Down-regulated clusterin expression enhances sensitivity of hepatoma cells to anti-cancer drugs / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between and underlying mechanistic pathway of clusterin (CLU) and chemo-resistance ofhepatocellular carcinoma (HCC) cells.</p><p><b>METHODS</b>CLU protein expression in HCC cell lines (Hep3B, SMMC7721, PLC, and HepG2) and HepG2/ADM cells was quantified by western blotting. Four short-hairpin (sh)RNAs designed to block CLU-mRNA were generated, screened by RT-PCR, and transfected into the cells to determine effects of CLU on cell viability and apoptosis. Effects of CLU blockade on drug efflux pump activity were measured by flow cytometry.</p><p><b>RESULTS</b>CLU was found to be over-expressed in HCC cell lines and HepG2/ADM cells. The four shRNAs inhibited CLU-mRNA as follows (vs. levels in untransfected cells): shRNA-1: 73.68% (q =23.011, P < 0.01), shRNA-2: 39.26% (q =11.991, P < 0.01), shRNA-3: 62.36% (q =19.392, P < 0.01), and shRNA-4: 55.35% (q =17.149, P < 0.01). shRNA-mediated depletion of CLU led to increased sensitivity to anti-cancer drugs and increased doxorubicin-induced apoptosis in HepG2/ADM cells, as evidenced by the apoptosis ratio of the shRNA-1 group of 39.28% vs. the apoptosis ratio of the untransfected control group of 4.92%. Silencing of CLU also decreased drug etflux pump activity, and the level of MDR1/P-gp expression was significantly reduced (shRNA-1 group vs.untransfected control group: q =14.604, P < 0.01).</p><p><b>CONCLUSION</b>CLU repression may enhance sensitivity of HCC cells to anti-cancers drugs and represents a potential molecular-target for reversal of multidrug-resistant HCC.</p>

Short hairpin RNA mediated glypican-3 silencing inhibits hepatoma cell invasiveness and disrupts molecular pathways of angiogenesis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms.</p><p><b>METHODS</b>GPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2, MHCC-97H, and Huh7. shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting, respectively. The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays, on migration by wound healing (scratch) assay, on invasion by transwell chamber assay, and on apoptosis by luminescence assay of caspase-3/7 activity. The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor, GLI1, and the beta-catenin Wnt signaling factor, b-catenin), immunofluorescent staining (for the insulin-like growth factor-II, IGF-II), and ELISA (for the vascular endothelial growth factor, VEGF). Pairwise comparisons were made by the independent sample t-test, and multiple comparisons were made by one-way ANOVA.</p><p><b>RESULTS</b>In all cell lines, transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (% reduction as compared to the non-target control shRNA: HepG2, 89.2+/-6.0%, t = -25.753, P less than 0.001; MHCC-97H, 75.3+/-4.9%, t = -26.487, P less than 0.001; Huh7, 73.6+/-4.6%, t = -27.607, P less than 0.001); the GPC-3 protein levels were similarly reduced. The GPC-3 shRNA-silenced cells showed significantly reduced proliferative, migratory and invasive capacities, as well as significantly increased apoptosis. The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of b-catenin mRNA (HepG2, 46.9+/-0.6%; MHCC-97H, 67.5+/-2.7%; Huh7, 56.3+/-8.4%) and significant up-regulation of GLI1 mRNA (HepG2, 49.2+/-28.6%; MHCC-97H, 54.6+/-24.4%; Huh7, 31.6+/-15.7%). At 72 h after transfection, the HepG2 cells showed significant down-regulation of VEGF protein (54.3+/-1.5%, t = 46.746, P less than 0.001).</p><p><b>CONCLUSION</b>GPC-3 contributes to migration, invasion, angiogenesis, and apoptosis of hepatoma cells, possibly through its interactions with the Wnt/b-catenin and Hedgehog signaling pathways. GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.</p>

