Abstract
Objective To explore the molecular mechanism of cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway in γ-aminobutyric acid inhibitory on the growth of cholangiocarcinoma QBC939 cell line. Methods QBC939 cells were cultured in different groups and treated withγ-aminobutyric acid (GABA), GABA+ 8 Br (cAMP agonists), GABA+ H89 (PKA antagonist) for 48 hours. (4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the proliferation of QBC939 cells. Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) binding assay was used to detect apoptosis in the QBC939 cells. Enzymelinkedimmunosorbentassay (ELISA) assay was detected to the expression of cAMP and PKA. Western blotting was applied to check the expression of PKAI and PKAII and extracellular regulated protein kinases (ERK) proteins in different groups of QBC939 cells. Animal models of cholangiocarcinoma bearing nude mice were established by subcutaneous injection of QBC939 cells and randomized into 2 groups: control and GABA-treated groups. The effect of GABA was evaluated after 5 weeks, including tumor volume. The expression of PKA Ⅰ and PKA Ⅱ and ERK was detected by Western blotting in xenograft tumors. Results MTT and flow cytometry (FCM) assays all showed that the effect of GABA inhibitory on the proliferation and induced apoptosis of QBC939 cells could be antagonized by H89, but not 8 Br. GABA significantly increased the content of cAMP and PKA, the content of cAMP was enhanced by 8 Br, the content of PKA was antagonized by H89. Western blotting analysis showed that GABA significantly down-regulated the expression of PAK I protein (0.087 8±0.003 0 vs. 0.152 1±0.003 0, t=29.687, P<0.05), up-regulated the expression of PKAⅡ, and also decreased the expression of ERK protein protein (0.368 3±0.007 0 vs. 0.468 7±0.010 0, t=13.647, P<0.05). Xenograft tumor volume [ (0.500±0.020 vs. 0.320±0.030) cm3,t=15.354, P<0.05]. The expression of PKAI and ERK were significantly decreased, and PKA II was increased in GABA-treated group as compared with control group. Conclusion GABA may inhibit the growth of cholangiocarcinoma cells QBC939 through cAMP/PKA, down-regulated PAK I, up-regulated PKA II, and decreased the expression of the ERK perhaps is one of its anti-tumor mechanisms. Key words: Bile duct neoplasms; Gamma-aminobutyric acid; Cyclic adenosine monophosphate; Protein kinase A; Extracellular regulated protein kinases
Published Version
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