MicroRNA-1269 promotes proliferation in human hepatocellular carcinoma via downregulation of FOXO1.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies and a major cause of cancer-related mortality in the world. MicroRNAs (miRNAs) are small, noncoding RNAs that play essential roles in various stages during cancer progression. The aim of the current study was to elucidate the role of miR-1269 in the pathogenesis of HCC. METHODS: The expression of miR-1269 in HCC cells and tissues were determined by Real-time PCR analysis. Cell viability, colony formation and anchorage-independent growth ability assays were performed to examine cell proliferative capacity and tumorigenicity. Flow cytometry analysis was conducted to determine cell cycle progression. The expression of p21, CyclinD1, phosphorylated Rb, Rb and FOXO1 were examined by Western blotting analysis. Luciferase assay was used to determine whether FOXO1 is the direct target of miR-1269. RESULTS: miR-1269 was upregulated in HCC cells and tissues. Ectopic miR-1269 expression promoted, but inhibition of miR-1269 reduced, proliferation, tumorigenicity and cell cycle progression of HCC cells. Furthermore, we demonstrated that FOXO1 was a direct target of miR-1269. Suppression of FOXO1 by miR-1269 was associated with dysregulation of p21, cyclin D1, phosphorylated Rb and Ki67 expression, thereby playing an essential role in the growth of HCC cells. CONCLUSIONS: Our study indicated that overexpression of miR-1269 promotes cell proliferation in HCC through directly suppressing FOXO1, and functions as an oncomiR in HCC.

[Transforming growth factor ß1 cooperates with stromal cell derived factor 1 to affect the proliferation of hepatic oval cells via ß-catenin inactivation].

OBJECTIVE: To investigate the role of stromal cell derived factor 1 (SDF-1) on the proliferation of hepatic oval cells, and the influencing factors. METHODS: Flow cytometry was used to detect the expression of CXCR4 on the cell surface when WB-F344 cells were growing in the culture medium with and without transforming growth factor ß1 (TGF-ß1) respectively. Western bolt was used to detect the expression of ß-catenin and its phosphorylation level. The translocation of ß-catenin was shown by confocal microscopy analysis. Q-RT-PCR was used in detecting the ß-catenin downstream gene expression such as Ccnd1 and c-Myc. MTT was used to detect the proliferation of WB-F344 cells which were treated by SDF-1 + TGF-ß1 and those cells exposed to SDF-1 or TGF-ß1 only, as well as of the negative control group. RESULT: WB-F344 cells rarely express CXCR4 under conventional circumstance, but this receptor can be up-regulated when the culture medium contain a modest amount of TGF-ß1 (the rate of CXCR4 positive cell increased by 39.5%). The bond of SDF-1 to CXCR4 results in the phosphorylation of ß-catenin, and its inactivation. SDF-1 alone didn't affect the proliferation of WB-F344 cells (0.512 ± 0.010 vs. 0.513 ± 0.008, t = 0.337, P > 0.05), while TGF-ß1 group show a slight decrease of cell population (0.393 ± 0.007,t = 28.001, P < 0.05). But when TGF-ß1 combined with SDF-1, the proliferation of WB-F344 was more weakened than TGF-ß1 group, and the difference was statistically significant (0.272 ± 0.009,t = 32.204, P < 0.05). CONCLUSIONS: TGF-ß1 can up-regulate the expression of CXCR4 in hepatic oval cells, and then inhibit the proliferation of hepatic oval cells via inactivating ß-catenin in vitro.

Rosiglitazone enhances 5-fluorouracil-induced cell growth inhibition in hepatocellular carcinoma cell line Hep3B.

