 staining. In addition, mycophenolate mofetil decreased the proportion of S phase cells and increased that of G0/G1 phase cells. [^3H]-Thymidine uptake assay indicated that the application of mycophenolate mofetil at different concentrations significantly inhibited the cell proliferation. RT-PCR identified the expression levels of ICAM-1 and VCAM-1 genes in liver cancer cells after cultured for 72 h with different concentrations of drug. An inverse relationship was found between the expressions of ICAM-1 and VCAM-1 and drug concentrations. Conclusion： Mycophenolate mofetil has remarkable inhibitory effect on hepatocarcinoma HepG-2 cells.

Hypoxia-induced enhancement of cell invasiveness in SMMC7721 hepatocellular carcinoma cells

Objective To explore the effects of hypoxia(1% O2)on the ability of cell invasiveness and expression of KAI1/CD82 in SMMC7721 hepatocellular carcinoma cells.Methods SMMC7721 hepatocellular carcinoma cells were cultured by hypoxia(1% O2)in vitro,and the ability of cell invasiveness was analyzed by cell invasion assay.Immunohistochemistry staining technique was used to evaluate the protein expression of KAI1/CD82.Results Cell invasion assay revealed that hypoxia enhanced the ability of invasiveness of hepatocellular carcinoma cells.In addition,KAI1/CD82 protein expression was positive in cultured SMMC7721 hepatocellular carcinoma cells,and it was located diffusedly in the cytoplasm and on the membrane.KAI1/CD82 protein expression was down-regulated when mediated by hypoxia;at the same time,it showed a time-effect relationship.Conclusion Hypoxia can enhance invasiveness of hepatocellular carcinoma cells.The down-regulation of KAI1/CD82 expression may play a certain role in those courses.

Cyclin L2, a novel RNA polymerase Ⅱ-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells

We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.

Construction,identification and biological feature study of human hepatocellular carcinoma cells stably expressing HBeAg

Objective: To construct a HepG2 cell line which stably expressing Hepatitis B e antigen(HBeAg)and investigate the effects of HBeAg on the proliferation,migration and invasion of HepG2 cells. Methods: The lentivirus carrying HBeAg gene was constructed and packaged. HepG2 cells were infected with the lentivirus and screened with puromycin to obtain HepG2 cells which stably expressing HBeAg(HepG2-HBeAg cells).The expression levels of HBeAg mRNA and protein were detected by RT-qPCR and Western blot,respectively. The content of HBeAg secretion in cell supernatant in both HepG2-HBeAg cells and control cells(HepG2-NC cells and HepG2 cells)were detected by IFMA assay. Furthermore,CCK-8 proliferation assay,colony formation assay and transwell migration and invasion assays were conducted to compare the abilities of cell proliferation,migration and invasion,respectively. Results: The expression of HBeAg in the HepG2-HBeAg cell was significantly higher than those in HepG2 cells and HepG2-NC cells. Secreted HBeAg in the supernatant of HepG2-HBeAg cells was 26. 33±2. 13 PEIU/mL but was undetectable in supernatant of control cells. The proliferation,migration and invasion were all significantly lower in HepG2-HBeAg cells compared to control cells(P<0. 01). Conclusion: A HepG2 cell line which stably expressing HBeAg was constructed successfully,and the over-expression of HBeAg could attenuate the proliferation,migration and invasion of hepatocellular carcinoma cells.

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.

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97

AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Holotransferrin Enhances Selective Anticancer Activity of Artemisinin against Human Hepatocellular Carcinoma Cells

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. （the blue-green herb） in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling （TUNEL） and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells （P〈0.05）, likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells （P〈0.01）. Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

Targeting of circulating hepatocellular carcinoma cells to prevent postoperative recurrence and metastasis

