【作者】 于春艳；

【导师】 孙连坤；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 以往的研究发现,抗肿瘤药物、氧化应激等诱导肿瘤细胞内质网应激,通过IRE1(inositol-requiring enzyme 1,肌醇需要酶1)和PERK(PKR-like ER kinase,PKR样内质网激酶)的未折叠蛋白反应及一个钙介导的信号级联反应激活自噬。被激活的自噬是发挥保护作用还是具有细胞毒性以及自噬调节细胞死亡的机制尚需要进一步探讨。本课题在维生素K3在诱导Hela细胞凋亡同时发生了自噬等前期工作的基础上,重点探讨内质网应激在氧化应激诱导细胞自噬中的作用及其相关机制,进一步揭示肿瘤细胞应答应激的能力及其调控机制。研究内容如下:利用维生素K3(Menadione)制作氧化应激模型,以人宫颈癌Hela细胞为研究对象,分别采用醌类药物代谢抑制剂(Dicoumarol),内质网应激诱导剂(Tunicamycin)和内质网应激抑制剂(TUDC),JNK抑制剂(SP600125),MEK抑制剂(U0126)。利用MTT法检测细胞存活率;DCFH-DA染色,共聚焦显微镜观察细胞内活性氧簇(reactive oxygen species,ROS);透射电镜观察内质网等细胞器形态学变化;Western blotting观察自噬相关蛋白LC3-II、Atg12-Atg5、内质网应激相关蛋白GRP78、IRE1α、PERK及其下游MAPK途径相关蛋白JNK、ERK的表达。结果表明,维生素K3可能通过其氧化还原反应产生活性氧簇、引起Hela细胞氧化损伤,维生素K3能够诱导细胞发生内质网应激,被激活的内质网应激通过JNK和ERK途径介导自噬发生。利用自噬抑制剂(3-MA),溶酶体抑制剂(NH4Cl,chloroquine),caspase特异性抑制剂(z-VAD-fmk),泛素-蛋白酶体抑制剂(LAC)。采用MTT法检测细胞存活率;光镜观察细胞形态,透射电镜观察内质网等细胞器形态学变化;采用Cathepsin D免疫荧光来检测溶酶体膜通透性;免疫荧光法观察LC3和LAMP1的共定位,检测自噬体与溶酶体融合过程;Western blotting观察自噬相关蛋白Atg12-Atg5、内质网应激相关蛋白GRP78、p62/SQSTM1及泛素和多泛素化蛋白的表达。结果表明,阻断自噬体与溶酶体融合,增加维生素K3诱导的细胞凋亡,伴有caspase激活及溶酶体膜通透性增加;p62/SQSTM1蛋白表达增加,错误折叠蛋白积聚,内质网应激反应延长。依据以上结果,本实验认为维生素K3诱导的氧化应激反应引起蛋白的错误折叠,错误折叠蛋白的积聚激活内质网应激,通过其下游JNK、ERK途径诱导自噬发生;抑制肿瘤细胞的自噬过程能够加重内质网应激,进而促进细胞凋亡,增加了维生素K3引起的氧化应激损伤。泛素化修饰和p62/SQSTM1参与维生素K3诱导的自噬过程,表明自噬在降解氧化应激诱导的错误折叠蛋白过程中可能具有特异性。可见,内质网应激-自噬反应参与了调控肿瘤细胞生存过程,进一步的实验研究能够为肿瘤的治疗提供新的理论依据。更多还原

【Abstract】 Kinds of stress may result in accumulation of misfolded proteins, which may be sorted to either the proteasome or the macroautophagy pathway for degradation. Macroautophagy can degrade all forms of misfolded proteins, whereas proteasomal degradation is likely limited to soluble ones. Unlike the bulk protein degradation that occurs during starvation, autophagic degradation of misfolded proteins induced by antitumor therapy and oxidative stress can have a degree of specificity, determined by ubiquitin modification and so on.Research during the last decade has contributed to highlight the important roles and modulation mechanisms on endoplasmic reticulum (ER) stress-autophagy response, in which activates a partial unfolded protein response involving IRE1 (inositol-requiring enzyme 1) and PERK (PKR-like ER kinase), and a calcium-mediated signaling cascade. While it still need to be investigated as to whether autophagy is initiated in response to ER stress plays a protective role or a detrimental role. To deal with the role and mechanisms that determine the final outcome of UPR and autophagy activation will be contributed to understanding the mechanisms of tumor, throwing light on the relationship between autophagy and apoptosis, sensitizing tumor cells to anti-tumor therapy.Quinones are widely distributed in nature and many clinically important antitumor drugs contain the quinone nucleus. Menadione is frequently used as oxidative stress model. It is indicated that Menadione could induce apoptosis and autophagy in Hela cells. In our study, wo deal with the role and mechanism of autophagy througy ER stress induction in Hela cell death induced by Menadione, and further reveal the ability to response to stress and modulation mechanismsMethodsTo establish injury model of cervical carcinoma Hela cells by Menadione, NAD(P)H: quinone oxidoreductase inhibitor (Dicoumarol), ER stress inducer Tunicamycin (TM), ER stress inhibtor Tauroursodeoxycholate (TUDC), JNK inhibitor SP600125, MEK1/2 inhibitor U0126, autophagy inhibitor 3-Methyladenine (3-MA), lysosomal inhibitors ammonium chloride (NH4Cl) and chloroquine (CQ), caspase special