葫芦素E诱导HeLa和MCF7细胞自噬及其分子机制研究
发布时间：2018-08-13 08:53
【摘要】：目的:葫芦素E(Cu E)是一类具有多种生物活性的天然三萜类化合物,包括抗炎、护肝、抗糖尿病和抗肿瘤作用。最近的研究表明葫芦素B、I能诱导肿瘤细胞发生自噬,而同属葫芦素家族的Cu E能否诱导肿瘤细胞自噬在国内外尚未见报道。本研究以乳腺癌MCF7和宫颈癌He La细胞为研究对象,探讨Cu E诱导细胞自噬的发生及其分子机制,为进一步研究Cu E发挥抗炎、抗糖尿病和抗癌作用提供实验依据。方法:1.以WST-1法检测Cu E分别对He La、MCF7和DU145细胞增殖的影响。2.免疫印迹法检测自噬标志物LC3-II、p62/SQSTM1蛋白的表达;通过检测m TORC1的底物p70S6K,4E-BP1和ULK1S758以及p70S6K的底物S6蛋白的磷酸化水平验证Cu E对m TORC1活性的影响;检测ULK1S555、RaptorS792、AktT308和AktS473的磷酸化水平验证Cu E对AMPK和Akt活性的影响;并检测ATG5和Beclin1的表达对Cu E诱导细胞自噬的影响。3.免疫荧光技术观察Cu E处理后细胞内LC3和LAMP2的共定位,检测自噬体和溶酶体的融合情况;并通过观察LAMP2和m TOR的共定位来检测m TORC1的活性;4.利用特异性抑制剂和小分子干扰RNA(Si RNA)技术研究相关蛋白和信号通路在Cu E诱导肿瘤细胞自噬过程中的作用。结果:1.Cu E剂量依赖性抑制He La、MCF7和DU145细胞增殖。Cu E处理He La细胞24 h后,其IC50为4.01μmol/L,处理48 h后,IC50为0.06μmol/L。2.Cu E处理He La和MCF7细胞后导致LC3-II的表达增加,利用氯喹(chloroquine)阻断自噬可导致LC3-II蛋白在胞内的累积,上调其蛋白水平;免疫荧光技术检测到自噬体标志蛋白LC3-II和溶酶体蛋白LAMP2高度共定位;而p62/SQSTM1蛋白能被降解。表明Cu E诱导了完整的细胞自噬过程。3.Cu E处理ATG5基因缺陷的DU145细胞24h后无LC3-II的表达;也没有发现有LC3斑与LAMP2的共定位;Si RNA技术敲低He La和MCF7细胞中ATG5基因表达后,胞内LC3-II的表达水平也被抑制;这些结果表明,Cu E诱导的细胞自噬依赖于ATG5的表达。4.通过对m TORC1底物磷酸化水平的检测发现,Cu E对p70S6K、S6的磷酸化作用有显著的剂量和时间依赖性抑制作用,但上调了4E-BP1T37/46和p-4E-BP1T37/46的表达。Cu E降低m TORC1下游底物ULK1S758磷酸化表达水平;免疫荧光技术发现m TOR与LAMP2斑点的共定位减少。这些结果表明Cu E能抑制m TORC1的活性,从而活化ULK1,诱导细胞自噬。5.Cu E处理He La、MCF7、和DU145细胞不同时间后,免疫印记结果显示AMPKα和AMPKβ的磷酸化水平在早期的时间点明显增加但在随后逐渐恢复到基础水平;而AMPK的底物ULK1S555和RaptorS792的磷酸化水平也呈现相应的变化,表明Cu E能激活AMPK的活性。AktT308的磷酸化不受Cu E处理的影响,m TORC2下游蛋白AktS473的磷酸化稍稍增强。6.如果Cu E和AMPK特异性抑制剂化合物C共同作用于He La细胞,其AMPK底物ULK1S555和RaptorS792的磷酸化可被完全抑制;p70S6K的磷酸化水平被完全颠倒,即p-p70S6K/p70S6K的比例增加;而不影响ULK1S758磷酸化水平,表明Cu E能通过活化AMPK而抑制m TORC1的活性。7.利用si RNAs干扰技术敲低AMPKα能减弱Cu E对m TORC1/p70S6K信号的抑制效应,而不影响m TORC1/ULK1S758信号的抑制;但明显降低LC3-II的表达水平。结论:葫芦素E通过抑制m TORC1的活性和上调AMPK活性诱导MCF7和He La细胞发生自噬。
[Abstract]:Objective: Cucurbitacin E (Cu E) is a kind of natural triterpenoids with a variety of biological activities, including anti-inflammatory, hepatoprotective, anti-diabetic and anti-tumor effects. Recent studies have shown that Cucurbitacin B and I can induce autophagy in tumor cells. Whether Cucurbitacin E can induce autophagy in tumor cells has not been reported at home and abroad. Objective: To investigate the mechanism of autophagy induced by Cu E in breast cancer MCF7 and cervical cancer He La cells, and to provide experimental basis for further study of anti-inflammatory, anti-diabetic and anti-cancer effects of Cu E. Methods: 1. WST-1 method was used to detect the effects of Cu E on proliferation of He La, MCF7 and DU145 cells. Expressions of autophagy markers LC3-II, p62/SQSTM1 protein, phosphorylation levels of substrates p70S6K, 4E-BP1, ULK1S758 and p70S6K were detected to verify the effect of Cu E on the activity of M TORC1, and phosphorylation levels of ULK1S555, RaptorS792, AktT308 and AktS473 to verify the effect of Cu E on the activities of AMPK and Akt. Effect of Beclin1 expression on Cu E-induced autophagy.3.Immunofluorescence technique was used to observe the co-localization of LC 3 and LAMP2 in the cells treated with Cu E and detect the fusion of autophagy and lysosome.The activity of M TORC1 was detected by co-localization of LAMP2 and m TOR. Results: 1. Cu E inhibited the proliferation of He La, MCF7 and DU145 cells in a