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.

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.

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.

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.

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.

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.

A novel TRPC6-dependent mechanism of TGF-β-induced migration and invasion of human hepatocellular carcinoma cells

Mechanisms on cancer cell migration and invasion have been major topics of cancer research and anti-cancer therapy development. Among the multiple cell signaling pathways involved in cell migration, those elicited by transforming growth factorβ（TGF-β） have attracted tremendous attention. The TGF-βpolypeptide cytokines include four isoforms：TGF-β1, TGF-β2, TGF-β3, and TGF-β4, which are secreted mainly from cells of white blood cell lineage, such as macrophages, T cells and platelets.

Transcriptomic changes associated with PCK1 overexpression in hepatocellular carcinoma cells detected by RNA-seq

Phosphoenolpyruvate carboxykinase 1(PCK1),a step limiting enzyme of gluconeogenesis,is downregulated in hepatocellular carcinoma(HCC).Overexpression of PCK1 has been shown to suppress hepatoma cell growth,but the underlying mechanism remains unclear.We used recombinant adenovirus overexpressing PCK1 or GFP in Huh7 cells,and the differentially expressed genes(DEGs)were identified by RNA-Seq.180 were upregulated by PCK1 overexpression,whereas 316 were downregulated.Pathway analysis illustrated that PCK1 was closely correlated with Wnt signaling pathway and TGF-beta signaling pathway.Hence,Wnt signaling pathway and its downstream component,FZD2,FZD6,FZD7 and b-catenin were confirmed by qRT-PCR and Western blot.In vivo we also observed that PCK1 had restrained tumor growth as a result of decreasing expression of b-catenin.Whole-transcriptomic profile analysis discovered that overexpression of PCK1 downregulates several oncogenic signaling pathways in HCC,providing potential therapeutic targets for improving HCC therapy.

Clear cell hepatocellular carcinoma: origin,metabolic traits and fate of glycogenotic clear and ground glass cells

Clear cell hepatocellular carcinoma(CCHCC)has hitherto been considered an uncommon, highly differentiated variant of hepatocellular carcinoma(HCC) with a relatively favorable prognosis. CCHCC is composed of mixtures of clear and/or acidophilic ground glass hepatocytes with excessive glycogen and/or fat and shares histology, clinical features and etiology with common HCCs. Studies in animal models of chemical, hormonal and viral hepatocarcinogenesis and observations in patients with chronic liver diseases prone to develop HCC have shown that the majority of HCCs are preceded by, or associated with, focal or diffuse excessive storage of glycogen(glycogenosis) which later may be replaced by fat(lipidosis/steatosis). In ground glass cells, the glycogenosis is accompanied by proliferation of the smooth endoplasmic reticulum, which is closely related to glycogen particles and frequently harbors the hepatitis B surface antigen(HBs Ag).From the findings in animal models a sequence of changes has been established, commencing with preneoplastic glycogenotic liver lesions, often containing ground glass cells, and progressing to glycogen-poor neoplasms via various intermediate stages, including glycogenotic/lipidotic clear cell foci, clear cell hepatocellular adenomas(CCHCA) rich in glycogen and/or fat, and CCHCC. A similar process seems to take place in humans, with clear cells frequently persisting in CCHCC and steatohepatitic HCC, which presumably represent intermediate stages in the development rather than particular variants of HCC. During the progression of the preneoplastic lesions,the clear and ground glass cells transform into cells characteristic of common HCC. The sequential cellular changes are associated with metabolic aberrations, which start with an activation of the insulin signaling cascade resulting in preneoplastic hepatic glycogenosis. The molecular and metabolic changes underlying the glycogenosis/lipidosis are apparently responsible for the dramatic metabolic shift from gluconeogenesis to the pentose phosphate pathway and Warburg-type glycolysis, which provide precursors and energy for an ever increasing cell proliferation during progression.

Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and underlying mechanism

AIM: To investigate the mechanism by which miR-204-3p inhibits the growth of hepatocellular carcinoma (HCC) tumor endothelial cells (TECs).

