Hepatocellular Carcinoma Cell Line SMMC-7721 Research Articles

In vivo Molecular targeting effects of anti-Sp17- ICG-Der-02 on hepatocellular carcinoma evaluated by an optical imaging system

BackgroundAs the expression of human sperm protein 17 (Sp17) in normal tissue is limited and the function is obscure, its aberrant expression in malignant tumors makes it to be a candidated molecular marker for tumor imaging diagnosis and targeting therapy of the diseases.The aim of this research is to evaluate the targeting effects of anti-sperm protein 17 monoclonal antibody (anti-Sp17) on cancer in vivo and investigate its usefulness as a reagent for molecular imaging diagnosis.MethodsImmunohistochemistry was used to identify the expression of Sp17 in a hepatocellular carcinoma cell line and tumor xenograft specimens. A near infrared fluorescence dye, ICG-Der-02, was covalently linked to anti-Sp17 for in vivo imaging. The immuno-activity of the anti-Sp17-ICG-Der-02 complex was tested in vitro by ELISA; it was then injected into tumor-bearing nude mice through the caudal vein to evaluate its tumor targeting effect by near infrared imaging system.ResultsOverexpression of Sp17 on the surface of the hepatocellular carcinoma cell line SMMC-7721 was demonstrated. Anti-Sp17-ICG-Der-02 with immuno-activity was successfully synthesized. The immuno-activity and photo stability of anti-Sp17- ICG-Der-02 showed good targeting capability for Sp17 expressing tumor models (SMMC-7721) in vivo, and its accumulation in the tumor lasted for at least 7 days.ConclusionsAnti-Sp17 antibody targeted and accumulated in Sp17 positive tumors in vivo, which demonstrated its capability of serving as a diagnostic reagent.

Effect of TPT1 transduction on the biological behaviors of liver cell lines

The aim of this study was to transfect TPT1 into cell lines SMMC-7721 and L-02, seperately, and to observe the changes of biological behaviors of the cell lines. Through lipofectamine, the eukaryotic report expression vector containing TPT1 ORF (open reading frame), pEGFP-N3TPT1, were transducted into hepatocarcinoma cell line SMMC-7721 cells and normal liver cell line L-02 cells, seperately. The transduction was repeated three times in 24 hrs. The differences of biological behaviors between the pEGFP-N3TPT1 and pEGFP-N3 groups were studied by RT-PCR, MTT assay, soft agar colony formation assay and cell cycle analysis. The pEGFP-N3TPT1 transfected cells had a high mRNA level in the two cell lines (P < 0.05) compared with the pEGFP-N3 controls. The ability of proliferation and the soft agar colony formation were enhanced in the SMMC-7721 transducted cells with pEGFP-N3TPT1 compared with that transducted with pEGFP-N3 (P < 0.05), and the cell cycle analysis showed that the cells in the phase G(2)+S/M increased after pEGFP-N3TPT1 transduction. In the L-02 cell line, we obtained similar results, pEGFP-N3TPT1 enhanced the colony formation in plate (P < 0.05), but not make it form colony in soft agar. TPT1 can enhance malignant phenotype of SMMC-7721 cells and promote the growth of L-02 cells, but not transform L-02 into malignant phenotype.

Effects of integrin α6β1 on migration of hepatocellular carcinoma cells

In this study, we applied specific blocking antibodies for integrin α6 or β1 subunit, and evaluated the in vitro effects of integrins α6β1 on the adhesion, chemotaxis and migration of hepatocellular carcinoma (HCC) cell line SMMC-7721 to type IV collagen. The adhesion force and cell migration, as measured by a micropipette aspiration system and Boyden chamber assay respectively, was dramatically reduced when either integrin subunits was blocked. The chemotaxis, as determined using a dual-micropipette system, was only affected by the antibody against β1 subunit. This study suggests that integrin α6β1 is an important cell surface receptor that mediates the adhesion of SMMC-7721 to type IV collagen. But the α6 subunit has minimal effect on pseudopod formation in response to type IV collagen. Therefore, the integrin α6β1-mediated cell migration is, at least in part, through the regulation on the cell adhesion step.

