【作者】 梁冰；

【导师】 刘晓冬；

【作者基本信息】 吉林大学 ， 放射医学， 2014， 博士

【摘要】 上皮型卵巢癌（epithelial ovarian cancer，EOC）是恶性程度极高的妇科肿瘤。据估计，2011年在美国有21990新发病例，并且有15460例死亡。铂/紫杉醇为基础的化疗是EOC分期手术和切除术后的现行治疗标准。尽管化疗在EOC的治疗上取得一定的成效，但其预后很差，存在腹部或盆腔复发现象，并可进一步发展耐药性。而耐药性的产生，是导致卵巢癌死亡率居高不下的重要原因。因此，如何提高肿瘤细胞对化疗药物的敏感性，是当前临床治疗EOC亟待解决的问题之一。肿瘤细胞通常涉及到多基因和多通路的激活，从而获得对化疗和放疗诱导的细胞死亡的抵抗能力。已知的几种细胞死亡方式包括坏死、凋亡和自噬性细胞死亡等。细胞凋亡又称Ⅰ型程序性细胞死亡，是指生物体为维持其内环境稳定，在特定信号诱导下，由多个基因调控的细胞自主地进行程序性死亡的过程，是放射治疗和其他癌症治疗方式的最重要的死亡机制。自噬性细胞死亡是除凋亡外的另一重要的细胞死亡调控机制，在癌症的发展中扮演重要的角色。细胞自噬是一种进化上保守的代谢过程，其主要作用是将细胞内功能受损的蛋白质以及多余的或有缺陷的细胞器降解，并对蛋白质和氨基酸等回收和再利用。除了这个关键作用，还发现自噬在其他方面也有重要的职能，特别是在有应激压力或损伤的情况下会导致自噬显著增强。自噬在肿瘤发生发展及耐药中发挥着重要的作用，并且依据肿瘤类型和环境条件不同而呈现促进或抑制两种相反的调控结局，即双向调控作用。因此，阐明自噬在肿瘤的治疗中发挥的作用和机制有利于针对不同类型的肿瘤，尤其放化疗抵抗的肿瘤，采取个体化的治疗方案而能够获得良好的治疗效果。目的：以人卵巢癌细胞株SKOV3及其耐药表型SKVCR为研究对象，探讨自噬在化疗及电离辐射诱导的细胞死亡中的作用及其机制，为指导临床肿瘤综合治疗方案的优化以及新治疗靶点的探索提供理论依据。方法：①选用人卵巢癌细胞株SKOV3及其多药耐药表型细胞株SKVCR；②应用深部X射线治疗机进行细胞照射，照射条件：电压180kV，电流18.0mA，滤板为0.25mm Cu和1.0mmAl，靶皮距60cm，剂量率0.40Gy· min–1；③药物选取：VCR（长春新碱）、THP（吡柔比星）、VP-16（依托泊苷）；④细胞存活检测采用CCK8（cell counting Kit-8）法和集落形成法；⑤细胞内mRNA的表达采用Real-Time PCR方法检测；⑥蛋白表达变化采用Western blot检测；⑦细胞自噬采用GFP-LC3转染和MDC染色；⑧细胞凋亡检测采用Hoechst33342染色检测；⑨细胞凋亡、自噬和细胞周期采用流式细胞术检测。结果：1.不同浓度VCR、VP-16、THP能够明显抑制SKOV3及SKVCR细胞的生长，相同药物浓度作用后，SKVCR细胞的存活率显著高于SKOV3细胞（P <0.05）。SKVCR中的P-gp蛋白表达量是SKOV3的50倍以上。MDC荧光染色、Real-time PCR及Western blot方法检测结果发现，两种细胞的基础凋亡水平接近，但耐药株SKVCR中自噬发生率明显高于SKOV3细胞（P <0.05）。2.不同浓度VCR作用4、8、24、48h可以明显抑制SKOV3及SKVCR细胞存活，且细胞毒性具有剂量依赖性和时间依赖性；选取0.02μg/ml VCR作用SKOV3细胞后，对细胞凋亡无明显影响，但细胞自噬率升高到2.63倍；选取10μg/ml VCR处理SKVCR细胞，能够诱导SKVCR细胞凋亡的发生（P <0.05），但对自噬无明显影响。3.分别取3MA、CQ、Rapamycin和ZVAD以最大无作用剂量处理两种细胞，发现3MA和CQ能够抑制自噬，Rapamycin可以诱导自噬，ZVAD对Caspase-3活化有明显抑制作用，并显著抑制凋亡（P <0.05）。4. VCR与3MA、CQ、Rapamycin、ZVAD单独或联合作用SKOV3细胞24h后发现：3MA和Rapamycin对VCR药物敏感性无影响；CQ与VCR联合作用能够增加低浓度VCR对SKOV3细胞的增值抑制率（P <0.05）；ZVAD能够降低0.02~0.2μg/ml VCR对SKOV3细胞的增值抑制作用，细胞生存率高于VCR单独作用组（P <0.05）。MDC染色、流式细胞术及Western blot结果表明，VCR诱导SKOV3细胞发生自噬，但对凋亡无影响，而预先给予自噬抑制剂3MA和CQ可以降低VCR诱导的细胞自噬（P <0.05），同时增加VCR诱导的SKOV3细胞凋亡，而预先加入ZVAD能够抑制VCR诱导的细胞凋亡并促进细胞存活（P <0.05）。5. VCR与3MA、CQ、Rapamycin、ZVAD单独或联合作用于SKVCR细胞24h后，与VCR单独作用相比，3MA/CQ与VCR共同作用能明显降低细胞生存率，起到化疗增敏作用（P <0.05），而Rapamycin则无增敏作用（P>0.05）；与VCR单独作用相比，ZVAD则提高VCR作用后SKVCR的生存率（P <0.05），降低SKVCR对VCR的药物敏感性。流式细胞术及Western blot结果表明，在VCR给药前加入自噬抑制剂3MA和CQ，能够降低VCR诱导的SKVCR细胞自噬（P <0.05），同时增加VCR诱导的SKVCR细胞凋亡，而预先加入ZVAD能够抑制VCR诱导的细胞凋亡和自噬（P <0.05）。6.辐射敏感性研究发现，电离辐射作用于SKOV3和SKVCR细胞可以显著降低细胞存活率，而且电离辐射对SKOV3及SKVCR的细胞毒性具有剂量依赖性，而在单次照射和分割照射之间没有差异；SKOV3细胞的辐射敏感性高于其耐药株（P <0.05）。7.电离辐射对细胞死亡影响的研究发现，辐射引起SKOV3细胞核固缩、碎裂、染色增强、浓缩，同时流式细胞术进一步证实不同辐射方式可以诱导SKOV3和SKVCR细胞的凋亡发生（P <0.05）。电离辐射作用后SKOV3细胞和SKVCR细胞MDC阳性百分率及MAPLC3II/I的比值明显增加（P <0.05），表明电离辐射能够诱导发生凋亡和自噬。8.调控剂对辐射敏感性的影响：凋亡抑制剂ZVAD对电离辐射诱导的SKOV3和SKVCR细胞死亡率没有影响，提示电离辐射诱导SKOV3和SKVCR细胞死亡可能是非凋亡依赖性的；而自噬抑制剂3MA使SKVCR细胞生存率显著下降（P <0.05），显示辐射增敏作用。9.调控剂对细胞死亡的影响：应用ZVAD后发现，与电离辐射单独作用相比，ZVAD抑制SKOV3和SKVCR细胞凋亡的同时伴随自噬抑制。应用3MA后发现，与单独电离辐射组相比，两种细胞MDC阳性百分率降低（P <0.05），3MA抑制了电离辐射诱导的自噬；3MA对电离辐射诱导的细胞凋亡作用不同，3MA抑制了辐射诱导的SKOV3细胞凋亡，却增加了SKVCR的凋亡率（P <0.05）。10.电离辐射对细胞周期的影响：辐射作用SKOV3和SKVCR细胞，可诱导G2/M期阻滞（P <0.05）；在SKVCR细胞中，S期细胞占比例较高，而且电离辐射引起S期时相明显延迟，但是在3MA作用后，发现辐射最不敏感的S期细胞明显减少，而相对敏感的G1/M期细胞比例增加（P <0.05）。表明3MA能够调节细胞周期再分布，协同影响辐射的杀伤作用。结论：1. SKVCR细胞中的自噬高表达现象是产生化疗耐药的重要机制之一。2. VCR化疗过程中发现，抑制自噬显著增加VCR诱导SKVCR细胞凋亡的能力，增加SKVCR细胞对VCR的化疗敏感性。3.电离辐射作用能够诱导两种卵巢癌细胞发生凋亡和自噬，自噬增加SKVCR对电离辐射的抗性。通过3MA抑制SKVCR细胞的自噬，可抑制电离辐射诱导S期延迟并增加辐射诱导的细胞凋亡，而提高放疗敏感性。自噬抑制剂有望成为耐药型卵巢癌放化疗的增敏剂。更多还原

