cGMP Phosphodiesterase, a Novel Chemoprevention Target

描述（由申请人提供）： 本提案的总体目标是验证环GMP磷酸二酯酶（cGMP PDE）作为癌症化学预防的新型药物靶点。以往的研究表明，非甾体抗炎药（NSAID）的化学预防功效涉及环氧合酶（考克斯）依赖性和独立的作用机制，尽管负责其凋亡诱导活性的精确靶点和途径尚未得到很好的定义。为支持COX非依赖性机制，在临床和临床前研究中已显示结构相关药物（如舒林酸砜）具有广泛的化学预防功效，且不抑制考克斯-1或-2同工酶。此外，高剂量的NSAID和考克斯-2抑制剂似乎是相对于其抗炎活性的化学预防功效所需的。将考克斯抑制活性与其抗肿瘤活性分离的可能性对于开发更安全和更有效的药物具有重要意义，因为NSAID（包括考克斯-2选择性抑制剂）的化学预防功效受到COX依赖性毒性的限制。最近，舒林酸砜被报道抑制cGMP PDE并升高细胞内cGMP水平以激活蛋白激酶G（PKG）。该途径通过一种新的机制与细胞凋亡诱导有关，该机制涉及PKG介导的磷酸化和β-连环蛋白降解，以抑制细胞存活基因的Tcf依赖性转录。我们假设某些NSAID和考克斯-2选择性抑制剂的化学预防活性也是由cGMP PDE抑制介导的，这可能为诱导携带Apc或β-连环蛋白突变的肿瘤细胞凋亡提供了高度选择性的机制。拟议的研究将集中在涉及Apc基因突变的结肠癌发生，并且可以通过非COX依赖性方式被NSAID抑制。提出了体外和体内研究，以确定cGMP PDE是否负责NSAID和考克斯-2抑制剂的化学预防功效。初步研究将确定一组结构不同的NSAID和考克斯-2选择性抑制剂的生长抑制和细胞凋亡诱导活性是否与cGMP PDE抑制相关，并将其与对cAMP PDE以及考克斯-1和考克斯-2的抑制作用进行比较。将测量PDE抑制性NSAID（PiN）对细胞内cGMP和cAMP水平的影响，以研究完整结肠肿瘤细胞中的潜在同工酶选择性。将研究PKG的激活和对PKA的潜在影响以及涉及β-连环蛋白降解和Tcf依赖性转录抑制的下游途径。将在结肠肿瘤发生的ApcMin小鼠模型中进行体内研究，以比较PIN和非COX抑制性cGMP PDE抑制剂的功效。最后，将测量来自本研究的肿瘤和血浆样品中的cGMP和cAMP水平，以确定体内是否发生PDE抑制以及环核苷酸作为生物标志物的效用。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to validate cyclic GMP phosphodiesterase (cGMP PDE) as a novel drug target for cancer chemoprevention. Previous studies have suggested that the chemopreventive efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) involve a cyclooxygenase (COX)-dependent and independent mechanism of action, although the precise target(s) and pathways responsible for their apoptosis inducing activity have not been well defined. In support of a COX-independent mechanism, structurally related drugs such as sulindac sulfone have been shown to have broad chemopreventive efficacy in clinical and preclinical studies without inhibiting COX-1 or -2 isozymes. Moreover, high dosages of NSAIDs and COX-2 inhibitors appear to be required for chemopreventive efficacy relative to their anti-inflammatory activity. The possibility of disassociating COX inhibitory activity from their antineoplastic activity has significant implications for developing safer and more efficacious drugs given that the chemopreventive efficacy of NSAIDs, including COX-2 selective inhibitors, is limited by COX-dependent toxicity. Recently, sulindac sulfone was reported to inhibit cGMP PDE and elevate intracellular cGMP levels to activate protein kinase G (PKG). This pathway has been linked to apoptosis induction by a novel mechanism involving PKG-mediated phosphorylation and degradation of beta-catenin to inhibit Tcf-dependent transcription of cell survival genes. We hypothesize that the chemopreventive activity of certain NSAIDs and COX-2 selective inhibitors is also mediated by cGMP PDE inhibition which may provide a highly selective mechanism for inducing apoptosis of neoplastic cells that harbor Apc or beta-catenin mutations. The proposed studies will focus on colon carcinogenesis that involves Apc gene mutations and can be inhibited by NSAIDs in a COX-independent manner. In vitro and in vivo studies are proposed to determine if cGMP PDE is responsible for the chemopreventive efficacy of NSAIDs and COX-2 inhibitors. Initial studies will determine if the growth inhibitory and apoptosis inducing activity of a panel of structurally diverse NSAIDs and COX-2 selective inhibitors correlate with cGMP PDE inhibition and will be compared to inhibitory effects on cAMP PDE as well as COX-1 and COX-2. The effects of PDE inhibitory NSAIDs (PiNs) on intracellular cGMP and cAMP levels will be measured to study potential isozyme selectivity in intact colon tumor cells. The activation of PKG and potential effects on PKA as well as a downstream pathway involving beta-catenin degradation and inhibition of Tcf-dependent transcription will be investigated. In vivo studies in the ApcMin mouse model of colon tumorigenesis will be performed to compare the efficacy of a PIN and a non-COX inhibitory cGMP PDE inhibitor. Finally, cGMP and cAMP levels will be measured in tumors and plasma samples from this study to determine if PDE inhibition occurs in vivo and the utility of cyclic nucleotides as biomarkers.