BACKGROUND AND OBJECTIVE: Rosiglitazone is a peroxisome proliferators-activated receptor gamma (PPARgamma) ligand, which inhibits tumor growth by activating PPARgamma signaling pathways. Fluorouracil (5-FU) is one of the commonly used chemotherapeutic drugs. However, patients develop drug resistance of 5-FU over time. The aim of this study was to investigate whether rosiglitazone can enhance 5-FU-induced cell growth inhibition and to explore its potential mechanisms. METHODS: Cell viability was measured using MTT assay. Protein expression levels were detected by Western blot analysis. Small interference RNA was utilized to knockout PPARgamma and PTEN in Hep3B cells. RESULTS: After 48 h of treatment with 10, 20, and 40 µmol/L rosiglitazone, the viability of Hep3B cells was (78.0 ± 2.7)%, (37.3 ± 8.1)%, and (19.8 ± 2.2)%, respectively (compared with control group, P values were all < 0.001). After 48 h of treatment with 10 µmol/L 5-FU, the viability of Hep3B cells was about (82.6 ± 3.9)%. When cells were treated with 10 µmol/L 5-FU in combination with either 10, 20 or 40 µmol/L rosiglitazone, the cell viability was (51.6 ± 5.4)%, (14.8 ± 4.2)%, and (8.5 ± 0.9)%, with corresponding q value of 1.36, 1.23, and 1.19, respectively. These data suggested that the two drugs had synergic effect in inhibiting Hep3B cell growth, which was further confirmed in an in vivo mice model. Subsequent investigations showed that rosiglitazone activated PPARgamma signaling pathways and increased the expression of PTEN. CONCLUSIONS: Rosiglitazone enhances 5-FU-induced cell growth inhibition of Hep3B cells.

Efficacy of combining temperature- and power-controlled radiofrequency ablation for malignant liver tumors.

BACKGROUND AND OBJECTIVE: Single mode of radiofrequency ablation (RFA) often leads to limited ablation in the zone of necrosis. This study clarifies the efficacy of combining temperature- and power-controlled RFA for malignant liver tumors. METHODS: Between April 2008 and August 2008, 58 patients with malignant liver tumors received RFA at Sun Yat-sen University Cancer Center. The patients were divided into 2 groups using a random number table: one group received combined temperature- and power-controlled RFA (the combination group), and the other group received power-controlled RFA alone (the control group). RESULTS: Three patients were lost to follow-up and 55 patients were included for evaluation. Twenty-five patients with 29 tumors were treated by the combination RFA, and 27 tumors (93.1%) achieved either complete response (CR) or partial response (PR). One patient had a seriously decreased heart rate. In the control group, 30 patients with 32 tumors received power-controlled RFA, and 29 tumors (90.6%) achieved CR or PR. There were no serious complications. There was no difference between the combination and control groups in treatment time ((13.3 +/- 1.3) min vs. (10.2 +/- 2.3) min, P = 0.459). The number of sessions of RFA for the combination group was less than that of control group (1.3 sessions vs. 2.4 sessions), but the difference was not significant (P = 0.579). CONCLUSION: RFA controlling both temperature and power is effective and safe for patients with malignant liver tumors, and the number of sessions of RFA for the combination group was less than that of the control group.

[Risk factors of survival for recurrent hepatocellular carcinoma treated by percutaneous radiofrequency ablation].

OBJECTIVE: To explore the efficacy and determine the risk factors of survival for recurrent hepatocellular carcinoma(HCC) treated by percutaneous radiofrequency ablation (PRFA). METHODS: From January 1999 to December 2008, 82 patients with recurrent HCC, with the diameter less than 7 cm for solitary tumor, or the largest tumor less than 5 cm for multiple tumors(the number of tumors less than 3), were treated by PRFA. The significance of 12 clinical or pathological variables in the risk factors of overall survival were assessed. RESULTS: The overall survival 1-, 3-, and 5-year survival rates were 75.8%, 43.9% and 34.5% (from the date of PRFA), and 95.1%, 63.2% and 46.6% (from initial hepatectomy), respectively. Univariate analysis indicated that tumor size before initial hepatectomy, recurrence interval from initial hepatectomy, number of recurrent tumors, diameter of largest recurrent tumor, serum glutamyl transpeptidase (GGT) and serum albumin (ALB) level were significant prognostic factors (P < 0.05, Kaplan-Meier Log-rank test). Multivariate analysis showed recurrence interval from initial hepatectomy, diameter of largest recurrence tumor, serum GGT and ALB level were significant prognostic (P < 0.05). CONCLUSION: PRFA is effective for recurrent HCC. Recurrence interval from initial hepatectomy, diameter of largest recurrent tumor, serum GGT and ALB level are significant prognostic factors.