【Abstract】 Epithelial ovarian cancer (EOC) is one of the highly malignant gynecologicaltumors. It is estimated that21,990newly diagnosed cases and15,460relationaldeaths established in the United States in2011. Currently, platinum/paclitaxel-basedchemotherapy is the standard strategy for EOC surgical staging and post-operationtreatment. Although chemotherapy achieved success on EOC treatment, theprognosis is still poor for the presence of abdominal or pelvic recurrence and furtherdeveloping drug resistance. The emergence of drug resistance is an essential causeof high mortality rate of ovarian cancer. Therefore, how to improve the sensitivity oftumor cells to chemotherapeutic agents is currently one of problems urgently to besolved for EOC treatment.The activations of multiple genes and pathways are normally involved in tumordevelopment consequently, to obtain the resistance to chemotherapy andradiotherapy-induced cell death occurr. Generally, cell death includes necrosis,apoptosis and autophagic cell death. Apoptosis, also called type I programmed celldeath, is the most important mechanism of radiotherapy and other therapy modalitiesto tumors. Autophagic cell death is another significant regulatory mechanism of celldeath in addition to apoptosis, which plays an important role in the development ofcancer. Autophagy is an evolutionarily conserved metabolic process, and its majorfunction is the degradation of impaired intracellular proteins, excess or defectiveorganelles and the reusing and recycling proteins and amino acids. Apart from this,autophagy is also found to enhance the response to stress or pressure. Autophagyplays a remarkable role in cancer development and drug resistance, and exhibits twoopposite outcomes, induce either adaptive response or cell death depending ontumor types and environmental conditions. Therefore, to clarify the function andmechanisms of autophagy in cancer treatment is beneficial for the individualized treatment of different types of cancers to acquire considerable therapeutic effect,especially the cancers resistant to radio-chemotherapy.Objective: In this study, we investigated the function and mechanism ofautophagy in chemotherapy and ionizing radiation-induced cell death in humanovarian cancer cell line SKOV3and its drug-resistant phenotype SKVCR in order tooptimize cancer treatment strategies and complement experimental theoretical basisof new therapeutic targets.Methods:(1) Human ovarian cancer cell line SKOV3and multi-drugresistance phenotype cell line SKVCR were used in this study;(2) X-irradiation wasused, irradiation conditions were as follows: voltage180kV, current18.0mA,filtration