NUCLEOSIDE TRANSPORT INHIBITORS FOR CANCER PREVENTION

描述（由申请人提供）： 最近的研究表明，在一些人乳腺癌、肝癌、胃癌和结直肠癌组织中核苷转运蛋白过表达，以及肿瘤中核苷转运蛋白亚型基因过表达的不同谱。这可能强调了在正常细胞转化为肿瘤细胞时观察到的摄取生理核苷的能力的显著增加。核苷转运蛋白基因属于延迟早期增殖基因。总之，这些观察结果有力地指出了核苷转运蛋白在致癌过程中的可能作用，至少在肿瘤促进和进展阶段。我们最近发现，核苷转运抑制剂有效地拮抗外源性核苷对脱氢表雄酮（DHEA）抗肿瘤促进活性的抑制作用，对12-O-十四烷酰基佛波醇-13-乙酸酯（TPA）诱导的肿瘤促进在JB6细胞癌变模型。这表明核苷转运蛋白可能参与了致瘤表型的建立，即促进锚定独立生长特征。这与化学预防剂如他莫昔芬下调核苷转运蛋白基因表达的报道一致。这项研究计划的长期目标是研究核苷转运抑制剂的化学预防潜力。本项目的具体目标是：1）探索核苷转运蛋白基因在JB6细胞致瘤转化过程中的表达变化，2）确定核苷转运蛋白阻断剂是否能与外源性核苷供应对化学预防活性的抑制作用竞争抑制致癌作用，3）确定关于化学预防和核苷转运抑制的结构-活性关系（SAR）。该研究计划涉及实验，以确定核苷转运抑制在调节DHEA和他莫昔芬等化学预防剂在体外JB6细胞致癌模型中的作用。这些研究将使用核苷转运抑制剂，即硝基苄硫代肌苷、双嘧达莫和地拉卓，以及将在PI实验室合成的新型四氢异喹啉嘌呤核苷抑制剂。使用锚定非依赖性生长作为终点的TPA诱导的JB6 P+细胞转化模型以及JB6 P +/AP-1-荧光素酶报告细胞肿瘤启动子模型将用于这些概念验证的体外研究。还将通过真实的时间定量RT-PCR对JB6致瘤转化过程中的核苷转运蛋白基因表达进行定量分析。还将进行辅助研究，包括通过HPLC分析细胞核苷酸水平，以及G6PDH活性及其被DHEA抑制的测定。关于致癌过程及其废除的机制的见解可能会导致识别新的分子靶点和癌症预防剂。

DESCRIPTION (provided by applicant): Recent studies have shown an overexpression of nucleoside transporters in some human breast, liver, stomach and colorectal cancer tissues, as well as varying profiles of nucleoside transporter subtype gene overexpression among tumors. This might underscore the marked increases in the capacity to take up physiological nucleosides that has been observed as normal cells are transformed into tumor cells. It has also been revealed that nucleoside transporter genes belong to the delayed early proliferative genes class. Taken together, these observations strongly point to a possible role of nucleoside transporters in the carcinogenesis process, at least at the tumor promotion and progression stages. We have recently found that nucleoside transport inhibitors potently antagonize the inhibitory effect of exogenous nucleosides on the antitumor promotion activity of dehydroepiandrosterone (DHEA) against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in the JB6 cell carcinogenesis model. This suggests that nucleoside transporters may be involved in the establishment of the tumorigenic phenotype, i.e. promotion of anchorage independent growth characteristics. This is consistent with reports that chemopreventive agents like tamoxifen downregulate nucleoside transporter gene expression. The long-term goal of this research program is to investigate the chemopreventive potential of nucleoside transport inhibitors. The specific aims of this project are: 1) to probe the changes in nucleoside transporter gene expression during JB6 cell tumorigenic transformation, 2) to determine whether nucleoside transport blockers can inhibit carcinogenesis compete against the inhibitory effects of exogenous nucleoside supplies on chemopreventive activity, and 3) to determine structure-activity relationships (SAR) regarding chemoprevention and inhibition of nucleoside transport. The research plan involves experiments to define the role of nucleoside transport inhibition in modulating the effects of chemopreventive agents like DHEA and tamoxifen, in the in vitro JB6 cell carcinogenesis model. Nucleoside transport inhibitors, namely, nitrobenzylthioinosine, dipyridamole and dilazep, as well as novel tetrahydroisoquinolinyl purine riboside inhibitors to be synthesized in the PI's laboratory will be used in these studies. The TPA-induced JB6 P+ cell transformation model using anchorage independent growth as end point, as well as JB6 P+/AP-l-luciferase reporter cell tumor promoter models will be employed for these proof-of-concept in vitro studies. Quantitative analysis of nucleoside transporter gene expression during JB6 tumorigenic transformation will also be carried out by means of real time quantitative RT-PCR. Ancillary studies involving analysis of cellular nucleotide levels by HPLC, and assays of G6PDH activity and its inhibition by DHEA will also be conducted. The mechanistic insights to be gained regarding the carcinogenesis process and its abrogation may lead to the identification of novel molecular targets and agents for cancer prevention.