inhibitor z-VAD-fmk, ubiquitin-proteasomal system inhibitor lactacystin (LAC).The survival rate of Hela cells was detected by MTT method. Intracellular ROS level is measured by DCFH-DA immunofluorescence. Observing celluar morphology by using light microscopy and ER morphology by transmission Electron Microscopy. Autophagy related proteins Atg12-Atg5, ER stress related proteins expressions GRP78、IRE1α、Phos-PERK and downstream MAPK pathway JNK, ERK proteins expressions were detected by Western blotting. Lysosomal membrane permeabiltiy is using Cathepsin D immunostaing. LAMP1和LC3 immunofl uorescence assay is used to study the fusion of autophagosomes with lysosomes. p62 /SQSTM1 and ubiquitin and polyubquitin proteins expressions are performed by Western blottin.Results(1) Dicoumarol increased the the effect of Menadione on inhibiting survival rate and DCFH-DA fluorescence in Hela cells.(2) Menadione induced ER dilation. Menadione increased the expressions of, IRE1α, Phos-PERK proteins, which the same to TM.(3) TM increased the expressions of LC3-II proteins. TUDC increased the effect of Menadione on inhibiting survival rate and downregulated the effect of Menadione on the expressions of GRP78 and LC3-II in Hela cells.(4) The expressions of Phos-ERK, phopho-JNK proteins were increased in Hela cells induced by Menadione. SP600125 and U0126 respectively increased the the effect of Menadione on inhibiting survival rate and inhibited the expression of LC3-II. Both SP600125 and U0126 downregulated GRP78 protein expression in Menadione-treated Hela cells.(5) 3-MA, NH4Cl and chloroquine increased the effect of Menadione on inhibiting cell viability in Hela cells. Inhibition of caspases with Z-VAD-fmk Z-VAD-fmk partially rescued cell death treated with combination of Menadione with NH4Cl. Cathepsin D-specific immunostaining revealed diffuse staining throughout the entire cell treated with combination of Menadione and NH4Cl. The level of Atg12-Atg5 complex under Menadione conditions did not change in the presence of NH4Cl. These LC3-positive structures, did not colocalize with LAMP1 positive vesicles in Hela cells treated with Menadione and NH4Cl, while LC3-positive. structures partially colocalized with LAMP1 positive vesicles in Menadione-treated cells.(6) NH4Cl increased cell viability in the Hela cells treated with LAC and Menadione. NH4Cl increased the ubiquitin and polyubquitin proteins and p62/SQSTM1 protein expressions in Hela cells induced by Menadione. Light microphagy showed that NH4Cl elicit a significant degree of cellular vacuolization compared to Menadione. Electron microscopic analysis indicated that the vacuoles consisted of dilated and expanded ER lumens.Combination of NH4Cl and Menadione increased p62/SQSTM1 protein expression, downregulated ERK activation.Conclusions(1) The mechanism of ROS is possibly mediated by one-electron reduction of Menadione, which induces oxidative stress injury.(2) Menadione induces ER stress during the oxidative stress injury.(3) Menadione induces autophagy through ER stress.(4) JNK and ERK pathway are involved in autophagy through ER stress.(5) Autophagy through ER stress has been proposed to protect against cell death.(6) Inhibition of autophagy cannot result in degradation of misfolded proteins, may exacerbate ER stress, which contribute to cell death. Autophagic degradation of misfolded proteins can have a degree of specificity, determined by ubiquitin modification and the interactions of p62/SQSTM1.更多还原