Hepatitis C virus core protein activates Wnt/β-catenin signaling through multiple regulation of upstream molecules in the SMMC-7721 cell line

The core protein of hepatitis C virus (HCV) has been implicated in HCV-induced liver pathogenesis. Previous data have shown that the HCV core protein has pleiotropic functions, including transcriptional regulation of a number of cellular genes, although the mechanism of gene regulation remains unclear. Wnt/β-catenin signaling is also involved in hepatocellular carcinoma (HCC) tumorigenesis. To elucidate the molecular mechanism of HCV pathogenesis, we examined whether HCV core protein activates Wnt/β-catenin signaling in the hepatoma cell line SMMC-7721. The effects of core protein on Wnt/β-catenin signaling cascades were investigated by luciferase reporter gene assay, immunofluorescence, western blot and RT-PCR analysis. Here, we demonstrate that HCV core protein plays an essential role in activating β-catenin/Tcf-4-dependent transcriptional activity and increases active β-catenin expression and nuclear accumulation in SMMC-7721 cells. An RT-PCR assay indicated that core protein upregulates gene expression of canonical Wnt ligands, such as Wnt2, Wnt3, Wnt3a, Wnt8b, Wnt10a, Wnt10b, frizzled receptors Fzd1, 2, 5, 6, 7, 9, and LRP5/6 co-receptors. However, Wnt antagonists SFRP3, 5 and Dkk1 were moderately repressed. Furthermore, ectopic expression of core protein markedly promoted cell proliferation. The soluble Fzd molecule FrzB or the β-catenin inhibitor siBC efficiently blocked cell growth stimulation by the core gene. Our present findings demonstrate that the HCV core protein activates canonical Wnt signaling through tight regulation of several important molecules upstream of β-catenin and presumably results in promotion of cell proliferation in the SMMC-7721 cell line. Taken together, these data suggested that the core protein may be directly involved in Wnt/β-catenin-mediated liver pathogenesis.

Cytotoxic constituents from the skin of the toad Bufo bufo gargarizans

To study the chemical composition of the skin of Bufo bufo gargarizans, various chromatographic methods were used in the isolation procedures and the structures of isolated compounds were determined based on NMR and MS analysis. As a result, two new compounds were isolated from its ethanolic extract and characterized as N-[2-hydroxy-2-(4-hydroxyphenyl)ethyl]-N-methylurea (1) and 19-oxocinobufotalin 3-adipoylarginine ester (2), together with 11 known compounds. Isolated bufadienolides showed significant inhibition effect against human hepatocellular carcinoma cell line SMMC-7721 in vitro.

Growth Inhibition and Apoptosis Induced by Lupeol, a Dietary Triterpene, in Human Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is the fifth most malignant tumor worldwide and is known to be resistant to conventional chemotherapy. New therapeutic strategies are urgently needed for treating HCC. Lup-20(29)-en-3H-ol (Lupeol), a novel dietary triterpene, is found in fruits, vegetables, and medicinal plants and possesses multiple bio-activities with very low toxicity. In the current study, we investigated its growth-inhibitory effects in HCC cell lines SMMC7721 and HepG2. In the in vitro studies, lupeol treatment alone caused decrease of cell viability in two HCC cell lines in a dose-dependent manner. It also induced apoptosis and caused cell accumulation in S phase. Further analysis revealed the induction of active caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage by lupeol treatment. In the in vivo studies, nude mice implanted with SMMC7721 cells subcutaneously were treated with lupeol three times a week and tumor development was significantly inhibited. We further investigated the combination anti-tumor effect of lupeol and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HCC, considering TRAIL treatment alone could not achieve high level of anti-tumor effect. The results demonstrated that lupeol could exert a combinational effect with TRAIL, resulting in chemosensitization of HCC. Our results suggested that lupeol alone or as an adjuvant to therapeutic agents could be developed as a potential agent for treating HCC.

Effects of metformin on proliferation of human hepatocellular carcinoma cell line SMMC7721

Effects of metformin on proliferation of human hepatocellular carcinoma cell line SMMC7721

Effects of over-expression of MAGE-A3 on human hepatocellular carcinoma cell line SMMC7721

