GSK-3ß suppresses the proliferation of rat hepatic oval cells through modulating Wnt/ß-catenin signaling pathway.

AIM: Glycogen synthase kinase 3ß (GSK-3ß) plays a crucial role in hepatic biology, including liver development, regeneration, proliferation and carcinogenesis. In this study we investigated the role of GSK-3ß in regulation of growth of hepatic oval cells in vitro and in liver regeneration in partially hepatectomized rats. METHODS: WB-F344 cells, the rat hepatic stem-like epithelial cells, were used as representative of oval cells. Cell viability was examined using a WST-8 assay. The cells were transfected with a recombinant lentivirus expressing siRNA against GSK-3ß (GSK-3ßRNAiLV) or a lentivirus that overexpressed GSK-3ß (GC-GSK-3ßLV). Adult rats underwent partial (70%) hepatectomy, and liver weight and femur length were measured at d 7 after the surgery. The expression of GSK-3ß, phospho-Ser9-GSK-3ß, ß-catenin and cyclin D1 was examined with immunoblotting assays or immunohistochemistry. RESULTS: Treatment of WB-F344 cells with the GSK-3ß inhibitor SB216763 (5 and 10 µmol/L) dose-dependently increased the levels of phospho-Ser9-GSK-3ß, but not the levels of total GSK-3ß, and promoted the cell proliferation. Knockout of GSK-3ß with GSK-3ßRNAiLV increased the cell proliferation, whereas overexpression of GSK-3ß with GC-GSK-3ßLV decreased the proliferation. Both SB216763 and GSK-3ßRNAiLV significantly increased the levels of ß-catenin and cyclin D1 in the cells, whereas GSK-3ß overexpression decreased their levels. In rats with a partial hepatectomy, administration of SB216763 (2 mg/kg, ip) significantly increased the number of oval cells, the levels of phospho-Ser9-GSK-3ß, ß-catenin and cyclin D1 in liver, as well as the ratio of liver weight to femur length at d 7 after the surgery. CONCLUSION: GSK-3ß suppresses the proliferation of hepatic oval cells by modulating the Wnt/ß-catenin signaling pathway.

[Regulation effect of adipose-derived mesenchymal stem cells on pancreatic cancer cells].

OBJECTIVE: To analyze the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer (PaCa) cells and the the possible mechanism involved. METHODS: ADSCs were isolated and co-cultured with PaCa cells. CCK-8 assay was used to detect the proliferation of PaCa cells. An ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. The proliferation of PaCa cells by SDF-1 was measured. AMD3100 regulated the co-culture of ADSCs and PaCa. The tumor growth of PaCa cells was assessed after treatment by ADSCs in vivo. RESULTS: ADSCs can promote the proliferation and invasion of PaCa cells (proliferation: SW1990: 1.535 ± 0.153; PANC-1: 1.370 ± 0.100; the value of control was 1; invasion: SW1990: 47.0 ± 2.6 vs. 28.3 ± 1.3; PANC-1: 40.3 ± 1.8 vs. 24.3 ± 1.3; t = 4.332-9.558, P < 0.05). The expression of SDF-1 was high in ADSCs, but not in PaCa cells (69 ± 5 vs. 0 and 0, F = 389.134, P < 0.01). The promotion of SDF-1 on PaCa cells depends on the concentration. AMD3100 significantly downregulates these growth-promoting effects of ADSCs on PaCa cells. ADSCs significantly promoted the growth of SW1990 in nude mice at the 5(th) week (volume: (1295 ± 102) mm(3) vs. (967 ± 81) mm(3), t = 5.614, P < 0.05) , but not in PANC-1 cell. CONCLUSION: ADSCs can promote the proliferation and invasion of PaCa cells, which may involve the SDF-1/CXCR4 axis.

Decreased expression of LKB1 correlates with poor prognosis in hepatocellular carcinoma patients undergoing hepatectomy.

AIM: To study any correlation of LKB1 expression with prognosis in hepatocellular carcinoma (HCC) cases. METHODS: A total of 70 HCC patients and 20 primary intrahepatic stone patients in the first affiliated hospital of Wenzhou Medical College were enrolled in this study. LKB1 expression was detected by immunohistochemistry. Patients were followed-up and prognostic factors were evaluated. RESULT: LKB1 expression was decreased in the HCC samples. Loss of LKB1 expression in HCC was significantly related to histologic grade (P=0.010), vascular invasion (P=0.025) and TMN stage (P=0.011). Patients showing negative LKB1 expression had a significantly shorter disease-free and overall survival than those with positive expression (P = 0.001, P=0.000, respectively). Multivariate Cox regression analysis indicated that LKB1 expression level was an independent factor of survival (P = 0.033). CONCLUSION: HCC patients with decreased expression LKB1 have a poor prognosis. The loss of LKB1 expression is correlated with a lower survival rate.

[Effects of glycogen synthase kinase 3ß overexpression in rat and glycogen synthase kinase 3ß inhibitor SB-216763 on proliferation of hepatic oval cells].

OBJECTIVE: To research the effects of glycogen synthase kinase (GSK3ß) overexpression and GSK3ß inhibitor SB-216763 on the proliferation of hepatic oval cells in rats and its regulatory mechanisms by Wnt signaling pathway. METHODS: The hepatic oval cells WBF-344 were divided into the blank control group, GSK3ß over-expression group, DMSO control group and GSK3ß inhibitor groups, while the inhibitor groups set up three concentration gradients, that was 1, 5, 10 µmol/L. Using the GSK3ß over-expression lentivirus, which had been identified correctly, and SB-216763 dealt with the cells WBF-344. The cells morphology of each group was observed under the phase contrast inverted microscope, and the expression of fluorescence in the lentivirus-transfected group was observed under the fluorescent microscope. The proliferation of each group cells was tested by CCK8 kits. The cells' apoptosis was detected by AnnexinV-FITC/PI kits. The expression of GSK3ß, ß-catenin and cyclin D1 were detected by Western blot. RESULTS: The cells of GSK3ß over-expression group were fewer and obvious aging. However, in each inhibitor added group, the cells' division and proliferation was vigorous, and the condition was good. Moreover, the cells' proliferation was getting stronger with the concentration of SB-216763 increasing. A large number of green fluorescence was expressed in the lentivirus-transfected cells. The cells' proliferation in GSK3ß over-expression group restrained (t = 7.178, P < 0.01, as compared with control), while the cells' proliferation was vigorous in inhibitor groups (F = 45.030, P < 0.01, as compared with control). Flow Cytometry showed that the cells apoptosis was significant in GSK3ß over-expression group. Western blot showed that the expression of GSK3ß was increased, while the expression of ß-catenin and cyclin D1 was decreased in the over-expression group. The expression of GSK3ß had no significant difference among the control group and inhibitor groups. However, the expression of ß-catenin and cyclin D1 was significantly increased with the concentration of SB-216763 increasing. CONCLUSIONS: The overexpression of GSK3ß can inhibit the Wnt signaling pathway, thus restrain the cells' proliferation and promotes apoptosis. SB-216763 can activate the Wnt pathway, thus promotes cells' proliferation.