【作者】 张博；

【导师】 周平坤；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 细胞死亡是多细胞生物生命过程中重要的生理或病理现象,也是恶性肿瘤治疗的细胞学响应基础,可分为凋亡(Apoptosis)、坏死(Necrosis)、自吞噬(Autophagy)和有丝分裂灾变死亡(Mitotic catastrophe),其中有丝分裂灾变死亡是发生在细胞有丝分裂期由于异常的细胞分裂而导致的死亡,它常常伴随着细胞周期检测点的异常或纺锤体结构的损伤而发生。有丝分裂灾变死亡的揭示为恶性肿瘤治疗、特别是对传统化疗药物有耐药性的肿瘤的治疗开辟了新的途径,因而也成为开发新的抗癌药物的分子靶向目标。DNA-PKcs是DNA依赖蛋白激酶的催化亚单位(DNA Dependent Protein Kinase Catalytic Subunit),与Ku70和Ku80共同构成DNA-PK复合物。主要功能是参与DNA双链断裂的非同源末端连接(NHEJ)和V(D)J重组,维持染色体端粒末端结构稳定性。DNA-PKcs具有丝氨酸-苏氨酸蛋白激酶活性,可磷酸化多种癌基因产物和转录因子(如c-fos、c-myc、oct-1、c-jun)、DNA损伤修复反应蛋白(如H2AX、p53、Chk2、Artemis、XRCC4),表明DNA-PKcs是一种具有多种功能的蛋白。近年来有研究表明,DNA-PKcs在人类多种肿瘤组织中异常高表达,并且与癌组织恶性度、耐辐射、预后差有密切关系。通过免疫组化分析显示90 %的肝癌患者的肿瘤组织中的DNA-PKcs蛋白水平呈强阳性,而正常肝组织中DNA-PKcs蛋白表达水平呈弱阳性,因此DNA-PKcs是一个很有价值的抗癌分子靶标。我们实验室前期从香兰素衍生物中发现DNA-PKcs的有效抑制剂BVAN08,并发现该化合物对多种组织来源的癌细胞都有杀伤效应,表明BVAN08是一种很有开发前景的抗肿瘤药物。本论文在前期研究结果的启示下,对BVAN08作为分子靶向抗肿瘤药物进行更深入研究,取得了如下主要进展:1)采用MTT法、台盼蓝染色法、Annexin V/PI染色法以及荧光染色法检测BVAN08对细胞增殖活性以及细胞凋亡的影响,发现BVAN08对HepG2细胞的生长有明显的抑制作用,并诱发细胞凋亡;通过单细胞电泳、脉冲电泳检测BVAN08对DNA损伤的影响,发现BVAN08可诱发HepG2细胞DNA双链断裂,且损伤程度与BVAN08作用时间呈正相关;通过流式细胞术检测BVAN08对细胞有丝分裂进程的影响以及激光共聚焦显微镜检测细胞纺锤体结构的变化,发现BVAN08诱发长时间的细胞有丝分裂中期阻滞,并且出现巨大细胞以及有丝分裂异常细胞,证实BVAN08诱发HepG2细胞有丝分裂灾变死亡;Western blot方法检测DNA损伤检测点相关蛋白的表达变化,发现BVAN08抑制DNA损伤修复蛋白DNA-PKcs以及G2/M期转换调控因子FoxM1蛋白的表达,激活检测点激酶Chk2蛋白。2)为了进一步研究BVAN08是通过何种信号调节通路导致细胞发生有丝分裂灾变死亡,以及是否与凋亡存在联系等,通过基因芯片技术以及蛋白质组学技术来研究有丝分裂灾变死亡的发生机制。鉴定出BVAN08作用后诱导表达上调的基因有CDKN1A,CCNE1,CDKN1C,CDKN2D,表达下调的基因有CDC2。蛋白质组学研究结果筛选到93个差异蛋白点(匹配137个肽段),其中表达上调的蛋白点有45个(匹配76个肽段),表达下调的蛋白点有48个(匹配61个肽段)。BVAN08作用后显著相关差异蛋白主要集中在14-3-3调节信号通路、PI3K/AKT信号通路、细胞骨架信号通路等,暗示这些通路参与细胞有丝分裂灾变死亡。3)为了研究DNA-PKcs与细胞有丝分裂灾变死亡的关系,采用RNA干扰手段构建DNA-PKcs的siRNA载体,筛选出DNA-PKcs蛋白表达抑制的肝癌细胞模型,并且比较三种化疗药物(DRB、DDP、VP16)对DNA-PKcs表达抑制的HepG2-H1细胞的影响。发现三种药物均能抑制HepG2-H1细胞生长,导致细胞周期紊乱,其中DRB是诱发G2-M期细胞阻滞,VP-16诱发S期阻滞。DNA-PKcs缺陷显著改变了HepG2细胞对上述药物的细胞周期变化反应性,其中显著加强了DDP诱发HepG2细胞的S阻滞,推动了VP-16诱发S期阻滞细胞进入G2-M。三种药物处理后HepG2-H1细胞都出现异常有丝分裂现象,多核细胞数目也明显增多,表现出有丝分裂灾变死亡的特征,表明DNA-PKcs在调控细胞有丝分裂过程中也发挥功能,BVAN08诱发的有丝分裂灾变死亡与抑制细胞中DNA-PKcs蛋白表达有关。4)通过建立表达绿色荧光蛋白的HepG2细胞系,并利用活体成像系统检测荷瘤鼠体内肿瘤的生长情况,研究发现BVAN08可抑制荷瘤鼠体内肿瘤生长,对荷瘤鼠体重以及外周血白细胞无明显的毒性作用。免疫组化实验显示BVAN08治疗组肿瘤组织中DNA-PKcs表达水平低于对照组,表明BVAN08具有荷瘤鼠体内的抗肿瘤活性,抑制肿瘤生长作用与降低组织中DNA-PKcs表达有关,与离体细胞学结果一致。5)研究BVAN08的辐射增敏效应以及对肿瘤的放疗效果,发现BVAN08与射线联合作用更加显著的抑制细胞存活,提高肿瘤细胞对射线的敏感性,BVAN08与射线联合作用显著抑制荷瘤鼠肿瘤的生长,表明BVAN08提高荷瘤鼠体内肿瘤的辐射增敏效应,并且对荷瘤鼠心、肝、肾无明显的毒性作用。6)研究BVAN08对细胞DNA损伤修复的影响,以及Western blot检测细胞DNA损伤修复蛋白DNA-PKcs、ATM、检测点激酶Chk2、G2期检测点的关键蛋白Plk1。结果发现BAN08抑制照射诱发的DNA双链断裂损伤的修复,抑制损伤修复蛋白DNA-PKcs的表达,揭示BVAN08通过激活ATM、Chk2通号通路提高细胞辐射敏感性。本课题提供了多方面证据表明BVAN08可以诱发细胞有丝分裂灾变死亡,可以抑制荷瘤鼠体内肿瘤生长,并且对正常组织毒性作用弱,本研究还揭示了DNA-PKcs在有丝分裂灾变死亡过程中以及提高荷瘤鼠辐射敏感性中的作用,也为DNA-PKcs作为抗肿瘤药物研发的一个有效分子靶标提供了有力的实验数据。综合结果表明,BVAN08具有开发成为一种有效的辐射增敏抗癌药物的价值。更多还原

