Induction of MGMT as a Strategy for Chemoprevention

描述（由申请人提供）： 调节DNA修复酶，导致促进消除致癌病变可能是一种新的和成功的癌症预防策略。MGMT（O 6-甲基鸟嘌呤-DNA-甲基转移酶）6可直接逆转鸟嘌呤-O 6烷基化，在保护细胞基因组免受内源性、食物来源、环境和治疗性烷基化致癌物的致突变和毒性作用方面发挥核心作用。大量的流行病学和研究数据表明，MGMT转基因小鼠中自发性和诱导性肿瘤的减少，MGMT-proficient细胞中Ras和p53突变的衰减以及正在进行的MGMT基因转导到骨髓干细胞中以实现烷化剂抗性的策略表明，MGMT是化学预防和可能的化学保护的一个极好的和合理的靶标。该试验（R 03）项目的长期目标是探索通过饮食方法在人类正常组织（包括造血系统）中使用无毒半胱氨酸前药和抗氧化剂植物化合物来增加MGMT水平的可能性。我们已经获得了强有力的证据表明，暴露于半胱氨酸前药（L-2氧代噻唑烷-4-羧酸或OTC）和抗氧化剂化合物（如水飞蓟素、姜黄素和几种药用植物的乙醇提取物）后，多种人类癌细胞系中MGMT活性显著且可重复增加（高达3倍）。MGMT mRNA水平的增加和MGMT蛋白合成速率的增加解释了DNA修复活性的增强。OTC是一种无毒化合物，通过细胞酶5-氧代脯氨酸酶代谢产生半胱氨酸，半胱氨酸的可用性反过来大大增加细胞内GSH水平。我们的假设，建立在这些观察结果是，OTC和选定的植物化学物质将提高MGMT在动物组织中的表达，并减弱临床使用的烷化剂的毒性。这项为期2年的试点项目的主要目的是通过以下具体目标在临床前环境中建立通过MGMT进行化学预防和化学保护的可行性：1）定量OTC和抗氧化剂（姜黄素、水飞蓟素和印楝素）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)描述OTC给药对BCNU水平和程度的影响，以及替莫唑胺诱导的小鼠突变和毒性。在具体目标1中，将在化学预防治疗后测量MGMT的DNA修复活性、肝、肺、结肠、脑、外周血淋巴细胞中的mRNA和蛋白水平以及肝mRNA水平。将评估修复蛋白持续升高所需的治疗持续时间。具体目标2将研究经OTC预处理的动物中BCNU和替莫唑胺诱导的脾T淋巴细胞Hprt基因突变频率、血液学和总体毒性参数。该研究有望为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益处。

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.