鸦胆子苦素D调控AMPK/ULK1通路对膀胱癌细胞糖酵解的影响

Effects of Bruceine D regulating the AMPK/ULK1 pathway on glycolysis in bladder cancer cells

关键词:  鸦胆子苦素D  膀胱癌  增殖  糖酵解  自噬  凋亡

Key words:  Bruceine D  bladder cancer  proliferation  glycolysis  autophagy  apoptosis

摘要:

[目的] 探讨鸦胆子苦素D调控AMP活化蛋白激酶（AMPK）/Unc-51样自噬激活激酶1（ULK1）通路对膀胱癌细胞糖酵解的影响。[方法] T24细胞随机分为膀胱癌组，鸦胆子苦素D低、中、高浓度组，AMPK激活剂（AICAR）组，鸦胆子苦素D高浓度+AMPK抑制剂（Compound C）组。克隆形成实验、CCK-8法检测细胞增殖；流式细胞术检测细胞凋亡；试剂盒检测乳酸生成量、葡萄糖摄取量、丙酮酸生成量；单丹磺酰戊二胺（MDC）染色检测细胞自噬小体相对含量；实时荧光定量逆转录聚合酶链反应（qRT-PCR）检测细胞中增殖细胞核抗原（PCNA）、活化的半胱天冬酶-3（Cleaved Caspase-3）、丙酮酸激酶M2型（PKM2）、己糖激酶2（HK2）mRNA水平；蛋白免疫印迹（Western blot）法检测细胞Beclin1、微管相关蛋白1轻链3（LC3）-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK、p-ULK1/ULK1蛋白表达。构建T24细胞裸鼠移植瘤模型，观察鸦胆子苦素D对移植瘤生长的影响，Western blot检测瘤组织AMPK/ULK1通路蛋白表达。[结果] 与膀胱癌组比较，鸦胆子苦素D低、中、高浓度组、AICAR组T24细胞克隆形成数、OD₄₅₀值、乳酸生成量、葡萄糖摄取量、丙酮酸生成量减少，PCNA、PKM2、HK2 mRNA水平降低，Cleaved Caspase-3 mRNA水平升高，细胞凋亡率、自噬小体相对含量及Beclin1、LC3-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK、p-ULK1/ULK1蛋白表达增加（P<0.05）；与鸦胆子苦素D高浓度组比较，鸦胆子苦素D高浓度+Compound C组T24细胞克隆形成数、OD₄₅₀值、乳酸生成量、葡萄糖摄取量、丙酮酸生成量增加，PCNA、PKM2、HK2 mRNA水平升高，Cleaved Caspase-3 mRNA水平降低，细胞凋亡率、自噬小体相对含量及Beclin1、LC3-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK、p-ULK1/ULK1蛋白表达降低（P<0.05）。与对照组比较，鸦胆子苦素D组瘤组织质量、体积降低，p-AMPK/AMPK、p-ULK1/ULK1蛋白表达升高（P<0.05）。[结论] 鸦胆子苦素D可能通过激活AMPK/ULK1通路抑制T24细胞增殖、糖酵解及裸鼠移植瘤的生长，诱导细胞自噬及凋亡。

Abstract:

[Objective] To investigate the effect of Bruceine D regulating the AMP-activated protein kinase（AMPK）/UNC-51-like autophagy-activated kinase 1（ULK1） pathway on glycolysis in bladder cancer cells. [Methods] The T24 cells were randomly divided into bladder cancer group，low-，medium-，and high-concentration Bruceine D groups，AMPK（AICAR activator） group，and high-concentration Bruceine D+AMPK inhibitor（Compound C） group. Cell proliferation was detected by clone formation assay and CCK-8 assay. Cell apoptosis was detected by flow cytometry. Kits were used to measure lactic acid production，glucose intake and amount of pyruvate production. The relative content of autophagosomes in cells was detected by monodansylcadaverine（MDC） staining. The mRNA levels of proliferating cell nuclear antigen（PCNA），Cleaved Caspase-3，pyruvate kinase M2（PKM2），and hexokinase 2（HK2） were measured by qRT-PCR. Western blot was used to examine the proteins of Beclin1，microtubule-associated protein 1 light chain 3（LC3）-Ⅱ/LC3-Ⅰ，p-AMPK/AMPK and p-ULK1/ULK1 in the cells. A nude mouse xenograft model of T24 cells was established to measure the effects of Bruceine D on the growth of xenograft tumors，and protein levels of AMPK/ULK1 pathway in tumor tissues by Western blot. [Results] Compared with the bladder cancer group，the number of T24 cell clone formation，OD₄₅₀ value，lactic acid production，glucose uptake，amount of pyruvate production in the low-，medium-，and high-concentration Bruceine D groups and the AICAR grou were decreased，the mRNA levels of PCNA，PKM2，and HK2 were reduced，while the Cleaved Caspase-3 mRNA level was raised. The apoptosis rate，the relative content of autophagosomes，and the protein expressions of Beclin1，LC3-Ⅱ/LC3-Ⅰ，p-AMPK/AMPK，and p-ULK1/ULK1 were increased（P<0.05）. Compared with the high concentration Bruceine D group，the number of clone formation，OD₄₅₀ value，lactic acid production，glucose uptake，amount of pyruvate production in the high concentration Bruceine D+Compound C group were added，PCNA，PKM2 and HK2 mRNA levels were increased，while Cleaved Caspase-3 mRNA level was reduced. The apoptosis rate，the relative content of autophagosomes，and the protein expressions of Beclin1，LC3-Ⅱ/LC3-Ⅰ，p-AMPK/AMPK，and p-ULK1/ULK1 were decreased（P<0.05）. Compared with the control group，the tumor tissue mass and volume in the Bruceine D group were decreased，while the p-AMPK/AMPK and p-ULK1/ULK1 protein levels in the tumor tissue were increased（P<0.05）. [Conclusion] Bruceine D may inhibit the proliferation of T24 cells，glycolysis，and the growth of xenograft tumors in nude mice，as well as induce cell autophagy and apoptosis，by activating the AMPK/ULK1 pathway.