Effects Of Recombinant Human Growth Hormone On GHR~+ Or GHR~- Human Liver Cancer Cell Lines With In Vitro

Objective:The study was designed to investigate the effects of recombinant human growth hormone (rhGH) on human liver cancer cell lines expressed different growth hormone receptor(GHR) and the ECV304 proliferation which co-cultured with GHR(+/-) hepatoma cells in vitro.Methords:Liver cancer cells line Bel-7402, HepG2, SMMC7721, QGY-77O1, Bel-7405, HCCLM3, and human vascular endothelial cells ECV304 were cultured in vitro. The expression of GHR in human liver cancer cell lines was detected by using immunocytochemistry techniques.①hree groups were assigned in each cell line:control group,50ng/ml rhGH1 group and 250ng/ml rhGH2 group. By ELISA, MTT assay, flow cytometry and fluorescent staining, effects of different concentrations of rhGH were investigated on the IGF-1 concentration, cell multiplication rate, rate of apoptosis and cell cycle.②Bel-7402 and SMMC-7721 cell at selected as two groups of hepatoma cells co-cultured with ECV304 with different concentrations of rhGH and Bevacizumab intervention. Cell proliferation curve and FCM cycle ratio were measured. To explore the rhGH induced factor which promotes the ECV304 proliferation, RT-PCR was used to display the VEGF mRNA expression of hepatoma cells, wheras ELISA was selected to display the VEGF concentration.③Four groups were assigned according to the cell line treatment modes: untreated group,50, 100and 200ng/ml rhGH treated group. The expression of GHR in human liver cancer cell lines was detected by immunohistochemistry, fluorescence and radioligand assays.Results:Bel-7402 and HepG2 cells expressed growth hormone receptors. Significant effects of rhGH were found on cell proliferation and IGF-1 concentration, but not rising with the increasing of rhGH concentration (P0.05); The proliferation parameters of co-cultured ECV304 with Bel-7402 or SMMC7721 cell were compared with that cultured alone (P<0.05). The rhGH accelerated the proliferation of ECV304 cell cultured with Bel-7402. The proportion of cells in S phase were significantly higher with rhGH dose-dependent manner (P<0.05). The VEGF mRNA expression and the VEGF secretion of Bel-7402 cell were significantly higher after rhGH intervention (P<0.05). However, we have not seen the manifestation in SMMC-7721 cell.After cutting down the signal pathway of VEGF by the Bevacizumab, the ECV304 proliferation was inhibited (P<0.05). Even if the high concentration of rhGH, the suppression of cell proliferation were still unable to reverse; By fluorescence, compared with the untreated group, the expressions of GHR with HepG-2 were significantly increased in the groups treated with 50,100,200ng/ml rhGH (P<0.05). By radioligand assays, the GHR site of the untreated group was 7.5137±0.5352(10-3cell), the sites of 50, 100,200ng/ml rhGH treated group were 8.4350±0.2396,9.3957±0.5143,8.3297±0.3133 (10-3cell) (P<0.05). But there was no remarkable effect on SMMC7721 and QGY-7701 cell (P>0.05).Conclusions:In vitro, rhGH stimulates cells proliferation, increase the expression of GHR, induces the VEGF,IGF-1 secretion, and promotes growth of human vascular endothelial cells co-cultured with them, in cell with expressed GHR but not in cell with low-expressed or no-expressed GHR.