Action In Hepatocellular Carcinoma Research Articles

DNA Methylation-mediated Repression of miR-941 Enhances Lysine (K)-specific Demethylase 6B Expression in Hepatoma Cells

MicroRNAs (miRNAs) have been shown to play important roles in carcinogenesis. However, their underlying mechanisms of action in hepatocellular carcinoma (HCC) are poorly understood. Recent evidence suggests that epigenetic silencing of miRNAs through tumor suppression by CpG island hypermethylation may be a common hallmark of human tumors. Here, we demonstrated that miR-941 was significantly down-regulated in HCC tissues and cell lines and was generally hypermethylated in HCC. The overexpression of miR-941 suppressed in vitro cell proliferation, migration, and invasion and inhibited the metastasis of HCC cells in vivo. Furthermore, the histone demethylase KDM6B (lysine (K)-specific demethylase 6B) was identified as a direct target of miR-941 and was negatively regulated by miR-941. The ectopic expression of KDM6B abrogated the phenotypic changes induced by miR-941 in HCC cells. We demonstrated that miR-941 and KDM6B regulated the epithelial-mesenchymal transition process and affected cell migratory/invasive properties.

Bortezomib effect on E2F and cyclin family members in human hepatocellular carcinoma cell lines

To evaluate the effects of the proteasome inhibitor bortezomib (BZB) on E2Fs and related genes in hepatocellular carcinoma (HCC) cells. The mRNA levels of the E2F family members (pro-proliferative: E2F1-3 and anti-proliferative: E2F4-8) and of their related genes cyclins and cyclin-dependent kinases (cdks) were evaluated in two HCC cell lines following a single BZB administration. mRNA levels of the epithelial-mesenchymal transition (EMT) genes were also measured in both cell lines after BZB treatment. The BZB concentration (40 nmol/L) used was chosen to stay well below the maximal amount/cm² recommended for in vivo application, and 2 d incubation was chosen as this time point has been found optimal to detect BZB effects in our previous studies. The HCC cell lines, HepG2 and JHH6, were chosen as they display different phenotypes, hepatocyte-like for HepG2 and undifferentiated for JHH6, thus representing an in vitro model of low and high aggressive forms of HCC, respectively. The mRNA levels of the target genes were measured by two-color microarray-based gene expression analysis, performed according to Agilent Technologies protocol and using an Agilent Scan B. For the E2F family members, mRNA levels were quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). Using small interfering RNA's, the effects of E2F8 depletion on cell number was also evaluated. After BZB treatment, microarray analysis of the undifferentiated JHH6 revealed a significant decrease in the expression of the pro-proliferative E2F member E2F2. Quantitative RT-PCR data were in keeping with the microarray analysis, and showed a significant increase and decrease in E2F8 and E2F2 mRNA levels, respectively. In contrast, BZB treatment of the hepatocyte-like HCC cell line HepG2 had a significant impact on mRNA levels of 5 of the 8 E2F members. In particular, mRNA levels of the pro-proliferative E2F members E2F1, E2F2, and of the anti-proliferative member E2F8, decreased over 80%. Notably, a reduction in E2F8 expression in HepG2 and JHH6 cells following siRNA treatment had no impact on cell proliferation. As observed with JHH6, BZB treatment of HepG2 cells induced a significant increase in mRNA levels of an anti-proliferative E2F member, E2F6 in this case. As was observed with E2F's, more dramatic changes in mRNA levels of the E2F related genes cyclins and Cdks and EMT genes were observed after BZB treatment of HepG2 compared to JHH6. The differential expression of E2Fs and related genes induced by BZB in diverse HCC cell phenotypes contribute to bortezomib's mechanism of action in hepatocellular carcinoma.

Downregulation of FGL2/prothrombinase delays HCCLM6 xenograft tumour growth and decreases tumour angiogenesis

Fibrinogen-like protein 2 (FGL2), which directly generates thrombin from prothrombin without activation of the conventional coagulation cascade, was shown to be overexpressed in various human malignant tumours. Herein, we aimed to investigate its expression pattern, biological function and mechanism of action in hepatocellular carcinoma (HCC). FGL2 expression and colocalization with fibrin was examined in 15 HCC tissues. FGL2 downregulation was performed by targeting microRNA in a HCCLM6 cell line in which FGL2 was highly expressed in xenografts of nude mice. The effects of FGL2 knockdown on tumour growth and angiogenesis were evaluated in vitro and in vivo. Cytometric bead arrays were employed to identify FGL2-regulated signalling pathways. FGL2 was overexpressed in HCC tissues and colocalized with fibrin deposition. Knockdown of FGL2 expression in HCCLM6 cells (hFGL2(low) HCCLM6) resulted in delayed xenografts tumour growth within an observation period of 42 days and decreased vascularization, which was accompanied by decreased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In vitro hFGL2(low) HCCLM6 cells exhibited decreased proliferation without significant induction of apoptosis. Overexpression of FGL2 in HCCLM6 cells or addition of recombinant hFGL2 protein induced phosphorylation of p38-MAPK and ERK1/2 involving protease-activated receptors (PARs).activation. FGL2 contributes to HCC tumour growth and angiogenesis in a thrombin-dependent manner, and downregulation of its expression might be of therapeutic significance in HCC.

