4.6 Article

The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line

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WORLD JOURNAL OF GASTROENTEROLOGY
卷 8, 期 3, 页码 469-475

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BAISHIDENG PUBLISHING GROUP INC
DOI: 10.3748/wjg.v8.i3.469

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AIM: The goal of this study was to characterize the AFP receptor, its possible signal transduction pathway and its proliferative functions in human hepatoma cell line Bel 7402. METHODS: Cell proliferation enhanced by AFP was detected by MTT assay, H-3-thymidine incorporation and S-stage percentage of cell cycle analysis. With radioactive labeled I-125-AFP for receptor binding assay; cAMP accumulation, protein kinase A activity were detected by radioactive immunosorbent assay and the change of intracellular free calcium ([ Ca2+](i)) was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncogenes N- ras, p53, and p21(ras) in the cultured cells in vitro were detected by Northern blotting and Western blotting respectively. RESULTS: It was demonstrated that AFP enhanced the proliferation of human hepatoma Bel 7402 cell in a dose dependent fashion as shown in MTT assay, H-3-thymidine incorporation and S-phase percentage up to 2-fold. Two subtypes of AFP receptors were identified in the cells with Kds of 1.3 x 10(-9) mol.L-1 and 9.9 x 10(-8) mol.L-1 respectively. Pretreatment of cells with AFP resulted. in a significant increase (625 %) in cAMP accumulation. The activity of protein kinase A activity were increased up to 37.5, 122.6, 73.7 and 61. 2 % at treatment time point 2, 6, 12 and 24 hours, The level of intracellular calcium were elevated after the treatment of alpha-fetoprotein and achieved to 204 % at 4 min, The results also showed that AFP (20 mg.L-1) could upregulate the expression of N-m oncogenes and p53 and p21(ras) in Bel 7402 cells. In the later case, the alteration were 81. 1 % (12 h) and 97.3 % (12 h) respectively compared with control. CONCLUSION: These results demonstrate that AFP is potential growth factor to promote the proliferation of human hepatoma Bel 7402 cells. Its growth-regulatory effects are mediated by its specific plasma membrane receptors coupled with its transmembrane signaling transduction through the pathway of cAMP-PKA and intracellular calcium to regulate the expression of oncogenes.

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