【摘要】：目的:葫蘆素E(Cu E)是一類具有多種生物活性的天然三萜類化合物,包括抗炎、護肝、抗糖尿病和抗腫瘤作用。最近的研究表明葫蘆素B、I能誘導(dǎo)腫瘤細(xì)胞發(fā)生自噬,而同屬葫蘆素家族的Cu E能否誘導(dǎo)腫瘤細(xì)胞自噬在國內(nèi)外尚未見報道。本研究以乳腺癌MCF7和宮頸癌He La細(xì)胞為研究對象,探討Cu E誘導(dǎo)細(xì)胞自噬的發(fā)生及其分子機制,為進(jìn)一步研究Cu E發(fā)揮抗炎、抗糖尿病和抗癌作用提供實驗依據(jù)。方法:1.以WST-1法檢測Cu E分別對He La、MCF7和DU145細(xì)胞增殖的影響。2.免疫印跡法檢測自噬標(biāo)志物L(fēng)C3-II、p62/SQSTM1蛋白的表達(dá);通過檢測m TORC1的底物p70S6K,4E-BP1和ULK1S758以及p70S6K的底物S6蛋白的磷酸化水平驗證Cu E對m TORC1活性的影響;檢測ULK1S555、RaptorS792、AktT308和AktS473的磷酸化水平驗證Cu E對AMPK和Akt活性的影響;并檢測ATG5和Beclin1的表達(dá)對Cu E誘導(dǎo)細(xì)胞自噬的影響。3.免疫熒光技術(shù)觀察Cu E處理后細(xì)胞內(nèi)LC3和LAMP2的共定位,檢測自噬體和溶酶體的融合情況;并通過觀察LAMP2和m TOR的共定位來檢測m TORC1的活性;4.利用特異性抑制劑和小分子干擾RNA(Si RNA)技術(shù)研究相關(guān)蛋白和信號通路在Cu E誘導(dǎo)腫瘤細(xì)胞自噬過程中的作用。結(jié)果:1.Cu E劑量依賴性抑制He La、MCF7和DU145細(xì)胞增殖。Cu E處理He La細(xì)胞24 h后,其IC50為4.01μmol/L,處理48 h后,IC50為0.06μmol/L。2.Cu E處理He La和MCF7細(xì)胞后導(dǎo)致LC3-II的表達(dá)增加,利用氯喹(chloroquine)阻斷自噬可導(dǎo)致LC3-II蛋白在胞內(nèi)的累積,上調(diào)其蛋白水平;免疫熒光技術(shù)檢測到自噬體標(biāo)志蛋白LC3-II和溶酶體蛋白LAMP2高度共定位;而p62/SQSTM1蛋白能被降解。表明Cu E誘導(dǎo)了完整的細(xì)胞自噬過程。3.Cu E處理ATG5基因缺陷的DU145細(xì)胞24h后無LC3-II的表達(dá);也沒有發(fā)現(xiàn)有LC3斑與LAMP2的共定位;Si RNA技術(shù)敲低He La和MCF7細(xì)胞中ATG5基因表達(dá)后,胞內(nèi)LC3-II的表達(dá)水平也被抑制;這些結(jié)果表明,Cu E誘導(dǎo)的細(xì)胞自噬依賴于ATG5的表達(dá)。4.通過對m TORC1底物磷酸化水平的檢測發(fā)現(xiàn),Cu E對p70S6K、S6的磷酸化作用有顯著的劑量和時間依賴性抑制作用,但上調(diào)了4E-BP1T37/46和p-4E-BP1T37/46的表達(dá)。Cu E降低m TORC1下游底物ULK1S758磷酸化表達(dá)水平;免疫熒光技術(shù)發(fā)現(xiàn)m TOR與LAMP2斑點的共定位減少。這些結(jié)果表明Cu E能抑制m TORC1的活性,從而活化ULK1,誘導(dǎo)細(xì)胞自噬。5.Cu E處理He La、MCF7、和DU145細(xì)胞不同時間后,免疫印記結(jié)果顯示AMPKα和AMPKβ的磷酸化水平在早期的時間點明顯增加但在隨后逐漸恢復(fù)到基礎(chǔ)水平;而AMPK的底物ULK1S555和RaptorS792的磷酸化水平也呈現(xiàn)相應(yīng)的變化,表明Cu E能激活A(yù)MPK的活性。AktT308的磷酸化不受Cu E處理的影響,m TORC2下游蛋白AktS473的磷酸化稍稍增強。6.如果Cu E和AMPK特異性抑制劑化合物C共同作用于He La細(xì)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顛倒,即p-p70S6K/p70S6K的比例增加;而不影響ULK1S758磷酸化水平,表明Cu E能通過活化AMPK而抑制m TORC1的活性。7.利用si RNAs干擾技術(shù)敲低AMPKα能減弱Cu E對m TORC1/p70S6K信號的抑制效應(yīng),而不影響m TORC1/ULK1S758信號的抑制;但明顯降低LC3-II的表達(dá)水平。結(jié)論:葫蘆素E通過抑制m TORC1的活性和上調(diào)AMPK活性誘導(dǎo)MCF7和He La細(xì)胞發(fā)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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R285
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本文編號：2180483