Currently,the main treatment for hepatocellular carcinoma(HCC)involves the surgical removal of tumors or liver transplantation.However,these treatments are often not completely curative,as they are associated with a risk for postoperative recurrence and metastasis.Circulating tumor cells(CTCs)are increasingly recognized as the main source for recurrence and metastasis after radical hepatectomies are performed.Many studies have demonstrated the association between the presence of either pre-or postoperative CTCs and an increased risk for HCC recurrence.To improve the therapeutic outcome of HCC,a personalized,comprehensive and multidisciplinary approach should be considered,involving the application of appropriate diagnostic and therapeutic measures targeting HCC CTCs in different stages throughout the course of treatment.This article proposes some HCC CTC-based strategies for the treatment of HCC,including the monitoring of HCC CTCs before,during and after radical hepatectomy,therapeutic targeting of HCC CTCs,prevention of the generation and colonization of CTCs,as well as the use of CTC indexes for the selection of indications,prediction of prognoses,and planning of individualized therapeutic regimens.Innovation and technological development of therapies targeting CTCs,as well as their translation into clinical practice,will help to effectively reduce postoperative recurrence and metastasis,and significantly prolong the survival of HCC patients.

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.

Micro RNA-548a-5p promotes proliferation and inhibits apoptosis in hepatocellular carcinoma cells by targeting Tg737

AIM: To investigate whether Tg737 is regulated by micro RNA-548a-5p(mi R-548a-5p), and correlates with hepatocellular carcinoma(HCC) cell proliferation and apoptosis.METHODS: Assays of loss of function of Tg737 were performed by the colony formation assay, CCK assay and cell cycle assay in HCC cell lines. The interaction between mi R-548a-5p and its downstream target, Tg737, was evaluated by a dual-luciferase reporter assay and quantitative real-time polymerase chain reaction. Tg737 was then up-regulated in HCC cells to evaluate its effect on mi R-548a-5p regulation. Hep G2 cells stably overexpressing mi R-548a-5p or mi R-control were also subcutaneously inoculated into nude mice to evaluate the effect of mi R-548a-5p up-regulation on in vivo tumor growth. As the final step, the effect of mi R-548a-5p on the apoptosis induced by cisplatin was evaluated by flow cytometry.RESULTS: Down-regulation of Tg737, which is a target gene of mi R-548a-5p, accelerated HCC cell proliferation, and mi R-548a-5p promoted HCC cell proliferation in vitro and in vivo. Like the downregulation of Tg737, overexpression of mi R-548a-5p in HCC cell lines promoted cell proliferation, increased colony forming ability and hampered cell apoptosis. In addition, mi R-548a-5p overexpression increased HCC cell growth in vivo. Mi R-548a-5p downregulated Tg737 expression through direct contact with its 3' untranslated region(UTR), and mi R-548a-5p expression was negatively correlated with Tg737 levels in HCC specimens. Restoring Tg737(without the 3'UTR) significantly hampered mi R-548a-5p induced cell proliferation, and rescued the mi R-548a-5p induced cell proliferation inhibition and apoptosis induced by cisplatin.CONCLUSION: Mi R-548a-5p negatively regulates the tumor inhibitor gene Tg737 and promotes tumorigenesis in vitro and in vivo, indicating its potential as a novel therapeutic target for HCC.

Guggulsterone induces apoptosis of human hepatocellular carcinoma cells through intrinsic mitochondrial pathway

AIM: To investigate the effects of guggulsterone on the proliferation and apoptosis of human hepatoma Hep G2 cells in vitro and relevant mechanisms.METHODS: Human hepatocellular carcinoma Hep G2 cells and normal human liver L-02 cells were treated with different concentrations of guggulsterone(5-100 μmol/L) for 24-72 h. Cell proliferation was tested by MTT assay. Cell cycle and apoptosis were investigated using flow cytometry(FACS). Bcl-2 and Bax m RNA and protein expression was detected by real-time PCR and Western blot, respectively. TGF-β1, TNF-α, and VEGF contents were determined by ELISA.RESULTS: Guggulsterone significantly inhibited Hep G2 cell proliferation in a dose- and time-dependent manner. FACS showed that guggulsterone arrested Hep G2 cell cycle at G0/G1 phase. Guggulsterone induced apoptosis was also observed in Hep G2 cells, with 24.91% ± 2.41% and 53.03% ± 2.28% of apoptotic cells in response to the treatment with 50 μmol/L and 75 μmol/L guggulsterone, respectively. Bax m RNA and protein expression was significantly increased and Bcl-2 m RNA and protein expression was decreased. ELISA analysis showed that the concentrations of TGF-β1 and VEGF were significantly decreased and TNF-α concentration was increased.CONCLUSION: Guggulsterone exerts its anticancer effects by inhibiting cell proliferation and inducing apoptosis in Hep G2 cells. Guggulsterone induces apoptosis by activation of the intrinsic mitochondrial pathway.