Objective To observe the effects of over-expression of target gene MAGE-A3 by transient transfection on proliferation and metastasis of human hepatocellular carcinoma (HCC) cell lines.Methods MAGE-A3 target gene that containing restriction enzyme cutting site of NheI and KpnI was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from HCC tissues.The target gene was cloned into the expression vector pcDNA3.1 ( - ) to construct MAGE-A3 eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3,which was checked by restrictive enzyme digestion and DNA sequencing,and then transiently transfected into human HCC cell line SMMC7721 with Lipofectamine 2000.The mRNA and protein expression of MAGE-A3 was detected by semi-quantitive RT-PCR and Western blotting assay respectively.MTT assay and Boyden chamber assay were performed to detect the effects of MAGE-A3 on colony formation and metastasis.Results MAGE-A3 target gene was amplified and the eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3 was correctly constructed,and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and Western blotting revealed a strongly increased level of MAGE-A3 mRNA and protein expression in transfection group as compared with untransfected group and empty vector-transfected group (for MAGE-A3 mRNA expression level: 7838,2847 and 1557 in three groups,and for MAGE-A3 protein levels: 30 938,24 088 and 29 654,respectively).Both the number of HCC cells transfected by peDNA3.1-MAGE-A3 in MTT assay and the number of cells penetrating matrigel were increased as compared with untransfected group and empty vector-transfected group (P ＜0.05 ).Conclusion The eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3 was successfully constructed.The number of cells and invasiveness of the HCC cells transfected by pcDNA3.1-MAGE-A3 were significantly increased,suggesting MAGE-A3 gene may play a significant role in proliferation and metastasis of human HCC. Key words: MAGE; Transfect; Carcinoma,hepatocellular; Proliferation

Osteopontin promotes hepatocellular carcinoma invasion by up-regulating MMP-2 and uPA expression

Osteopontin (OPN) is over-expressed in a variety of cancers, but its role in hepatocellular carcinoma (HCC) progression has not been clarified. In this study, weakly tumorigenic, non-metastastic human HCC cell line SMMC-7721 cells were forced to over-express OPN via stable transfection. A series of functional assays were performed to assess the effects of OPN on tumor cell behaviors and cDNA microarray was used to identify the genes regulated by OPN. The results showed that OPN significantly enhanced the migration and invasion of SMMC-7721 cells in vitro. In addition, CD44v6 antibody could significantly inhibit the invasion of OPN over-expressing SMMC-7721 cells. Moreover, MMP-2 and uPA expressions were significantly up-regulated in OPN over-expressing SMMC-7721 cells. Together, these findings indicate that OPN enhanced HCC cells invasion through interaction with its receptor CD44v6 and increased MMP-2 and uPA expressions, providing at least one mechanism for OPN-mediated HCC progression and metastasis.

Caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside 1 induced apoptosis through caspase 3‐dependent pathway in human hepatoma cell line SMMC7721

Caudatin-2,6-dideoxy-3-O-methy-β-D-cymaropyranoside (CDMC), the C-21 steroidal glycoside recently extracted from the traditional Chinese medicinal plant, the root of Cynanchum auriculatum Royle ex Wight (Asclepiadaceae), has been shown to possess potent antitumor properties. However, the bioactivities of CDMC are still largely unknown, especially the antitumor effect and its mechanism. This study investigated the CDMC antitumor effects on human hepatoma cell line SMMC7721 cells by analysis of cell viability, cell cycle phases and apoptosis. The results showed that CDMC inhibited the growth of SMMC7721 cells in a time- and dose-dependent manner and resulted in cell cycle arrest in G(0)/G(1) phase. Furthermore, CDMC induced SMMC7721 cell apoptosis rather than necrosis through caspase 3 activation, and a caspase 3 inhibitor, Ac-DEVD-CHO, could attenuate the apoptosis induced by CDMC. The results suggested that the anticancer activity of CDMC could be attributed partially to its inhibition of cell proliferation and induction of apoptosis associated with caspase 3 activation.

CIDE-3 interacts with lipopolysaccharide-induced tumor necrosis factor, and overexpression increases apoptosis in hepatocellular carcinoma

Cell death-inducing DFF45-like effector-3 (CIDE-3) is a novel member of an apoptosis-inducing protein family, but its function is unknown. CIDE-3 shows a different distribution pattern in hepatocellular carcinoma (HCC) tissues and normal adjacent tissues. Therefore, this work tested the hypothesis that CIDE-3 induces apoptosis in HCC cells, inhibiting oncogenesis and tumor development. We used immunohistochemistry to evaluate the expression of CIDE-3 in 82 HCC samples and 51 adjacent liver tissues. Overexpression of CIDE-3 induced apoptosis, as detected by flow cytometry, in the HCC cell line SMMC-7721, which had undetectable levels of CIDE-3 in the absence of CIDE-3 overexpression. A yeast two-hybrid system was employed to screen for proteins that interact with CIDE-3. The expression of CIDE-3 was decreased in HCC tissue, compared to adjacent normal tissues, and CIDE-3 expression and HCC differentiation were positively correlated. CIDE-3 expression levels were lower in poorly differentiated HCC tissue than in well-differentiated HCC tissue. Overexpressed CIDE-3 inhibited proliferation and induced apoptosis in HCC cells. We found that lipopolysaccharide-induced tumor necrosis factor (LITAF) interacted with CIDE-3 in hepatic cells. This is the first demonstrated interaction between CIDE-3 and LITAF, and the first report that CIDE-3 induces apoptosis in hepatocellular carcinoma.

