γ-氨基丁酸抑制胆管癌细胞生长的机制

The mechanism of γ- aminobutyric acid inhibitory on the growth of cholangiocarcinoma cell line

目的探讨γ-氨基丁酸（GABA）通过环磷酸腺苷／蛋白激酶A（cAMP／PKA）通路抑制胆管癌细胞株QBC939生长的分子机制。方法分别以GABA、GABA＋环磷酸腺苷（cAMP）激动剂（8Br）、GABA＋蛋白激酶A（PKA）拮抗剂（H89）孵育胆管癌细胞株QBC93948h，采用噻唑蓝（UYr）法及膜联蛋白V-异硫氰酸荧光素／碘化丙锭（AnnexinV—FITC／PI）流式细胞术检测细胞的增殖及凋亡，酶联免疫吸附试验（ELISA）法检测cAMP、PKA表达，Westernblot法检测蛋白激酶AI（PKAI）、蛋白激酶AⅡ（PKAⅡ）及细胞外调节蛋白激酶（ERK）表达。构建人胆管癌裸鼠皮下种植瘤模型，随机分为对照组和GABA治疗组，用药5周后观察肿瘤体积，Westernblot法检测种植瘤PKAI、PKAⅡ及ERK的表达。结果MTr及流式细胞术检测结果显示8Br协同GABA所导致的增殖抑制和凋亡效应，而H89拮抗此效应；EHSA检测结果显示GABA明显上调cAMP及PKA的含量，8Br协同GABA增加cAMP含量，H89拮抗GABA所导致PKA的含量；Westernblot检测结果显示，GABA较对照组能明显下调PKAI（0．0878±0．0030比0．1521±0．0030，t=29．687，P〈0．05），上调PKAⅡ（0．2042±0．0120比0．1461±0．0720，t=8．152，P〈0．05）蛋白的表达，且下调ERK蛋白的表达（0．3683±0．0070比0．4687±0．0100，t=13．647，P〈0．05）；与对照组比较，治疗组的种植瘤体积明显缩小[（0．500±0．020）cm^3比（0．320±0．030）cm^3，t=15．354，P〈0．05]，PAKI、ERK蛋白表达下降，PKAⅡ表达上升。结论GABA抑制胆管癌细胞株Qac939生长可能由cAMP／PKA信号通路介导，通过下调PKAI及上调PKAⅡ的表达发挥调控作用，而下调ERK蛋白的表达可能发挥抗肿瘤作用。

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 trea- ted with γ- 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） as- say 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. Enz- ymelinkedimmunosorbentassay （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 ex- pression 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 in- creased 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 ofPAK I protein （0.087 8 ＋0.003 0 vs. O. 152 1 ±0.003 0, t =29.687, P〈0.05）, up - regulated the expression of PKA Ⅱ , and also decreased the expression of ERK protein protein （0. 368 3 ± 0. 007 0 vs. 0. 468 7 ＋0. O10 0, t = 13. 647, P 〈0.05）. Xenograft tumor volume [ （0. 500 ±0. 020 vs. 0. 320 ±0. 030） cm^3, t = 15. 354, P 〈0.05]. The expression of PKAI and ERK were significantly de- creased, 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.