Effects of inhibited IGF-IR expression on proliferation and apoptosis of human hepatocellular carcinoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic value of inhibiting the expression of insulin-like growth factor-I receptor (IGF-IR) using picropodophyllin (PPP) by studying the effects on proliferative and metastatic potentials of human hepatocellular carcinoma (HCC) using an in vitro cultured cell system.</p><p><b>METHODS</b>IGF-IR expression in human HCC cell lines (Bel-7404, Bel-7402, HepG2, and Huh-7) and human hepatocytes (L02) was assessed at baseline (pre-treatment) and after PPP treatment by western blotting. Changes in cell cycle were analyzed by flow cytometry and in cell viability by sulforhodamine B staining. Early apoptosis was detected by annexin-V/FITC and propidium iodide double-staining assay. Caspase-3/7 activity was suppressed by z-VAD-FMK and analyzed by homogeneous luminescence assay. Effects on cell motility were tested by wound-scratch test. Between-group differences were assessed by t-test or one-way analysis of variance.</p><p><b>RESULTS</b>IGF-IR was markedly up-regulated in all HCC cell lines (vs. non-hepatoma hepatocytes). HCC cells with PPP-inhibited IGF-IR showed time-dependent decreases in cell motility and viability. After treatment with PPP for 24 hours, the proportion of HCC cells in G1 phase was 2.1% +/- 0.4%, in S phase was 11.0% +/- 0.7%, and in G2/M phase was 87.1% +/- 0.6%, and no healing was observed in the wound-scratch assay. The PPP treatment induced cell apoptosis, as evidenced by enhanced caspase-3/7 activity; the proportion of annexin-V+/PI- cells was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (16.4% +/- 0.4% vs. 5.8% +/- 0.2%, t = 14.05, P less than 0.01). After z-VAD-FMK treatment, the apoptosis rate was significantly higher in the HepG2 cells than in the non-hepatoma hepatocytes (11.3% +/- 0.7% vs. 5.8% +/- 0.2%, t = 11.83, P less than 0.01).</p><p><b>CONCLUSION</b>IGF-IR is associated with proliferation, cell motility, and apoptosis of HCC cells, and may be a promising molecular target for HCC.</p>

Correlation between epigenetic alterations in the insulin growth factor-II gene and hepatocellular carcinoma / 中华肝脏病杂志

To investigate whether epigenetic alterations in the insulin-like growth factor-II (IGF-II) gene that cause differential transcription or expression are correlated with onset and severity of hepatocellular carcinoma (HCC). Patient-matched specimens of HCC, paracancerous, and non-cancerous tissues were collected from 40 primary liver cancer patients. Epigenetic alterations in the promoter (P3) sequence of the IGF-II gene were analyzed by methylation-specific PCR (MSP) and IGF-II transcription was measured by RT-PCR. IGF-II protein expression and clinicopathological features were assessed by immunohistochemistry and microscopic observation. The rate of IGF-II P3 methylation was significantly lower in HCC tissues (0%) than in paracancerous tissues (vs. 47.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 100%; x2 = 80.000, P less than 0.001). IGF-II mRNA expression was significantly higher in HCC tissues (100%) than in paracancerous tissues (vs. 52.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 80.000, P less than 0.001). IGF-II protein expression was significantly higher in HCC tissues (82.5%) than in paracancerous tissues (vs. 45.0%; x2 = 12.170, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 56.170, P less than 0.001). IGF-II overexpression in HCC was significantly associated with degree of differentiation, extent of infiltrated serosa, size of tumor, and HBV-positive infection status. Epigenetic alterations in the IGF-II gene regulate its transcription and expression and are closely associated with HCC development and progression.

Expression features of glypican-3 and its diagnostic and differential values in hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression features of glypican-3 (GPC-3) and its diagnostic and differential values in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Rat hepatoma models were made and the dynamic expression features of GPC-3 protein and its gene were investigated by Western blotting and RT-PCR respectively. Liver specimens from 36 HCC patients were collected by self-control method and the expression and clinicopathological features of GPC-3 were analyzed by immunohistochemistry. Serum GPC-3 levels were quantitatively detected by ELISA and its efficiency for HCC diagnosis was evaluated in patients with liver diseases.</p><p><b>RESULTS</b>The incidence of GPC-3 was 0% in control, 83.3% in degeneration, 100% in precancerosis and 100% in canceration during dynamic formation of rat hepatoma, respectively. The positive GPC-3 was brown granule- like staining localized in membrane and cytoplasm in human HCC.</p><p><b>CONCLUSIONS</b>The GPC-3 positive rates were 80.6% in HCC, 41.7% in surrounding tissues and none in distal tissues (P < 0.01), respectively. No positive relationship presented between GPC-3 and differentiation grade or the number of tumor except of tumor size (Z = 2.941, P < 0.01). The incidence of serum GPC-3 was 52.8% in HCC patients except of one patient with cirrhosis. No significant differences were found between GPC-3 and sex, age, AFP, tumor number, Child classification or extrahepatic metastasis except of tumor size (χ² = 6.318, P < 0.05) and HBV infection (χ² = 23.362, P < 0.01). Combined detection of GPC-3 and AFP could rise up diagnosis of HCC. GPC-3 expression closely associated with HCC and might be useful for early diagnosis of HCC.</p>