The MicroRNA miR-139 Suppresses Metastasis and Progression of Hepatocellular Carcinoma by Down-regulating Rho-Kinase 2

We investigated mechanisms of hepatocellular carcinoma (HCC) metastasis and identified an antimetastatic microRNA (miRNA), miR-139, that is down-regulated in human HCC samples. Effects of stable and transient expression of miRNA-139 and its inhibitors were studied in the human HCC cell lines SMMC-7721 and BEL7402; cells were analyzed for migration and invasion. Liver samples from patients with metastatic HCC were analyzed for levels of miRNA-139; data were compared with survival data using the Kaplan-Meier method and compared between groups by the log-rank test. Tumor formation and metastasis from human HCC MHCC97L cells that did or did not express miR-139 were analyzed in mice. Down-regulation of miR-139 in HCC was associated significantly with poor prognosis of patients and features of metastatic tumors, including venous invasion, microsatellite formation, absence of tumor encapsulation, and reduced differentiation. miR-139 expression was reduced in metastatic HCC tumors compared with primary tumors. Overexpression of miR-139 in HCC cells significantly reduced cell migration and invasion in vitro and the incidence and severity of lung metastasis from orthotopic liver tumors in mice. miR-139 interacted with the 3' untranslated region of Rho-kinase 2 (ROCK2) and reduced its expression in HCC cells. Levels of miR-139 were correlated inversely with ROCK2 protein in human HCC samples. Overexpression of miR-139 did not inhibit HCC cell motility when ROCK2 was knocked down. The microRNA miR-139 interacts with ROCK2 and reduces its expression in HCC cells. Down-regulation of miR-139 increased the invasive abilities of HCC cells in vitro and HCC metastasis in vivo. Expression of miR-139 is reduced in human metastatic HCC samples and correlates with prognosis.

The complete preventive effect of homologous tumor vaccines – Based on a 5-year experimental study in mice

By using a frozen-thaw method, we developed homologous and heterologous cell-based tumor vaccines, which were derived from mouse H22 hepatoma, S180 sarcoma and human A549 lung carcinoma, SK-OV-3 ovarian, and SMMC-7721 hepatoma cell lines. The prophylactic and therapeutic effects of those vaccines were evaluated in mice challenged with live H22 or S180 cells. The result demonstrated that homologous vaccines and heterologous vaccines had no therapeutic effect on tumor growth. However, homologous vaccines showed a complete prevention against live H22 and S180 cell challenge and they could stimulate cross-immune response of anti-tumor in mice. Furthermore, these tumor-free mice immunized with homologous vaccines showed full protection against the repeat challenge every 3 months for 5 years. The study also revealed that tumor-free female, not male, mice transferred anti-tumor ability to some of their offsprings. Heterologous vaccines exhibited no protective effect on tumor development. Immunological analysis discovered that activities of CTLs and NK were enhanced and the levels of IL-2, IL-12 and IFN-γ were significantly increased. Our results demonstrated that homologous tumor vaccines could elicit complete cross-protection against the lethal challenge of tumor cells through enhancing cell-mediated immune response, which lasted for 5 years in mice. These observations may provide a new vaccine strategy for tumor prevention.

Inhibition of tumorigenesis and invasion of hepatocellular carcinoma by siRNA-mediated silencing of the livin gene

Livin, a member of the inhibitor of apoptosis protein family, is overexpressed in a variety of solid tumors and cancer cell lines. Livin overexpression has been reported in hepatocellular carcinoma (HCC), suggesting the biological significance of livin in HCC progression. However, the mechanisms of livin and the consequence of its down-regulation in HCC have not been fully elucidated. A small interfering RNA (siRNA) eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. It was then transfected into the human HCC cell line SMMC-7721. RT-PCR and Western blotting were used to validate the gene-silencing efficiency of livin in SMMC-7721 cells. Stable clones were obtained by G418 screening. Using multiple cellular and molecular approaches, such as an apoptosis assay, MTT assay, flow cytometry, Western blotting and a migration assay, the effects of livin inhibition on cell growth, migration and the induction of apoptosis in SMMC-7721 cells were observed. The siRNA eukaryotic expression vector specific to livin was constructed by gene recombination, and efficiently decreased the mRNA and protein expression of livin. The targeted inhibition of livin strongly sensitized SMMC-7721 cells to pro-apoptotic stimuli, and was associated with caspase-3 activation. In addition, the MMT assay indicated that the silencing of livin inhibited the cell growth of SMMC-7721 cells by specifically inhibiting cell mitosis. The results also showed that the silencing of livin strongly reduced the invasive capacity of SMMC-7721 cells. The findings suggest that livin expression not only provides HCC cells with increased resistance to apoptotic stimuli, but also contributes significantly to the proliferation and invasive capacity of HCC cells. Inhibition of livin may be a potential targeted approach for the treatment of HCC.