plate0.25mm Cu and1.0mm Al, distance between target and X source60cm, dose rate0.40Gy/min;(3) VCR (vincristine), THP (pirarubicin) and VP-16(etoposide) were used for chemotherapy resistance experiments;(4) CCK8(cellcounting Kit-8) method and colony formation assay were used for cell survivaldetection;(5) Real-time PCR method was used for mRNA detection;(6) Westernblot was used for protein expression;(7) GFP-LC3Cell transfection and MDCstaining was used for cell autophagy detetion;(8) Hoechst33342staining was usedfor apoptosis detection;(9) Flow cytometry was used for apoptosis, autophagy andcell cycle detection.Results:1. The different concentrations of VCR, VP-16and THP inhibited the growthof SKOV3and SKVCR cells, at the same concentration of drug, the survival rate ofSKVCR cells was higher than that of SKOV3cells (P <0.05). P-gp expression inSKVCR cells was50times higher than that in SKOV3cells. Based on the results ofMDC staining, real-time PCR and Western blot tests, the apoptosis level was foundto be the same in two cell lines, but autophagy in SKVCR cells was significanthigher than that in SKOV3cells (P <0.05).2. The different concentration of VCR can inhibit the survival rate of SKOV3and SKVCR cells in time-and-dose-dependent manner;0.02μg/ml VCR had no effect on apoptosis in SKOV3cells, but autophagy rate increased2.63times;10μg/ml VCR induced apoptosis in SKVCR cells without any effect on autophagy (P <0.05).3.3MA、CQ、Rapamycin and ZVAD at the maximal non-effect concentrationwere used in SKOV3and SKVCR cells,3MA and CQ inhibited but Rapamycininduced autophagy, and ZVAD had an effect on the activation of caspase-3andinduced apoptosis (P <0.05).4. SKOV3was treated with VCR alone or in combination with3MA, CQ,Rapamycin and ZVAD for24h:3MA and Rapamycin had no effect on the drugsensitivity to VCR; CQ combined with VCR enhanced VCR inhibition ofproliferation (P <0.05); ZVAD diminished the inhibition of0.02-0.2μg/ml VCR onSKOV3proliferation, and cell survival rate was higher than that in the VCR alonegroup (P <0.05). Based on the results of MDC staining, flow cytometry andWestern blot, VCR induced autophagy in SKOV3cells and had no effect onapoptosis, but treatment of3MA and CQ prior to VCR reduced the autophagy andincreased apoptosis induced by VCR (P <0.05), whereas pretreatment of ZVADinhibited apoptosis induced by VCR and promoted cell survival (P <0.05).5. SKVCR was treated with VCR alone or in combination with3MA, CQ,Rapamycin and ZVAD for24h:3MA/CQ combined with VCR significantlydecreased the cell survival rate and played a role in sensitizing chemotherapy, ascompared with VCR alone (P <0.05), but Rapamycin had no sensitizing effect (P>0.05)；ZVAD significantly increased the cell survival rate and diminished the drugsensibility to VCR in SKVCR cells (P <0.05). Based on the results of flowcytometry and Western blot, pretreatment of3MA and CQ reducedautophagy andincreased apoptosis induced by VCR(P <0.05),whereas pretreatment