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Upregulation of PD-L1 expression promotes epithelial-to-mesenchymal transition in sorafenib-resistant hepatocellular carcinoma cells

Background:The epithelial-to-mesenchymal transition(EMT)status is associated with programmed death-1 ligand 1(PDL1)expression in various cancers.However,the role and molecular mechanism of PD-L1 in the EMT of sorafenib-resistant hepatocellular carcinoma(HCC)cells remain elusive.In this study,we aimed to investigate the regulation of PD-L1 on the EMT in sorafenib-resistant HCC cells.Methods:Initially,the sorafenib-resistant HCC cell lines HepG2 SR and Huh7 SR were established.Western-blot assays were used to detect the expression of PD-L1,E-cadherin,and N-cadherin.The intervention and overexpression of PD-L1 were used to explore the role of PD-L1 in the regulation of EMT in HepG2 SR and Huh7 SR cells.Cell migration and invasion were assessed by transwell assays.PD-L1 or Sterol regulatory element-binding protein 1(SREBP-1)overexpression and knockdown were performed in order to study the mechanism of PD-L1 in sorafenib-resistant HCC cells.Results:PD-L1 expression was upregulated,whereas E-cadherin levels were downregulated and N-cadherin expression was increased in HepG2 SR and Huh7 SR cells.The cell viabilities of HepG2 and Huh7 cells were lower than those of HepG2 SR and Huh7 SR cells.PD-L1 overexpression reduced E-cadherin expression and increased N-cadherin levels,whereas PD-L1 knock-down increased E-cadherin expression and decreased N-cadherin expression.PD-L1 expression promoted EMT and the migratory and invasive abilities of HepG2 SR and Huh7 SR cells.PD-L1 promoted the EMT of sorafenib-resistant HCC cells via the PI3K/Akt pathway by activating SREBP-1 expression in HepG2 SR and Huh7 SR cells.Conclusions:The findings reveal that PD-L1 expression promotes EMT of sorafenib-resistant HCC cells.

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.

Expression of nuclear factor-KB in hepatocellular carcinoma and its relation with the X protein of hepatitis B virus

AIM In this study we investigated the relationship of the X protein of HBV and nuclear factor-κB(NF-κB)and the expression of NF-κB in human hepatocellular carcinoma tissues. METHODS Immunohistochemistry SP method was used to detect the expression of NF-κB and the X protein of HBV in human hepatocellular carcinoma tissues of 52 cases.Gene transfection mediated by lipofectamine was used to transfect the eukaryotic expression vector pCDNA3.1-HBX of HBV x gene into human hepatocellular carcinoma cell line HCC-9204 and NF-κB was detected. RESULTS NF-κB was widely expressed in human hepatocellular carcinoma tissues in a total of 52 cases and its expression was related to the X protein of HBV.NF-κB was localized both in the cytoplasm and the nuclei of hepatocellular carcinoma cells in 11 cases which were positive for the X protein of HBV while in 41 cases negative for the X protein of HBV,NF-κB was only localized in the cytoplasm of hepatocellular carcinoma cells but translocated to the nuclei of hepatocellular carcinoma cells after the eukaryotic expression vector pCDNA3.1-HBX was transfected into HCC-9204 cells. CONCLUSION This study strongly suggests that the nuclear factor NF-κB is widely expressed in hepatocellular carcinoma tissues in different styles according to the expression of the X protein of HBV.NF-κB is abnormally activated in hepatocellular carcinoma,which is probably rélated to the X protein of HBV.The X protein of HBV can activate NF-κB to translocate into nuclei of hepatocellular carcinoma 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.

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.

Calcium channel α2δ1 subunit as a novel biomarker for diagnosis of hepatocellular carcinoma

Objective:Hepatocellular carcinoma(HCC)is the fifth most common malignancy worldwide.The identification of new simple,inexpensive and highly accurate markers for HCC diagnosis and screening is needed.This case-control study evaluates the role of annexin A2 and voltage-gated calcium channelsα2δ1 subunit as serum biomarkers for HCC diagnosis.Methods:The study comprised three groups:group 1,50 patients with an initial diagnosis of HCC associated with chronic hepatitis C virus infection;group 2,25 patients diagnosed with chronic hepatitis C virus infection and cirrhosis without any evidence of HCC;and group 3,15 healthy controls.All participants were subjected to clinical and laboratory investigations,and radiological scanning.The serum levels of alpha-fetoprotein(AFP),annexin A2,and theα2δ1 subunit were evaluated by using ELISA technique.Results:The serum levels of annexin A2 significantly increased in patients with HCC(10.4±2.5 ng/m L;P<0.001)or with cirrhosis(9.31±1.8 ng/m L;P<0.001)comparing to that of healthy controls(0.296±0.09 ng/m L).However,there was no significant difference in serum annexin A2 levels in patients with HCC comparing to those with cirrhosis.Serumα2δ1 subunit significantly increased in patients with HCC(20.12±3.7 ng/m L)comparing to that in patients with cirrhosis(10.41±3.4 ng/m L,P<0.001)and healthy controls(10.2±2.9 ng/m L,P<0.001).Conclusions:The serumα2δ1 subunit may function as a new biomarker for HCC diagnosis.Conversely,serum annexin A2 has low diagnostic value as an HCC marker,especially in patients with underlying cirrhosis.