三七总皂苷通过调控Hippo-YAP信号通路对膀胱癌细胞生物学行为的影响

Panax notoginseng saponins affect the biological behavior of bladder cancer cells by regulating Hippo-YAP pathway

目的观察三七总皂苷(PNS)对人膀胱癌T24细胞增殖、迁移、侵袭及凋亡的影响,并探讨其诱导凋亡的机制。方法2019年9月至2020年7月,选取人膀胱癌T24细胞株(购自中国科学院上海生命科学研究院)作为研究对象。将T24细胞分为对照组(加PBS)、实验组(低浓度组100 mg/L、高浓度组200 mg/L),检测其被不同浓度的PNS处理12、24、48 h后的细胞活力;在经过PNS(100、200 mg/L)处理48 h后,使用细胞划痕实验、Transwell评估处理后T24细胞迁移、侵袭能力的变化,通过流式细胞仪检测干预后T24细胞凋亡状况,利用蛋白质印迹法(Western blot)法检测T24细胞在处理后活化的半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved Caspase-3)、B细胞淋巴瘤/白血病-2(bcl-2)和Hippo信号通路关键蛋白TEA结构域转录子1(TEAD1)、Yes协同蛋白1(YAP1)和大肿瘤抑制基因1(LATS1)的表达水平。组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果PNS处理48 h后,实验组细胞增殖活力显著低于对照组[(63.28±5.81)%、(39.38±3.34)%比(103.00±3.30)%,F=126.045,P<0.05];细胞划痕实验提示PNS处理后,实验组膀胱癌T24细胞系的迁移率显著降低[(25.99±2.25)%、(9.79±0.44)%比(52.50±2.29)%,F=398.345,P<0.05];Transwell结果说明PNS(100、200 mg/L)能够显著减弱T24细胞株的侵袭性[(249.67±28.01)、(152.67±7.64)个比(397±30.12)个,F=77.856,P<0.05];流式细胞仪结果显示,PNS(100、200 mg/L)处理48 h后,实验组细胞凋亡率[(9.55±0.39)%、(15.08±0.94)%]相对于对照组[(6.86±0.68)%]明显升高(F=105.821,P<0.05);Western blot结果表明,实验组中抗凋亡蛋白bcl-2(0.43±0.06、0.17±0.04比0.80±0.08)和Hippo信号通路中TEAD1(0.38±0.05、0.17±0.04比0.62±0.06)和YAP1(0.40±0.04、0.23±0.06比0.53±0.02)等蛋白的表达量显著降低,而cleaved Caspase-3(0.39±0.04、0.50±0.03比0.24±0.02)、LATS1(0.51±0.07、0.73±0.04比0.28±0.05)的表达明显上调(F=87.713、55.109、38.325、53.658、47.578,P<0.05)。结论三七总皂苷能够抑制人膀胱癌T24细胞的增殖、迁移和侵袭等能力,并能诱导T24细胞发生凋亡,其中的机制可能与激活Hippo-YAP信号通路相关。

Objective To observe the effects of panax notoginseng saponins(PNS)on proliferation,migration,invasion and apoptosis of huamn bladder cancer T24 cells,and to research the mechanism of T24 apoptosis by PNS.Methods The study was conducted between September 2019 and July 2020.The human bladder cancer cell line T24 was purchased from Shanghai Institutes for Biological Sciences.Divided into control(with PBS)and experimental group(low concentration group 100mg/L、high concentration group 200 mg/L),the proliferation of huamn bladder cancer T24 cells were evaluated after 12,24 and 48 hours of PNS treatment.After PNS(100,200 mg/L)treatment for 48 h,The effect on migration and invasion of human bladder cancer T24 cells was estimated via Wound Healing and Transwell.After 48 h of PNS administration,the apoptosis of human bladder cancer T24 cells were detected by flow cytometry.In human bladder cancer T24 cells,the expression levels of Cleave cysteinyl aspartate-specific protease(Caspase)-3,B cell lymphoma/leukemia-2(bcl-2)proteins and TEA domain 1(TEAD1),Yes-associated protein 1(YAP1),large tumor suppressor 1(LATS1)proteins of Hippo-YAP signaling pathway were observed by Western blotting after T24 cells were cultured with PNS.The t test was used for comparison between the two groups.ANOVA was adopted for comparison between multiple groups.Results After PNS involvement for 48 h,the cell proliferation vitality of experimental group[(63.28±5.81)%,(39.38±3.34)%vs.(103.00±3.30)%,F=126.045,P<0.05].The results of Wound Healing indicated that migration rate of huamn bladder cancer T24 cells in the experimental group was significantly reduced after PNS treatment[(25.99±2.25)%,(9.79±0.44)%vs.(52.50±2.29)%,F=398.345,P<0.05].Based on the results of Transwell,we found that the invasion ability was reduced because of PNS(249.67±28.01,152.67±7.64 vs.397.00±30.12,F=77.856,P<0.05).The apoptosis rates of experimental group were obviously increased compared with control group by flow cytometry after 48 h of PNS(100,200 mg/L)regulation[(9.55±0.39)%,(15.08±0.94)%vs.(6.86±0.68)%,F=105.821,P<0.05].In Western blotting,we could learn that the expressions of bcl-2(0.43±0.06,0.17±0.04 vs.0.80±0.08),TEAD1(0.38±0.05,0.17±0.04 vs.0.62±0.06)and YAP1(0.40±0.04,0.23±0.06 vs.0.53±0.02)proteins in the experimental group were significantly reduced,at the same time cleaved Caspase-3(0.39±0.04,0.50±0.03 vs.0.24±0.02)and LATS1(0.51±0.07,0.73±0.04 vs.0.28±0.05)was upregulated(F=87.713,55.109,38.325,53.658,47.578,P<0.05).Conclusion The proliferation vitality,migration and invasion of huamn bladder cancer T24 cells were inhibited by PNS.In the meantime,apoptosis of T24 cells were induced by PNS,it relates to activation of Hippo-YAP signaling pathway.