Rosiglitazone sensitizes hepatocellular carcinoma cell lines to 5-fluorouracil antitumor activity through activation of the PPARgamma signaling pathway.

AIM: Resistance to 5-fluorouracil (5-FU) is a major cause of chemotherapy failure in advanced hepatocellular carcinoma (HCC). Rosiglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has a crucial role in growth inhibition and induction of apoptosis in several carcinoma cell lines. In this study, we examine rosiglitazone-induced sensitization of HCC cell lines (BEL-7402 and Huh-7 cells) to 5-FU. METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. Western blotting analysis was performed to detect the protein expression (PPARgamma, PTEN, and COX-2) in BEL-7402 cells. Immunohistochemistry staining was used to examine the expression of PTEN in 100 advanced HCC tissues and paracancerous tissues. In addition, small interfering RNA was used to suppress PPARgamma, PTEN, and COX-2 expression. RESULTS: Rosiglitazone facilitates the anti-tumor effect of 5-FU in HCC cell lines, which is mediated by the PPARgamma signaling pathway. Activation of PPARgamma by rosiglitazone increases PTEN expression and decreases COX-2 expression. Since distribution of PTEN in HCC tissues is significantly decreased compared with the paracancerous tissue, over-expression of PTEN by rosiglitazone enhances 5-FU-inhibited cell growth of HCC. Moreover, down-regulation of COX-2 is implicated in the synergistic effect of 5-FU. CONCLUSION: Rosiglitazone sensitizes hepatocellular carcinoma cell lines to 5-FU antitumor activity through the activation of PPARgamma. The results suggest potential novel therapies for the treatment of advanced liver cancer.

Green tea polyphenol epigallocatechin-3-gallate enhances 5-fluorouracil-induced cell growth inhibition of hepatocellular carcinoma cells.

AIM: 5-Fluorouracil (5-FU) is one of the most commonly used chemotherapeutic drugs. Resistance to 5-FU is a major cause of chemotherapy failure in advanced-stage hepatocellular carcinoma (HCC). Green tea polyphenol Epigallocatechin-3-gallate (EGCG) plays a critical role in growth inhibition and apoptotic induction in HCC cell lines. The aim of this study is to investigate whether EGCG can enhance 5-FU-induced cell growth inhibition and to explore its potential mechanisms. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cell growth. Western blotting analysis was performed to detect the proteins expression in Hep3B cells. Small interfering RNA was used to suppress cyclooxygenase-2 (COX-2) expression. Furthermore, enzyme linked immunosorbent assay was used to test the prostaglandin E(2) (PGE(2) ) production in cell cultures. RESULTS: Epigallocatechin-3-gallate augmented the anti-tumor effect of 5-FU in Hep3B cells. Significant difference was observed between the treated groups and the control group (P < 0.05). EGCG (its concentrations at over 5 µmol/L) combined with 5-FU presented a synergic effect. Furthermore, the combination of EGCG and 5-FU abrogated the COX-2 overexpression and PGE(2) secretion induced by 5-FU. The upregulation of COX-2 expression decreased the phosphorylation of Akt (Thr(308) ) expression. These appeared to be followed by the AMPK hyperactivation. CONCLUSION: Epigallocatechin-3-gallate sensitizes HCC cells to 5-FU antitumor activity, and the combination of EGCG and 5-FU exhibits synergism in chemo-resistant cancer cells. The results suggest potential novel therapies for the treatment of advanced-stage liver cancer.