of ZVADdecreased apoptosis and autophagy (P <0.05).6. Radiosensitivity testing showed that ionizing radiation significantly reducedthe survival of SKOV3and SKVCR cells in a dose-dependent manner, whereasthere was no difference between single irradiation and fractionated irradiation; SKOV3cells radiosensitivity was higher than that in its drug-resistant cell lines (P <0.05).7. Effects of ionizing radiation on cell death: Hoechst staining showed thatirradiation induced SKOV3cell nuclear karyopyknosis, fragmentation, stainingenhancement and enrichment; Meanwhile flow cytometry further confirmed thatirradiation induced apoptosis in SKOV3and SKVCR cells (P <0.05). After ionizingradiation, the percentage of MDC staining positive cells and MAPLC3II/I ratiosignificantly increased in SKOV3and SKVCR cells (P <0.05), suggesting thationizing radiation can induce apoptosis and autophagy.8. Influence of modulators on radiation sensitivity: Apoptosis inhibitor ZVADhad no effect on the ionizing radiation-induced cell death in SKVCR and SKOV3cells. This suggests that SKVCR and SKOV3cell death induced by ionizingradiation may be apoptosis-independent; But autophagy inhibitor3MA significantlydecreased SKVCR survival and showed radiosensitization effect (P <0.05).9. Effects of modulators on cell death: ZVAD inhibited SKVCR and SKOV3apoptosis accompanied by autophagy inhibition as compared with that of ionizingradiation alone, while3MA reduced the percentage of MDC staining positive inSKVCR and SKOV3cells as compared with that of ionizing radiation alone,confirming that3MA could inhibit ionizing radiation-induced autophagy (P <0.05).Simultaneously,3MA inhibits ionizing radiation-induced apoptosis in SKOV3cells,but increased SKVCR cells apoptotic rate (P <0.05).10. Effects of ionizing radiation on cell cycle: Ionizing radiation induced G2/Marrest in SKVCR and SKOV3cells (P <0.05); a higher percentage of S phase cellswas found in SKVCR cells, and ionizing radiation induced significantly S delay;3MA decreased the percentage of S phase in cells which have lower sensitive toionizing radiation), but increased that of G1/M phase in which cells have highersensitive to ionizing radiation (P <0.05). The result shows that3MA can regulatecell cycle redistribution and might have synergistic effect of ionizing radiation. Conclusion:1. The high autophagic level in SKVCR consists of one of the importantmechanism of chemotherapy resistance.2. The inhibition of autophagy can increase apoptosis induced by VCR andenhance the chemosensitivity to VCR in SKVCR cells.3. Ionizing radiation can induce apoptosis and autophagy in ovarian cancer celllines, and autophagy prompts the resistance of SKVCR cells to ionizing radiation;3MA inhibits S phase delay and apoptosis induced by ionizing radiation, andenhances radiosensitivity through the inhibition of autophagy of SKVCR cells.Autophagy inhibitors may become the chemotherapy sensitizer for drug-resistant ovarian cancer in future.更多还原