cGMP Phosphodiesterase, a Novel Chemoprevention Target

描述(由申请人提供)： 这项建议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药物靶点。以前的研究表明，非甾体抗炎药的化学预防作用涉及环氧合酶(COX)依赖和独立的作用机制，但其确切的靶点(S)和诱导细胞凋亡的途径尚未很好地确定。为了支持COX非依赖机制，结构相关的药物，如舒林酸磺酸，已被证明在临床和临床前研究中具有广泛的化学预防效果，而不抑制COX-1或-2同工酶。此外，与抗炎活性相关的化学预防效果似乎需要大剂量的非甾体类抗炎药和环氧合酶-2抑制剂。考虑到包括COX-2选择性抑制剂在内的NSAIDs的化学预防效果受到COX依赖毒性的限制，将COX抑制活性与其抗肿瘤活性分离的可能性对于开发更安全、更有效的药物具有重要意义。最近研究发现，舒林酸磺酸能抑制cGMP-PDE，提高细胞内cGMP水平，从而激活蛋白激酶G(PKG)。这一途径与一种新的机制有关，该机制涉及PKG介导的磷酸化和β-连环蛋白的降解，以抑制依赖于Tcf的细胞生存基因的转录。我们推测，某些NSAIDs和COX-2选择性抑制剂的化学预防活性也是通过抑制cGMP PDE介导的，这可能为诱导携带APC或β-catenin突变的肿瘤细胞的凋亡提供了一种高度选择性的机制。拟议的研究将集中在涉及APC基因突变的结肠癌发生，并可以非COX依赖的方式被非类固醇抗炎药抑制。建议进行体外和体内研究，以确定cGMP PDE是否与非类固醇抗炎药和环氧合酶-2抑制剂的化学预防效果有关。初步研究将确定一组结构不同的NSAIDs和COX-2选择性抑制剂的生长抑制和诱导凋亡活性是否与cGMP PDE抑制相关，并将与对cAMP PDE以及COX-1和COX-2的抑制作用进行比较。PDE抑制NSAIDs(Pins)对细胞内cGMP和cAMP水平的影响将被测量，以研究完整的结肠肿瘤细胞中潜在的同工酶选择性。将研究PKG的激活和对PKA的潜在影响，以及涉及β-连环蛋白降解和抑制Tcf依赖的转录的下游途径。体内研究将在结肠癌发生的ApcMin小鼠模型中进行，以比较PIN和非COX抑制cGMP PDE抑制剂的疗效。最后，本研究将测量肿瘤和血浆样本中的cGMP和cAMP水平，以确定PDE抑制是否在体内发生，以及环核苷酸作为生物标志物的用途。

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.