Artemisinin inhibits in vitro and in vivo invasion and metastasis of human hepatocellular carcinoma cells

Artemisinin (ART) is isolated from the medicinal plant Artemisia annua L. To determine its effects on the invasion and metastasis of tumors, the human hepatocellular carcinoma (HCC) cell lines HepG2 and SMMC-7721 were treated with different concentrations of ART. Starting at 12.5μM, ART had inhibitory effects in migration and invasion assays that increased at higher concentrations. The inhibitory effect also became stronger with time, from 24 to 72h. ART significantly inhibited the in vivo metastatic abilities of the HepG2 HCC cell line. ART inhibited the invasion and metastasis of HCC cells both in vitro and in vivo by reducing the level of the MMP2 metalloproteinase, and by inducing the TIMP2 protein. ART activated Cdc42, which enhanced E-cadherin activity, resulting in greater cell-cell adhesion, and significantly reduced metastasis.

Increased hsp70 of glucocorticoid receptor complex induced by scald and heat stress and its possible effect on the affinity of glucocorticoid receptor

Background Glucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro. Methods The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting. Results Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols. Conclusions The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity. Chin Med J 2010;123(13):1780–1785

Chemical constituents from seeds of Aplinia katsumadai, inhibition on NF-κB activation and anti-tumor effect

To study the chemical constituents from the seeds of A. katsumadai, and their inhibition on NF-kappaB activation and antitumor effect. Tweleve compounds were isolated from the seeds of Aplinia katsumadai on repeated column chromatography on silica gel, and Sephadex LH-20, and their structures were determined mainly by means of MS and NMR techniques; and their inhibition on NF-kappaB activation and antitumor effect were also tested by High-Content Screening (image-based) with immunofluorescent probe and MTT method, respectively. From the EtOAC fraction of the seeds of A. katsumadai 12 compounds were isolated and identified as follows: (3S,5S)-trans-3,5-dihydroxy-1 ,7-diphenyl-hept-1-ene (1), (3R,5S)-trans-3,5-dihydroxy-1,7-diphenyl-hept-1-ene (2), 5-hydroxy-1,7-diphenyl-hepta-6-en-3-one (3), cardamonin (4), alpinetin(5), pinocembrin(6), pinostrobin(7), naringenin (8), (+)-catechin(9), chrysin(10), rutin(11) and 2,4- dihydroxy-6-phenethl-benzinic acid methyl ester (12). Compound 14 showed inhibitory effect on NF-kappaB activation with the IC50 values as 14.8, 16.5, 23.2 and 7. 5 micromol x L(-1), respectively. Compound 4 displayed cytotoxicity against leukemia K562 cells and human hepatoma cell line SMMC-7721 with IC50 values as 3.2 and 3.5 mg x L(-1), and compound 6 showed moderate cytotoxicity against SMMC-7721 with the IC50 value as 18.3 mg x L(-1). Compounds 7-12 were isolated from A. katsumadai for the first time and Compound 12 were isolated from the genus Aplinia for the first time; compound 4 has the activity of anti-tumor and NF-kappaB activation inhibition, compounds 1-3 have the activity NF-KB activation inhibition.

