Induction of MGMT as a Strategy for Chemoprevention

描述(由申请人提供)： 调节DNA修复酶，促进致癌病变的消除，很可能是一种新的、成功的癌症预防策略。MGMT(O6-甲基鸟嘌呤-DNA-甲基转移酶)6可以直接逆转鸟嘌呤-O6烷基化反应，在保护细胞基因组免受内源性、食源性、环境和治疗性烷基化致癌物的诱变和毒性作用方面发挥着核心作用。大量的流行病学和研究数据表明，MGMT转基因小鼠的自发和诱导肿瘤的减少，MGMT熟练细胞中RAS和P53突变的减弱，以及正在进行的将MGMT基因转导到骨髓干细胞以实现烷化耐药的策略，表明MGMT是一个很好的和合理的化学预防和化学保护的靶点。这个试点项目(R03)的长期目标是探索通过饮食方法提高人类正常组织中MGMT水平的可能性，包括使用无毒的半胱氨酸前体药物和抗氧化剂植物化合物的造血系统。我们已经获得了强有力的证据表明，在暴露于半胱氨酸前体药物(L-2氧并噻唑-4-羧酸或OTC)以及几种药用植物的抗氧化剂化合物(如水飞蓟素、姜黄素和乙醇提取物)后，多种人类癌细胞株的MGMT活性显著和可重复性增加(最高可达3倍)。MGMT mRNA水平的升高和MGMT蛋白合成速率的增加是DNA修复活性增强的原因。OTC是一种无毒的化合物，由细胞内的5-羟基脯氨酸酶代谢生成半胱氨酸，半胱氨酸的可利用性大大提高了细胞内的GSH水平。我们的假设建立在这些观察的基础上，即非处方药和选定的植物化学物质将增强动物组织中MGMT的表达，并减轻临床使用的烷化剂的毒性。这个为期两年的试点项目的主要目标是通过以下具体目标在临床前环境中建立MGMT进行化学预防和化学保护的可行性：1)量化非处方药和抗氧化剂(姜黄素、水飞蓟素和尼姆比丁)给药后小鼠肝脏和非肝脏组织(包括外周血淋巴细胞)中MGMT表达的变化。2)研究非处方药给药对小鼠BCNU水平和程度、替莫唑胺致突变及毒性的影响。在特定目的1中，将检测MGMT的DNA修复活性，肝、肺、结肠、脑、外周血淋巴细胞的mRNA和蛋白水平，以及肝组织的mRNA水平。将评估持续升高修复蛋白所需的治疗时间。具体目的2研究OTC对小鼠脾T淋巴细胞HPRT基因突变频率、BCNU和替莫唑胺整体毒性及血液学指标的影响。这项研究有望为MGMT靶向化学预防策略的临床试验铺平道路。此外，由于GSH和GST-pi可能被诱导，这些方法将提供多种好处。

DESCRIPTION (provided by applicant): Modulation of DNA repair enzymes, leading to facilitated elimination of carcinogenic lesions is likely to be a 6 novel and successful strategy for cancer prevention. MGMT (O6-methylguanine-DNA-methyltransferase), 6 which elicits a direct reversal of guanine-O6 alkylations, plays a central role in protecting the cellular genome from the mutagenic and toxic actions of endogenous, food-derived, environmental, and therapeutic alkylating carcinogens. A vast amount of epidemiological and research data on the reduction of spontaneous and induced tumors in MGMT transgenic mice, attenuation of Ras and p53 mutations in MGMT-proficient cells and the ongoing strategy of MGMT gene transduction into the bone marrow stem cells for achieving alkylator resistance suggest that MGMT is an excellent and rational target for chemoprevention and perhaps chemoprotection. The long-term objective of this pilot (R03) project is to explore the possibility of augmenting MGMT levels through a dietary approach in human normal tissues including the hematopoietic system using a non-toxic cysteine prodrug and antioxidant plant compounds. We have obtained strong evidence for a marked and reproducible increase of MGMT activity (up to 3-fold) in a variety of human cancer cell lines after exposure to a cysteine prodrug (L-2oxothiazolidine-4-carboxylic acid or OTC), and antioxidant compounds such as silymarin, curcumin and ethanol extracts of several medicinal plants. Increased levels of MGMT mRNA and increased rate of MGMT protein synthesis accounted for the enhanced DNA repair activity. OTC is a non-toxic compound, metabolized by the cellular enzyme, 5-oxoprolinase to generate cysteine, whose availability in turn, increases the intracellular GSH levels greatly. Our hypothesis, built on these observations is that OTC and selected phytochemicals will enhance MGMT expression in animal tissues and attenuate the toxicity of clinically used alkylating agents. The major objective of this 2-year pilot project is to establish the feasibility of chemoprevention and chemoprotection via MGMT in a preclinical setting through the following Specific Aims: 1) Quantitate alterations in MGMT expression in hepatic and non-hepatic tissues including the peripheral blood lymphocytes in mice after OTC and antioxidant (curcumin, silymarin, and nimbidin) administrations. 2) Characterize the effect OTC administration on the levels and extent of BCNU, and temozolomide-induced mutations and toxicity in mice. In Specific Aim 1, The DNA repair activity of MGMT, mRNA and protein levels in liver, lung, colon, brain, peripheral blood lymphocytes, and hepatic mRNA levels will be measured after chemopreventative treatments. The treatment duration required for sustained elevation of the repair protein will be assessed. Specific Aim 2 will investigate the mutation frequencies in the Hprt gene of splenic T-lymphocytes, parameters of hematological and overall toxicities induced by BCNU and temozolomide in animals pretreated with OTC. The study promises to pave way for clinical trials of MGMT-targeted chemopreventative strategies. Further, because GSH and GST-pi, likely to be induced, these approaches will provide multiple benefits.