Dehydroepiandrosterone supplement increases malate dehydrogenase activity and decreases NADPH-dependent antioxidant enzyme activity in rat hepatocellular carcinogenesis

Beneficial effects of dehydroepiandrosterone (DHEA) supplement on age-associated chronic diseases such as cancer, cardiovascular disease, insulin resistance and diabetes, have been reported. However, its mechanism of action in hepatocellular carcinoma in vivo has not been investigated in detail. We have previously shown that during hepatocellular carcinogenesis, DHEA treatment decreases formation of preneoplastic glutathione S-transferase placental form-positive foci in the liver and has antioxidant effects. Here we aimed to determine the mechanism of actions of DHEA, in comparison to vitamin E, in a chemically-induced hepatocellular carcinoma model in rats. Sprague-Dawley rats were administered with control diet without a carcinogen, diets with 1.5% vitamin E, 0.5% DHEA and both of the compounds with a carcinogen for 6 weeks. The doses were previously reported to have anti-cancer effects in animals without known toxicities. With DHEA treatment, cytosolic malate dehydrogenase activities were significantly increased by ~5 fold and glucose 6-phosphate dehydrogenase activities were decreased by ~25% compared to carcinogen treated group. Activities of Se-glutathione peroxidase in the cytotol was decreased significantly with DHEA treatment, confirming its antioxidative effect. However, liver microsomal cytochrome P-450 content and NADPH-dependent cytochrome P-450 reductase activities were not altered with DHEA treatment. Vitamin E treatment decreased cytosolic Se-glutathione peroxidase activities in accordance with our previous reports. However, vitamin E did not alter glucose 6-phosphate dehydrogenase or malate dehydrogenase activities. Our results suggest that DHEA may have decreased tumor nodule formation and reduced lipid peroxidation as previously reported, possibly by increasing the production of NADPH, a reducing equivalent for NADPH-dependent antioxidant enzymes. DHEA treatment tended to reduce glucose 6-phosphate dehydrogenase activities, which may have resulted in limited supply for de novo synthesis of DNA via inhibiting the hexose monophophaste pathway. Although both DHEA and vitamin E effectively reduced preneoplastic foci in this model, they seemed to function in different mechanisms. In conclusion, DHEA may be used to reduce hepatocellular carcinoma growth by targeting NADPH synthesis, cell proliferation and anti-oxidant enzyme activities during tumor growth.

The tamoxifen-induced suppression of telomerase activity in the human hepatoblastoma cell line HepG2: a result of post-translational regulation

Patients with advanced hepatocellular carcinoma (HCC) have shown to benefit from tamoxifen treatment. The mechanisms of tamoxifen action in HCC, however, are not yet clearly understood. Results from studies on the human hepatoblastoma cell line HepG2 provide evidence that estrogen-receptor-alpha-independent antiproliferative actions of tamoxifen in HCC are mediated by the suppression of telomerase activity [5]. We investigate the pathway of the tamoxifen-induced down-regulation of telomerase activity, using HepG2 cells incubated over 24 h or 48 h in the presence of 20 microM tamoxifen. The transcriptional levels of the three telomerase core components-human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT) (all variants), and telomerase-associated protein (TP1)-did not change during tamoxifen treatment, as revealed by RT-PCR analysis. Furthermore, the hTERT splice pattern was not shifted from the active full-length variant (+alpha/+beta) to the inactive deletion variants (-alpha; -beta; -alpha/-beta) and the level of the 120 kDa hTERT full-length protein remained constant, as shown by Western blot analysis. Protein kinase C (PKC) activity has been suggested to be crucial for post-translational up-regulation of telomerase activity. In HepG2 cells, we observed a tamoxifen-induced suppression of the total protein kinase C (PKC) activity (cytosolic and membrane-bound). Inhibition of PKC with bisindolylmaleimide I resulted in a reduction of telomerase activity, as revealed by TRAP-assay. Alpha-tocopherol (vitamin E) diminished the effects of tamoxifen on PKC-activity as well as on telomerase activity. We conclude that the tamoxifen-induced decrease of telomerase activity in HepG2 cells is mediated post-translationally via suppression of PKC-activity.