Inhibitory effect of miRNA silencing hypoxia-inducible factor alpha subunit gene on the proliferation of HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of miRNA silencing HIF-1α gene on the proliferation of HepG2 cells.</p><p><b>METHODS</b>The eukaryotic expression plasmids of HIF-1α miRNA and report gene containing hypoxia-reponse element were constructed and transfected into HepG2 cells. The expressions of HIF-1α gene and protein were determined by real time-PCR and Western blotting. The expressions of HIF-1α, vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were quantitatively detected by ELISA. The alterations of cell cycles and apoptosis rate were quantitatively measured by flow cytometry and Annexin V-FITC/PI double dyeing assay.</p><p><b>RESULTS</b>72 h after transfection the down regulations of HIF-1α mRNA and protein were 87% and 56% respectively, and the decrease of target gene was 46% in the report gene, 54% in VEGF and 36% in Ang-2, respectively. The apoptotic ratio of HepG2 cells was 22.46+/-0.61% (P < 0.01). The cell cycle changed greatly at the ratio of G1 (61.49+/-1.12%) and S (22.40+/-0.58%, P < 0.01). After being combined with doxorubicin, the apoptotic ratio increased to 36.99+/-0.88% and the ratios of G1 and S phases were upregulated to 65.68+/-0.91% and 19.47+/-1.34% respectively.</p><p><b>CONCLUSIONS</b>HIF-1α miRNA or / and doxorubicin can regulate the growth cycles of HepG2 cells, promote the cell apoptosis and inhibit the cell proliferation.</p>

Effect of siRNA-mediated inhibition of nuclear transcription factor-kappa B on apoptosis of hepatocarcinoma cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of siRNA-mediated inhibition of NF-κB on apoptosis of hepatocarcinoma cells.</p><p><b>METHODS</b>Specific small interfering RNA Targeting NF-κB gene was synthesized and transfected into HepG2 cells by liposomes. Nested RT-PCR and quantitative RT-PCR were used to detect the mRNA expression of NF-κB. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blot were performed to examine the protein expression of NF-κB. Annexin V-FITC was used to test cell apoptosis.</p><p><b>RESULTS</b>The expression of NF-κB in HepG2 cells (1.13+/-0.03) was significantly higher (t=27.02, P<0.05) than that in normal hepatocytes (0.29+/-0.07). The down-regulation of NF-κB expression was depended on the dosage of siRNA and the time after transfection. 72 h after siRNA transfection, NF-κB expression reduced by 93% and 62% at the mRNA and protein levels, respectively. The apoptosis of HepG2 cells increased by 85% with NF-κB inhibition.</p><p><b>CONCLUSIONS</b>NF-κB is abnormally active in HepG2 cells and NF-κB inhibition mediated by siRNA promotes HepG2 cells apoptosis. It suggested that NF-κB could be a potential target for HCC prevention gene therapy.</p>

Expression characteristics of hypoxia inducible factor-1a and its clinical values in hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic expression of hypoxia inducible factor-1alpha (HIF-1alpha) and its clinical values in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The dynamic changes of liver pathology, HIF-1alpha transcription and expression were observed through the hepatoma model. The self-control specimens from 35 human HCC patients were collected and the expression, cellular distribution, and clinicopathological features of HIF-1alpha and its gene was analyzed by immunohistochemistry, western blotting and nested- PCR, respectively.</p><p><b>RESULTS</b>Both levels of hepatic HIF-1alpha and HIF-1alpha mRNA expression increased during the HCC development course. The incidence of HIF-1alpha and the ratio of HIF-1alpha to beta-actin was 0% and 0.16+/-0.02 in the control rats, 77.8% and 0.29+/-0.04 in the denatured rats, 88.9% and 0.52+/-0.03 in the precancerous rats, and 100% and 0.84+/-0.02 in the cancerous rats respectively, with significant difference between the control group and any of the experimental groups (P = 0.000). The positive HIF-1alpha was brown and granule-like and mainly presented in cytoplasm and few in nucleus. The incidence of HIF-1alpha was 80% (28/35) in HCC and 100% (35/35) in its surrounding tissues. The clinical pathological features indicated HIF-1alpha expression associated with tumor size and differentiation degree the of HCC. No correlation was found between HIF-1alpha and tumor numbers or positive-HBsAg.</p><p><b>CONCLUSIONS</b>HIF-1alpha expression is associated with occurrence and development of HCC, and is perhaps a target molecule for HCC therapy.</p>

