溶瘤腺病毒调控MGMT表达对葡萄膜黑色素瘤化疗敏感性影响的实验研究

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults; however, current therapeutic modalities,including chemotherapy, have not been successful. Oncolytic viruses serve as an emerging gene therapy tool for cancer treatment because they specifically kill tumor cells while sparing normal cells. H101, a recombinant human type-5 adenovirus with E1B and portions of the E3 region deleted, has been approved for the clinical treatment of head and neck malignancies by the Chinese State Food and Drug Administration. Unfortunately, the monotherapy of adenovirus has demonstrated limited efficacy in a clinical setting. Thus, novel treatment strategies in which an oncolytic virus is combined with existing chemicals are advancing toward potential clinical use. Previous studies demonstrated that O6-methylguanine DNA methyltransferase (MGMT) expression was related to resistance against alkylating agent, and adenovirus E1A could inhibit MGMT promoter activity. In the previous studies, we found out high MGMT expression in UM cell lines. Then we chose the combination of oncolytic virus H101 and the alkylating agent dacarbazine (DTIC) to treat UM cells in vitro. Our results demonstrated that combination of H101 and alkylating agent (dacarbazine,DTIC) exerted a synergistic anti-tumor effect and a down regulation of MGMT expression. In this research, we intend to further validate the synergistic anti-tumor effect of the combination therapy in vivo, uncover the underline molecular mechanisms involved in the combination therapy, and further investigate the regulation of MGMT expression by H101, including the change of MGMT promoter methylation ,histone acetylation and p53 expression. The study is expected to provide new ideas for UM molecular targeted therapy, and provide experimental basis for combination therapy.

葡萄膜黑色素瘤（Uveal Melanoma，UM）是成人最常见的眼内原发性恶性肿瘤，对化疗耐药性高。生物治疗联合化疗是UM治疗的新方向。重组人5型腺病毒（H101）注射液是基于多数肿瘤细胞缺乏p53活性而研发的溶瘤腺病毒，已被批准应用于临床。研究表明，DNA修复蛋白MGMT的高表达导致肿瘤细胞对烷化剂类耐药，腺病毒E1A蛋白能降低MGMT活性。课题组前期研究发现，当H101与烷化剂达卡巴嗪（DTIC）联用时对UM有协同杀伤作用，且使细胞内MGMT表达下调。本研究拟在前期研究基础上，通过体内实验进一步证实H101联合化疗对UM生长的作用，深入探讨H101对MGMT表达的调控，包括对MGMT启动子甲基化水平变化、相应组蛋白的乙酰化水平变化、细胞内p53水平变化的影响等，探索其作用的分子机制。该研究可望为UM分子靶向治疗提供新思路，并为联合治疗的临床研究提供实验基础。

葡萄膜黑色素瘤（Uveal Melanoma， UM）是成人最常见的眼内原发性恶性肿瘤，对化疗耐药性高。生物治疗联合化疗是 UM 治疗的新方向。重组人 5 型腺病毒（H101）注射液是基于多数肿瘤细胞缺乏 p53 活性而研发的溶瘤腺病毒，已被批准应用于临床。研究表明，DNA 修复蛋白 MGMT 的高表达导致肿瘤细胞对烷化剂类耐药，而腺病毒能够通过降低MGMT活性增加化疗药物对肿瘤细胞的杀伤力。在前期的体外实验中我们发现，当 H101 与烷化剂达卡巴嗪（DTIC）联用时对 UM 细胞有协同杀伤作用。这种协同抗瘤作用在本课题的动物实验中得到了验证——H101与DTIC的联合治疗无论是在抑制肿瘤生长方面还是在延长动物生存期方面都优于单药治疗。我们关于作用机制的研究表明，加入了H101后，UM细胞中耐药蛋白MGMT的表达显著降低，而其他蛋白如P300、H3K9等则无明显变化。体外实验还提示，H101可能是通过影响p53的表达而对MGMT蛋白进行调控，进而起到增加UM细胞对化疗敏感性的作用。本研究中，我们通过人UM的SP6.5、VUP 等细胞系，采用PCR，Western blot，siRNA转染等手段，从分子、细胞、动物整体水平等多方面验证了H101联合DTIC的协同抗瘤作用，探讨了p53介导的MGMT调控机制，为 UM 分子靶向治疗提供新思路，并为联合治疗的临床研究提供实验基础。