羧胺三唑乳清酸盐抑制人胰腺癌吉西他滨耐药细胞株增殖及线粒体能量代谢  

Carboxyamidotriazole-orotate inhibits proliferation and mitochondrial metabolism in human pancreatic cancer gemcitabine-resistant cell strain

作　　者：陈秋霞 杨黎星 马瑞 赵永晶 王钰铖 鞠瑞[1] 郭磊[1] CHEN Qiu-xia;YANG Li-xing;MA Rui;ZHAO Yong-jing;WANG Yu-cheng;JU Rui;GUO Lei(Department of Pharmacology,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所北京协和医学院基础学院药理系,北京100005

出　　处：《基础医学与临床》2022年第4期570-576,共7页Basic and Clinical Medicine

基　　金：国家自然科学基金(81872897,82002094)。

摘　　要：目的研究羧胺三唑乳清酸盐(CTO)对人胰腺癌耐吉西他滨细胞株(AG细胞)增殖、代谢和凋亡的影响。方法体外培养AG细胞。将细胞分为对照组和不同浓度CTO组;用磺酰罗丹明B(SRB)检测细胞活力;用annexin V/PI双染色及流式细胞测量术检测细胞凋亡;用羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)染色及流式细胞测量术检测细胞分裂速度;通过Seahorse生物能量仪检测细胞耗氧率(OCR);MTT法检测胞内NAD+、NADH含量,计算NAD+/NADH比率;Western blot检测细胞内自噬相关蛋白表达。结果与对照组相比,CTO处理组中AG活细胞数目明显减少,细胞凋亡比例增加,药物作用呈时间-剂量依赖性。20μmol/L CTO组AG细胞内CFSE染色荧光强度明显升高(P<0.05);与对照组相比,不同浓度CTO处理后细胞内烟酰胺腺嘌呤二核苷酸的还原态(NADH)含量升高(P<0.05或P<0.01)、烟酰胺腺嘌呤二核苷酸的氧化态(NAD+)含量无明显变化、NDA+/NADH比值降低(P<0.01);与对照组相比,CTO处理组中AG细胞内的OCR明显降低,自噬相关蛋白的表达水平明显降低(P<0.05)。结论CTO通过诱导AG细胞凋亡,损伤AG细胞线粒体呼吸作用,并下调细胞自噬相关蛋白表达水平从而达到抑制AG增殖的作用。Objective To investigate the effects of carboxyamidotriazole-orotate(CTO)on proliferation,apoptosis and metabolism of human pancreatic cancer gemcitabine-resistant cell line(ASPC-1-GEM,AG cells).MethodsCell viability was detected by sulforhodamine B(SRB);apoptosis was detected by annexin V/PI staining and flow cytometry;cell division rate was examined by CFSE microscopy and flow cytometry;The oxygen consumption rate(OCR)was detected by Seahorse bio-energy assay;intracellular NAD+and NADH contents were detected with MTT and NAD+/NADH ratio was calculated.Autophagy-related protein expression was detected by Western blot.Results Comparing with the control group,the living AG cells were significantly reduced and the proportion of apoptotic cells was increased in CTO-treated group.The drug effect was time-dose dependent.The fluorescence intensity of CFSE staining of AG cells was significantly increased in the 20μmol/L CTO group(P<0.05);Comparedwith the control group,intracellular nicotinamide adenine dinucleotidehydrogen(NADH)content was increased after incubation with different concentrations of CTO treatment(P<0.05 or P<0.01)and no significant change in the content of nicotinamide adenine dinucleotide(NAD+)NDA+/NADH ratio was decreased(P<0.01).The OCR in AG cells was significantly reduced and the expression of autophagy-related proteins was significantly inhibited in the CTO-treated group(P<0.05).Conclusions CTO inhibits the proliferation of gemcitabine-resistant pancreatic cancer cells by inducing apoptosis,impairing mitochondrial respiration,and down-regulating the expression of autophagy-related proteins in AG cells.

关 键 词：羧胺三唑 胰腺癌 吉西他滨耐药 凋亡 

分 类 号：R96[医药卫生—药理学]