【Abstract】 Cell death is an important physiological and pathological phenomenon, which is also the fundamental basis of cellular response to cancer therapy. Cell death can be mechanistically and morphologically categorized into apoptosis, necrosis, autophagy and mitotic catastrophe. Mitotic catastrophe occurs in mitotic cells as the consequence of abnormal cell division, it is often accompanied by abnormal cell cycle checkpoint or the destroyed spindle structure. Mitotic catastrophe has been taken as a new strategy for the development of new anticancer drugs, especially against the cancers with the resistant to traditional chemotherapy treatment.DNA-PKcs is the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex, inincluding another two regulatory components, i.e, Ku70 and Ku80. DNA-PKcs was firstly identified in the pathways of the nonhomolgous end joining (NHEJ) of DNA double-strand breaks and V(D)J recombination. In addition, DNA-PKcs is important for maintaining telomere length and stability, and it can phosphorylate a lot of essential proteins in vitro and in vivo, such as oncogenes products and signal pathway factors (e.g.c-fos, c-myc, oct-1, c-jun), DNA damage response proteins (e.g. H2AX、p53、Chk2、Artemis、XRCC4) and so on.Lately, overexpression of DNA-PKcs has been reported in a extensive variety of human tumour. The immunohistochemistry of liver tumour indicated that the DNA-PKcs level was higher than normal cells. Our laboratory work has found an effective inhibitor of DNA-PKcs by the vanillin derivative—BVAN08, which shows a killing effect on various cancer cells, and has the potential of development for a new anticancer drug. In order to confirm the anticancer target potential of DNA-PKcs and develop BVAN08 as a new anticancer drugs, we have made the following major experimental research achievements:(1) MTT assay, Typan blue staining, Annexin V/PI staining and fluorescent staining were used to analyze the effect of BVAN08 on cell proliferation and apoptosis of HepG2 cells, single cell gel electrophoresis and pulsed-field gel electrophoresis were used to analyze DNA damage and repair. The results indicated that BVAN08 exhibited an effective antiproliferation against HepG2 cells, induced apoptosis and DNA double strand break. On the base of observation of G2/M arrest induced by BVAN08, and the mitotic arrest, spindle structure destruction (aberrant multi-polar spindles) and multinucleated cells were further demonstrated by laser confocal microscopic observation of BVAN08-treated cells after immunostained with the anti-γ-tubulin antibody and anti-?-tubulin antibody. Western blot analysis indicated that cell cycle control associated transcription regulation factor FoxM1 and DNA-PKcs were down-regulated, while the checkpoint kinase Chk2 was activated by BVAN08. Our results indicated that BVAN08 induced mitotic catasphophe of HepG2 cells. Inactivation of FoxM1and DNA-PKcs proteins could involve in the mechanistic pathway of mitotic catastrophe induced by BVAN08.(2) In order to further reveal the signal pathway which is responsible for the induction of mitotic catastrophe by BVAN08, and to study the relationship between mitotic catastrophe and apoptosis, we employed the gene chip technology and proteomics technologies to investigate the mechanism of mitotic catastrophe. The results showed that CDKN1A, CCNE1, CDKN1C, CDKN2D were upregulated and CDC2 was decreased in HepG2 cells treated with BVAN08. The proteomic analyses have identified 93 differentially expressed protein spots which matched 137 peptides, including these 45 increased protein spot(matching 76 peptides), and 48 protein decreased spots(matching 61 peptides). These results suggested that a number of cell cycle-related genes participate in mitotic catastrophe, 14-3-3 signal pathway, and PI3K/AKT signal pathway involve in mitotic catastrophe.