dose-dependent manner. After 24 hours of cuE treatment, the IC50 of He La cells was 4.01 micromol/L. After 48 hours of treatment, the expression of LC3-II was increased in He La and MCF7 cells treated with IC50 of 0.06 micromol/L.2. (chloroquine) blocked autophagy could lead to the accumulation of LC3-II protein in cells and up-regulate its protein level; immunofluorescence assay showed that autophagy marker protein LC3-II and lysosomal protein LAMP2 were highly co-located; and p62/SQSTM1 protein could be degraded. It indicated that Cu E induced the complete autophagy process. 3. Cu E treated the fine DU145 of ATG5 gene defect. There was no LC3-II expression after 24 hours; no co-localization of LC3 spots and LAMP2 was found; the expression of LC3-II was also inhibited by knocking down ATG5 gene expression in He La and MCF7 cells by Si RNA technology; these results indicated that autophagy induced by Cu E was dependent on ATG5 expression. The phosphorylation of p70S6K and S6 was inhibited in a dose-and time-dependent manner, but up-regulated the expression of 4E-BP1T37/46 and p-4E-BP1T37/46. Cu E decreased the phosphorylation level of ULK1S758, the downstream substrate of M TORC1. Immunofluorescence assay showed that the co-localization of M TOR and LAMP2 spots was decreased. These results indicated that Cu E could inhibit the activity of M TORC1. The results of immunoblotting showed that the phosphorylation levels of AMPK alpha and AMPK beta increased significantly at the early stage but gradually returned to the basal level, and the phosphorylation levels of AMPK substrates ULK1S555 and RaptorS792 also changed correspondingly. The phosphorylation of AMPK substrate ULK1S555 and RaptorS792 was completely inhibited if Cu E and AMPK inhibitor C acted together on He La cells. Total inversion, i.e. the increase of p-p70S6K/p70S6K ratio without affecting the phosphorylation level of ULK1S758, indicates that Cu E can inhibit the activity of M TORC1 by activating AMPK. 7. Inhibiting AMPK alpha with Si RNAs interference technique can attenuate the inhibitory effect of Cu E on M TORC1/p70S6K signal without affecting the inhibition of M TORC1/ULK1S758 signal, but significantly reduce the LC3-II surface. CONCLUSION: Cucurbitacin E can induce autophagy of MCF7 and He La cells by inhibiting the activity of M TORC1 and up-regulating the activity of AMPK.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285
，
本文编号：2180483
[Abstract]:Objective: Cucurbitacin E (Cu E) is a kind of natural triterpenoids with a variety of biological activities, including anti-inflammatory, hepatoprotective, anti-diabetic and anti-tumor effects. Recent studies have shown that Cucurbitacin B and I can induce autophagy in tumor cells. Whether Cucurbitacin E can induce autophagy in tumor cells has not been reported at home and abroad. Objective: To investigate the mechanism of autophagy induced by Cu E in breast cancer MCF7 and cervical cancer He La cells, and to provide experimental basis for further study of anti-inflammatory, anti-diabetic and anti-cancer effects of Cu E. Methods: 1. WST-1 method was used to detect the effects of Cu E on proliferation of He La, MCF7 and DU145 cells. Expressions