Abstract 243: Characterization of CDK5RAP3 in hepatocellular carcinoma

Abstract CDK5 regulatory subunit associated protein 3 (CDK5RAP3, also called C53 and LZAP) is a novel cyclin-dependent kinase 5 (CDK5) activator interacting protein that has been shown to regulate apoptosis induced by genotoxic stress. More recently, CDK5RAP3 has also been found to regulate p53 and NF-κB signaling. Here, we have characterized the role of CDK5RAP3 in hepatocellular carcinoma (HCC) cell lines. Using real-time quantitative RT-PCR, we found that 58% (39/67) of human HCC samples had significantly higher expression (at least 2-fold) of CDK5RAP3 transcripts as compared to their corresponding nontumorous livers. The overexpression of CDK5RAP3 was statistically correlated with clinicopathological feature of more metastatic phenotype. Using stable expression clones, we demonstrated that knockdown of CDK5RAP3 in HCC cell line inhibited anchorage-independent growth and tumorigenicity of cells while overexpression of CDK5RAP3 remarkably promoted tumorigenicity of cells. Our western blotting data indicated that knockdown of CDK5RAP3 in HCC cell line SMMC-7721 resulted in an upregulation of p14ARF. Using luciferase reporter assay, we showed that ectopic expression of CDK5RAP3 suppressed p14ARF promoter activity in a dose-dependent manner. Taken together, our data provide the first evidence that CDK5RAP3 is overexpressed in HCC and overexpression of CDK5RAP3 may contribute to the development of HCC via downregulation of p14ARF. In summary, CDK5RAP3 is a potential oncoprotein in promoting hepatocarcinogenesis, the regulation behind may attribute to its regulation on p14ARF expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 243.

Influence of Recombinant Human Growth Hormone (rhGH) on Proliferation of Hepatocellular Carcinoma Cells with Positive and Negative Growth Hormone Receptorsin Vitro

Recombinant human growth hormone (rhGH) is increasingly used in the clinic because it promotes the synthesis of proteins. However, rhGH is able to increase malignant transformation and tumor recurrence. The aim of this study was to investigate the effects of rhGH on hepatocellular carcinoma (HCC) cells with positive and negative growth hormone receptors (GHR) in order to guide its clinical application. Cells of the human HCC cell lines Bel-7402 (GHR+) and SMMC-7721 (GHR-) as well as human umbilical vein endothelial cell line ECV304 cells in the exponential growth phase were harvested and divided into experimental and control groups. After the human HCC cells were cultured alone or co-cultured with ECV304 cells under the different treatments, cell cycle phase, proliferation index, and expression levels of vascular endothelial growth factor (VEGF) mRNA and proteins were determined. In the Bel-7402 GHR+ cells treated with rhGH, both the percentage of cell in G2-M phase and the proliferation index were higher than those of controls (P < 0.05); this was not the case in the SMMC-7721 GHR- cells treated with rhGH (P > 0.05). Although there was no difference in the cell doubling times between ECV304 cells co-incubated with Bel-7721 GHR-cells treated with rhGH and without rhGH, the doubling times of ECV304 cells co-incubated with Bel-7402 GHR+ cells, when treated with rhGH, were significantly shortened compared to those of controls (P < 0.05). The cell doubling times of ECV304 cells co-incubated with Bel-7721 GHR- or Bel-7402 GHR+ cells which were treated with bevacizumab were longer than those of controls and of cells with rhGH (P < 0.05). The VEGF mRNA and protein expression levels were higher in Bel-7402 GHR+ cells treated with different doses of rhGH than controls (P < 0.05 or P < 0.01); however, there was no statistically significant difference in the expression levels of VEGF mRNA and proteins between SMMC-7721 GHR- cells treated with rhGH and controls. rhGH can induce VEGF secretion and stimulate proliferation of Bel-7402 GHR+ cells in vitro, but has little effect on the proliferation of SMMC-7721 GHR-cells, suggesting that rhGH may be applied safely to treatment for the catabolic state in patients with GHR-negative HCC.

Effect of polypeptides in bee venom on growth inhibition and apoptosis induction of the human hepatoma cell line SMMC-7721 in-vitro and Balb/c nude mice in-vivo

Polypeptides in bee venom (PBV) produced a significant growth inhibition against SMMC-7721 human hepatoma cell line. Analysis of the mechanisms of cell death indicated that PBV induced an apoptotic cell death. SMMC-7721 cells exposed to PBV (10.0 microg mL(-1)) produced an insignificant morphological change. Analysis of the cytotoxicity with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay confirmed that the cytotoxic effects of PBV were dose- and timedependent. The result of Ki67 immunohistochemistry demonstrated that the proliferation of SMMC-7721 cells treated with PBV (10.0 mug mL(-1)) was inhibited. The apoptotic cell death was then confirmed by annexin V, propidium iodide staining and DNA fragmentation analysis. In in-vivo experiments, treatment with PBV (1.5 or 3 mg kg(-1)) resulted in a significant retardation of SMMC-7721 cell growth in Balb/c nude mice. These findings suggested that PBV could be used as a chemotherapeutic agent against tumours.