Hepatopoietin Cn suppresses apoptosis of human hepatocellular carcinoma cells by up-regulating myeloid cell leukemia-1

AIM:To investigate the role of hepatopoietin Cn(HPPCn) in apoptosis of hepatocellular carcinoma(HCC)cells and its mechanism. METHODS:Two human HCC cell lines,SMMC7721 and HepG2,were used in this study.Immunostaining, Western blotting and enzyme linked immunosorbent assay were conducted to identify the expression of HPPCn and the existence of an autocrine loop of HPPCn/ HPPCn receptor in SMMC7721 and HepG2.Apoptotic cells were detected using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide.RESULTS:The HPPCn was highly expressed in human HCC cells and secreted into culture medium(CM). FITC-labeled recombinant human protein(rhHPPCn) could specifically bind to its receptor on HepaG2 cells. Treatment with 400 ng/mL rhHPPCn dramatically increased the viability of HCC-derived cells from 48.1% and 36.9%to 85.6%and 88.4%,respectively(P< 0.05).HPPCn silenced by small-interfering RNA reduced the expression and secretion of HPPCn and increased the apoptosis induced by trichostatin A.Additionally, HPPCn could up-regulate the expression of myeloid cell leukemia-1(Mcl-1)in HCC cells via mitogen-activated protein kinase(MAPK)and sphingosine kinase-1. CONCLUSION:HPPCn is a novel hepatic growth factor that can be secreted to CM and suppresses apoptosis of HCC cells by up-regulating Mcl-1 expression.

Ethanol extract of Kalopanax septemlobus leaf inhibits HepG2 human hepatocellular carcinoma cell proliferation via inducing cell cycle arrest at G_1 phase

Objective:To investigate the effects of an ethanol extract of Kalopanax septemlobus(Thunb.)Koidz.leaf(EEKS) on cell proliferation in human hepatocellular carcinoma cells and its mechanisms of action.Methods:Cells were treated with EEKS and subsequently analyzed for cell proliferation and flow cytometry analysis.Expressions of cell cycle regulators were determined by reverse transcriptase polymerase chain reaction analysis and Western blotting,and activation of eyclin-associaled kinases studied using kinase assays.Results:The EEKS suppressed cell proliferation in both HepG2 and Hep3 B cells,but showed a more sensitive anli-proliferative activity in HepG2 cells.Flow cytometry analysis revealed an association between the growth inhibitory effect of EEKS and with G_1 phase cell cycle arrest in HepG2 cells,along with the dephosphorylation of retinoblastoma protein(pRB) and enhanced binding of pRB with the E2 F transcription factor family proteins.Treatment with EEKS also increased the expression of cyclin-dependent kinase(CDK) inhibitors,such as p21WAF1/CIP1 and p27KIP1.without any noticeable changes in G_1 cyclins and CDKs(except for a slight decrease in CDK4).Treatment of HepG2 cells with EEKS also increased the binding of p21 and p27 with CDK4 and CDK6.which was paralleled by a marked decrease in the cyclin D- and cyclin E-associated kinase activities.Conclusions:Overall,our findings suggest that EEKS may be an effective treatment for liver cancer through suppression of cancer cell proliferation via G_1,cell cycle arrest Further studies arc required to identify the active compounds in EEKS.