of autophagy markers LC3-II, p62/SQSTM1 protein, phosphorylation levels of substrates p70S6K, 4E-BP1, ULK1S758 and p70S6K were detected to verify the effect of Cu E on the activity of M TORC1, and phosphorylation levels of ULK1S555, RaptorS792, AktT308 and AktS473 to verify the effect of Cu E on the activities of AMPK and Akt. Effect of Beclin1 expression on Cu E-induced autophagy.3.Immunofluorescence technique was used to observe the co-localization of LC 3 and LAMP2 in the cells treated with Cu E and detect the fusion of autophagy and lysosome.The activity of M TORC1 was detected by co-localization of LAMP2 and m TOR. Results: 1. Cu E inhibited the proliferation of He La, MCF7 and DU145 cells in a dose-dependent manner. After 24 hours of cuE treatment, the IC50 of He La cells was 4.01 micromol/L. After 48 hours of treatment, the expression of LC3-II was increased in He La and MCF7 cells treated with IC50 of 0.06 micromol/L.2. (chloroquine) blocked autophagy could lead to the accumulation of LC3-II protein in cells and up-regulate its protein level; immunofluorescence assay showed that autophagy marker protein LC3-II and lysosomal protein LAMP2 were highly co-located; and p62/SQSTM1 protein could be degraded. It indicated that Cu E induced the complete autophagy process. 3. Cu E treated the fine DU145 of ATG5 gene defect. There was no LC3-II expression after 24 hours; no co-localization of LC3 spots and LAMP2 was found; the expression of LC3-II was also inhibited by knocking down ATG5 gene expression in He La and MCF7 cells by Si RNA technology; these results indicated that autophagy induced by Cu E was dependent on ATG5 expression. The phosphorylation of p70S6K and S6 was inhibited in a dose-and time-dependent manner, but up-regulated the expression of 4E-BP1T37/46 and p-4E-BP1T37/46. Cu E decreased the phosphorylation level of ULK1S758, the downstream substrate of M TORC1. Immunofluorescence assay showed that the co-localization of M TOR and LAMP2 spots was decreased. These results indicated that Cu E could inhibit the activity of M TORC1. The results of immunoblotting showed that the phosphorylation levels of AMPK alpha and AMPK beta increased significantly at the early stage but gradually returned to the basal level, and the phosphorylation levels of AMPK substrates ULK1S555 and RaptorS792 also changed correspondingly. The phosphorylation of AMPK substrate ULK1S555 and RaptorS792 was completely inhibited if Cu E and AMPK inhibitor C acted together on He La cells. Total inversion, i.e. the increase of p-p70S6K/p70S6K ratio without affecting the phosphorylation level of ULK1S758, indicates that Cu E can inhibit the activity of M TORC1 by activating AMPK. 7. Inhibiting AMPK alpha with Si RNAs interference technique can attenuate the inhibitory effect of Cu E on M TORC1/p70S6K signal without affecting the inhibition of M TORC1/ULK1S758 signal, but significantly reduce the LC3-II surface. CONCLUSION: Cucurbitacin E can induce autophagy of MCF7 and He La cells by inhibiting the activity of M TORC1 and up-regulating the activity of AMPK.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285
，
本文编号：2180483
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