(3) In order to further investigate the function of DNA-PKcs in mitotic catastrophe, RNA interference technique was used to decrease the protein level of DNA-PKcs. Stable HepG2 cells line which transfected with DNA-PKcs siRNA vector were selected. In order to compare effect of three anticancer drugs (DRB, DDP, VP-16) on the growth inhibition of DNA-PKcs-depleted HepG2-H1 cells. The results showed that these anticancer drugs inhibited the growth of HepG2-H1 cells and significantly disturbed the cell cycle progression. For example, DRB induces G2-M arrest, while VP-16 induces S phase arrest. However, DNA-PKcs depletion augments the induction of S phase arrest by DDP, and could promote the entrance of the VP-16-arrested S phase cells into G2 phase. The three drugs induced abnormal mitosis phenomenon in BVAN08 treated HepG2-H1 cells, indicated the function of DNA-PKcs in regulating the process of cell mitosis, mitotic catastrophe induced by BVAN08 was related to inactivation of DNA-PKcs protein.(4) In order to investigate the anticancer activity in vivo, HepG2 cell line expressing green fluorescent protein were established for the in vivo tumor growth monitoring of molecular image system. The results demonstrated that BVAN08 has antitumor activity on tumor-bearing mice, and no obvious side effect to body weight and peripheral white blood. Immunohistochemistry demonstrated that the expression of DNA-PKcs protein was decreased in BVAN08 treated group. These results indicated that DNA-PKcs may play a role in inhibiting tumor growth.(5) The sensitization of BVAN08 on radiotherapy of tumors has been further investigated. The results showed that combined treatment BVAN08 andγ-rays significantly decreased the cancer cells survival, increased radiosensitivity of cancer cells. Combined treatment of BVAN08 and andγ-rays remarkably inhibited the growth of tumor in vivo. The results indicated that BVAN08 has radiosensitization effect both in vivo and in vitro. Moreover, BVAN08 has less toxic to the normal tissues.(6) To study the mechanism of BVAN08 on radiosensitization and the expression of a number of critical DNA damage repair proteins, including DNA-PKcs, ATM, checkpoint kinase2 (Chk2) and Plk1 proteins. The results showed that BAN08 inhibited DNA double-strand break repair after irradiation, decreased expression of DNA-PKcs protein, while activated ATM, Chk2 sigaling pathway to increase cell radiosensitivity.This research provided the broad evidence showed that BVAN08 can induce cancer cell death in term of mitotic catastrophe. BVAN08 inhibited tumor growth in vivo, enhance the sensitivity of tumors to radiotherapy but less toxicity to normal tissues. DNA-PKcs can be used as an effective molecular target for cancer therapy and BVAN08 has the value for cancer developing as an effective anticancer drug.更多还原