γ-氨基丁酸对胆管癌QBC939细胞系生长的抑制作用分子机制

[Molecular mechanism of γ-aminobutyric acid inhibitory on the growth of cholangiocarcinoma QBC939 cell line].

作者信息

Zhu Cheng-lin, Huang Qiang, Liu Chen-hai, Xie Fang, Zhu Kai

机构信息

Department of General Surgery, Affiliated Provincial Hospital of Anhui Medical University, Hefei 230001, China.

出版信息

Zhonghua Wai Ke Za Zhi. 2013 Mar;51(3):261-5.

PMID:23859331

Abstract

OBJECTIVE

To explore the molecular mechanism of γ-aminobutyric acid (GABA) inhibitory on the growth of cholangiocarcinoma cell line (QBC939).

METHODS

QBC939 cells were cultured in different groups and treated with GABA, GABA + bicuculine (A receptor antagonist), GABA + phaclofen (B receptor antagonist) for 48 hours. MTT assay was used to determine the proliferation of QBC939 cells. Annexin V-FITC/PI binding assay was used to detect apoptosis in the QBC939 cells. Western blot was applied to check the expression of signal transducer and activator of transcription 3 (STAT3) and phosphorylation-STAT3 (p-STAT3) 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 the body weight and tumor volume. The expression of p-STAT3 was detected by immunohistochemistry and Western blot in xenograft tumors.

RESULTS

MTT and FCM assays both showed that the effect of GABA inhibitory on the proliferation (15.30% ± 0.80% vs. 2.66% ± 0.74%, t = 23.15, P = 0.00) and induced apoptosis (23.15% ± 0.21% vs. 4.30% ± 0.69%, t = 52.40, P = 0.00) of QBC939 cells could be antagonized by phaclofen, but not bicuculine. The expression of STAT3 and p-STAT3 proteins were all observed in the QBC939 cells and GABA significantly down-regulated p-STAT3 protein expression (0.77 ± 0.00 vs. 0.45 ± 0.01, t = 63.14, P = 0.00), this action was also antagonized by phaclofen (0.45 ± 0.01 vs. 0.76 ± 0.01, t = 56.25, P = 0.00). Xenograft tumor volume ((0.62 ± 0.03) cm³ vs. (0.34 ± 0.03) cm³, t = 13.45, P = 0.00) and the expression of p-STAT3 protein were significantly decreased in GABA-treated group as compared with control group.

CONCLUSIONS

GABA may inhibit the growth of cholangiocarcinoma cells QBC939 through GABA(B) receptor, and down-regulation of the p-STAT3 expression perhaps is one of its anti-tumor mechanisms.

摘要

目的

探讨γ-氨基丁酸（GABA）抑制胆管癌细胞系（QBC939）生长的分子机制。

方法

将QBC939细胞分组培养，分别用GABA、GABA + 荷包牡丹碱（A受体拮抗剂）、GABA + 巴氯芬（B受体拮抗剂）处理48小时。采用MTT法检测QBC939细胞的增殖情况。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶结合法检测QBC939细胞的凋亡情况。应用蛋白质免疫印迹法检测不同组QBC939细胞中信号转导子和转录激活子3（STAT3）及磷酸化STAT3（p-STAT3）蛋白的表达。通过皮下注射QBC939细胞建立荷胆管癌裸鼠动物模型，并随机分为2组：对照组和GABA处理组。5周后评估GABA的作用，包括体重和肿瘤体积。通过免疫组织化学和蛋白质免疫印迹法检测移植瘤中p-STAT3的表达。

结果

MTT法和流式细胞术检测均显示，巴氯芬可拮抗GABA对QBC939细胞增殖的抑制作用（15.30% ± 0.80% vs. 2.66% ± 0.74%，t = 23.15，P = 0.00）及诱导凋亡的作用（23.15% ± 0.21% vs. 4.30% ± 0.69%，t = 52.40，P = 0.00），而荷包牡丹碱无此作用。QBC939细胞中均观察到STAT3和p-STAT3蛋白的表达，GABA可显著下调p-STAT3蛋白表达（0.77 ± 0.00 vs. 0.45 ± 0.01，t = 63.14，P = 0.00），巴氯芬也可拮抗此作用（0.45 ± 0.01 vs. 0.76 ± 0.01，t = 56.25，P = 0.00）。与对照组相比，GABA处理组移植瘤体积（（0.62 ± 0.03）cm³ vs. （0.34 ± 0.03）cm³，t = 13.45，P = 0.00）及p-STAT3蛋白表达明显降低。