3-溴丙酮酸通过抑制糖酵解阻止A549肺腺癌细胞增殖并诱导其凋亡

3-Bromopyruvic acid arrests proliferation and induces apoptosis of A549 lung adenocarcinoma cells by inhibiting glycolysis

目的探究3-溴丙酮酸(3-BP)对人肺腺癌A549细胞的增殖和凋亡的影响及机制。方法采用噻唑蓝(MTT)法检测(10、20、40、80、160)μmol/L3-BP处理24、48 h,对肺腺癌A549细胞存活率的影响;采用(0、50、100、150)μmol/L 3-BP处理肺腺癌A549细胞24 h,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双染法结合流式细胞术检测A549细胞的凋亡率;试剂盒检测A549细胞ATP水平、2′,7′-二氯二氢荧光黄二乙酸酯(DCFH-DA)染色结合流式细胞术检测细胞活性氧(ROS)水平变化,Western blot法检测糖酵解及凋亡相关蛋白己糖激酶2(HK-2)、单羧酸转运蛋白1(MCT1)、丙酮酸脱氢酶激酶1(PDK1)和凋亡相关B细胞淋巴瘤分子2(Bcl2)、Bcl2相关X蛋白(BAX)的表达。近红外荧光蛋白(iRFP)和荧光素酶双标记的A549细胞接种至裸鼠皮下,当皮下肿瘤体积达到100 mm^(3)时,将所有裸鼠随机分成PBS组、5 mg/kg顺铂(DDP)组、7 mg/kg 3-BP组;每组5只,采用腹腔注射方式进行给药。HE染色检测肿瘤病变情况,免疫组织化学染色检测KI-67抗原(ki67)表达确定对肿瘤细胞增殖的影响,原位末端转移酶标记技术(TUNEL)检测肿瘤细胞凋亡情况,评估3-BP的体内抗肿瘤效果并进行肝肾功能检测确定3-BP肝肾毒性。结果3-BP可以抑制A549细胞增殖,诱导其凋亡;与对照组相比,3-BP处理组ATP水平明显下降,ROS水平明显升高,HK-2、MCT1、PDK1、Bcl2蛋白表达明显降低,BAX表达明显增加。动物模型结果显示,与PBS组相比,3-BP可以抑制裸鼠肺腺癌移植瘤的生长,3-BP可以抑制肿瘤细胞的增殖并诱导其凋亡,3-BP对裸鼠肝肾毒性较为轻微,安全性较高。结论3-BP通过抑制糖酵解抑制A549人肺腺癌细胞增殖并诱导其凋亡,对荷肺癌裸鼠的抗肿瘤效果较好且肝肾毒性较低。

Objective To observe the effects of 3-bromopyruvic acid(3-BP)on the proliferation and apoptosis of A549 human lung adenocarcinoma cells and the underlying mechanism.Methods MTT assay was used to detect the effects of(10,20,40,80,160)μmol/L 3-BP treatment for 24 and 48 hours on the survival rate of A549 cells.After(0,50,100,150)μmol/L 3-BP was used to treat A549 cells for 24 hours,fluorescein isothiocyanate labeled annexinⅤ/propidium iodide(annexinⅤ-FITC/PI)double staining method combined with flow cytometry were used to detect apoptosis rate of A549 cells;the kit to detect the ATP level of A549 cells;2′,7′-dichlorofluorescein diacetate(DCFH-DA)staining combined with flow cytometry to detect cellular reactive oxygen species(ROS)levels;Western blot analysis to detect hexokinase 2(HK-2),monocarboxylic acid transporter 1(MCT1),pyruvate dehydrogen kinase 1(PDK1)and Bcl2,BAX.A549 cells double-labeled with near-infrared fluorescent protein(iRFP)and luciferase were inoculated into nude mice subcutaneously.When the subcutaneous tumor volume reached 100 mm^(3),all nude mice were randomly divided into PBS group,5 mg/kgcisplatin(DDP)group,and 7 mg/kg 3-BP group,with 5 rats in each group,and they were administered by intraperitoneal injection.HE staining was performed to check the lesions of tumor tissue;immunohistochemical staining to test the expression of antigen KI-67(ki67)in tumor cells for evaluating the proliferation;and TUNEL to detect tumor cell apoptosis.To evaluate the anti-tumor effects of 3-BP in vivo,we examined liver and kidney functions of the mice and determine 3-BP liver and kidney toxicity.Results 3-BP inhibited the proliferation of A549 cells and induced their apoptosis.Compared with the control group,the ATP level in the 3-BP treatment group significantly decreased,the ROS level significantly increased,and the protein expression of HK-2,MCT1,PDK1,and Bcl2 significantly dropped,while the expression of BAX went up significantly.Compared with the PBS group,3-BP inhibited the growth of lung adenocarcinoma xenografts in the nude mice,inhibited the proliferation of tumor cells and induced their apoptosis.3-BP was little toxic to the liver and kidney in the nude mice.Conclusion 3-BP can inhibit the proliferation and induce apoptosis of A549 human lung adenocarcinoma cells by inhibiting glycolysis.3-BP has good anti-tumor effects and lower liver and kidney toxicity in nude mice bearing lung cancer.