Dynamic expression and quantitative analysis of hepatic nuclear factor-kappa B and its gene during the development of hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the kinetic expression and alteration of nuclear transcription factor-kappa B (NF-kappaB) and its gene in hepatocellular carcinoma (HCC) development.</p><p><b>METHODS</b>A hepatoma model was established with N-(2-fluorenyl) acetamide (2-FAA) using male SD rats. Morphological changes and dynamic alterations of NF-kappaB and NF-kappaB mRNA of the rat livers at different stages of HCC development were observed by pathological examinations. The liver specimens from HCC patients were collected by self-control method. The expression of NF-kappaB was quantitatively analyzed by ELISA.</p><p><b>RESULTS</b>Hepatocytes showed vacuole-like denaturation, atypical hyperplasia, and transformation into highly differentiated cancerous hepatocytes with increasing tendencies of liver NF-kappaB and NF-kappaB mRNA expressions. The NF-kappaB positive material was granule-like and stained brown, with dot-nest-like staining localized in the nuclei and cytoplasm of HCC cells, but only in the cytoplasm of the cells of park cancer tissues. Its expression in HCC cells was stronger than that in their surrounding tissues (chi2 = 13.1, P less than 0.01). No positive relationship was found between NF-kappaB expression and histological grades, the number of tumors, or size of the tumors.</p><p><b>CONCLUSION</b>The expression of NF-kappaB and its gene are associated with the development of HCC. To inhibit the expression may be useful to HCC therapy.</p>

An evaluation of transforming growth factor-beta 1 in diagnosing hepatocellular carcinoma and metastasis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic expressions of TGF-beta 1 and TGF-beta 1 mRNA at different stages of hepatocellular carcinoma (HCC) development and their use in clinical diagnosis.</p><p><b>METHODS</b>Hepatoma models were developed with 2-FAA using male Sprague-Dawley (SD) rats. Morphological changes of the rat liver histological preparations (H and E stained) were studied. The fragment of TGF-beta 1 gene obtained was amplified by nested RT-PCR. Dynamic change of TGF-beta 1 level was quantitatively analyzed by ELISA. The distribution of TGF-beta 1 in the cells and its gene expression were detected in human HCC tissues.</p><p><b>RESULTS</b>The progressive increases of hepatic TGF-beta 1 and TGF-beta 1 mRNA were observed in rat hepatocytes which progressed from granular degeneration, atypical hyperplasia and finally to HCC development induced by 2-FAA. The expression levels in HCC tissues were significantly higher than those in the normal and degenerative ones. TGF-beta 1 was shown in rat hepatocytes by immunohistochemistry. Plasma TGF-beta 1 was detected in 89.5% of all the patients with HCC, but it was detected in 93.3% of them who had an AFP less than 400 microg/L. TGF-beta 1 mRNA showed a stronger expression in HCC tissues. TGF-beta 1 mRNA was found in peripheral blood mononuclear cells from all HCC patients with extrahepatic metastasis.</p><p><b>CONCLUSION</b>TGF-beta 1 may participate in hepatocyte canceration. The overexpression of TGF-beta 1 and TGF-beta 1 mRNA could be useful markers for early diagnosis and predicting prognosis of HCC.</p>

Clinical values of vascular endothelial growth factor expression and microvascular density analysis in liver cancer specimens / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the roles of vascular endothelial growth factor (VEGF) in microvessel angiogenesis, development and metastasis of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The cellular distributions of VEGF expression and microvascular density (MVD) in 36 HCCs were investigated, and the levels of total RNA and VEGF were detected in HCCs, Para cancerous, and distal cancerous tissues, respectively.</p><p><b>RESULTS</b>The incidence of VEGF was 63.9% in 36 cases of HCCs, 78.3% in non-encapsulated HCCs, and 90.9% in HCCs with extrahepatic metastasis, respectively. The VEGF expression was tightly correlated with MVD (t=4.49, P<0.01). No significant difference was found between VEGF or MVD and tumor diameter or differentiation degree. The level of total RNA in HCCs was lower but the VEGF level significantly higher than those of Para cancerous or distal cancerous ones (q=6.10, P<0.01).</p><p><b>CONCLUSION</b>The present data suggest that VEGF over expression and MVD abnormality are useful markers for vascular invasion and metastasis of liver tumors.</p>