组蛋白去乙酰化酶抑制剂丙戊酸致乳腺肿瘤放射增敏作用及其分子机制的研究

Histone deacetylase inhibitors (HDACis) are a kind of compounds that can inhibit the histone deacetylase to induce histone hyperacetylation，valproic acid (VPA) is one from HDACis. It was demonstrated that the status of histone acetylation is associated with transcriptional activity and chromosomal remodeling. And it has been further found HDACis are not only strong anticancer agents but also powerful sensitizer to radiation. However, the molecular mechanism of HDACi-induced radiation sensitization to tumor cells is not clear. DNA double strand breaks (DSB) are the most dangerous form of DNA lesions caused by ionizing radiation (IR).Our previous studies have suggested whether the damaged DSB are repaired effectively, this would be directly related to the sensitivity of breast cancer cells to IR. To repair DSB, eukaryotes have developed two repair pathways: homologous recombination (HR) and non-homologues end joining (NHEJ). VPA is the antiepileptic drug commonly used in clinic, and found the therapeutic dose of VPA showed anti-tumor properties. Our preliminary experiments have indicated that VPA can significantly decrease HR frequencies, increase the accumulation of more DSB and synthetic lethality, suggesting that DNA repair mechanism may involve in HDACi-induced sensitization to breast cancer cells. Therefore, it will be explored whether DNA repair mechanism is required for the radiosensitization induced by HDACis through working models: different tumor cell lines, animal model with breast cancer and primary culture cells from tumor tissue of patients with breast cancer. The research goal in this project would find the new mechanism to understand HDACis-induced sensitization in order to provide the strong and reliable evidence for tumor-therapy in clinic.

组蛋白去乙酰化酶抑制剂（HDACis）是引起组蛋白过度乙酰化的一类化合物，丙戊酸（VPA）是其中一种。HDACis可增加细胞对放射的敏感性，但其增敏机制尚不清楚。我们研究揭示放射导致的DNA双链断裂（DSB）是否被有效地修复关系到乳腺癌细胞对放射的敏感性，修复DSB的分子机制包括同源重组和非同源末端连接。VPA为抗癫痫药物，发现其治疗安全剂量就显示出抗肿瘤特性；我们预实验表明VPA可显著地下调同源重组分子机制和增加乳腺癌细胞对放射的敏感性。提示DNA修复机制在VPA所致的放射增敏作用中发挥重要作用。由此本项目以VPA为HDACis代表，并以其对DSB修复机制的影响为切入点，利用乳腺癌细胞系和动物模型、及原代培养的活体乳腺肿瘤组织细胞，从基因、蛋白分子水平探讨HDACis放射增敏作用的机制，发现其特异打靶的损伤修复因子，旨在系统阐述HDACis抗癌新理论，为应用其治疗肿瘤患者提供有力的证据。

丙戊酸（VPA）是组蛋白去乙酰化酶抑制剂（HDACis）中具有代表性的化合物，也是临床上常用的抗癫痫病药物。 近年来发现HDACis类化合物可增加肿瘤细胞对放射（IR）的敏感性，但其增敏机制尚不清楚。我们既往研究揭示IR导致的DNA双链断裂（DSB）损伤是否被有效地修复关系到乳腺癌细胞对放射的敏感性，修复DSB的分子机制包括同源重组（HR）和非同源重组（NHEJ）。本项目应用几种不同细胞系（乳腺癌的细胞、骨肉瘤细胞及原代培养的活体乳腺肿瘤组织细胞）以及原发性乳腺癌动物模型等研究体系，从细胞与动物水平上研究VPA对肿瘤组织的放射增敏作用及其机制。研究成果如下：（1）临床治疗癫痫病安全剂量(0.5 mM)与临界剂量(1 mM)的VPA具有增加乳腺癌细胞MCF7与骨肉瘤细胞U2OS对IR的敏感性，其放射增敏机制与干扰BRCA1-Rad51介导的HR与KU80介导的NHEJ两种DNA损伤修复能力有关；VPA与IR联合能增加肿瘤细胞基因组的不稳定性；抑癌基因p53状态能够影响VPA对肿瘤细胞的放射敏感性，表现为p53表达缺欠细胞对VPA的放射增敏作用减低。（2）应用成功建立的雌性SD大鼠原发性乳腺癌动物模型，通过观察肿瘤大小、组织病理形态表现以及B超形态检查等发现VPA能显著地增加放射对肿瘤组织的杀伤作用，在动物水平进一步证明VPA与IR联合对乳腺肿瘤组织具有杀伤作用。（3）在所建立的原发性乳腺癌大鼠模型基础上，获取的活体乳腺肿瘤组织进行原代细胞培养，发现VPA对原代培养的肿瘤细胞也具有放射增敏作用以及降低细胞对DNA损伤的修复能力，为应用VPA进行抗肿瘤放射治疗提供直接的实验依据。综上，本课题阐述了HDACis抗癌新理论，为临床上应用VPA治疗肿瘤患者提供有力的科学证据。