Role of iNOS, Nitric Oxide & Arginase in Statin-Mediated Toxicity in Cancer Cells

项目总结/摘要。 长期目标：他汀类药物选择性抑制羟甲基戊二酰辅酶A（HMG-CoA） 还原酶导致胆固醇生物合成减少。几种天然和合成他汀类药物增强 在人淋巴母细胞、骨髓瘤和乳腺癌细胞中的细胞凋亡。这种影响与他们的 抑制HMG CoA还原酶的能力，其阻断异戊二烯化的小GTP酶的合成，而不是通过 角鲨烯，胆固醇的直接前体。这项提议是基于他汀类药物导致 通过增加诱导型一氧化氮的表达增加对乳腺癌细胞的细胞毒性 合成酶（iNOS）和一氧化氮（NO）和/或降低的腺苷酸酶表达。他汀介导的细胞死亡是 1400 W（一种更特异的iNOS（NOS II）抑制剂）和甲羟戊酸（一种立即抑制iNOS的药物）可部分逆转 乙酰辅酶A/HMG-CoA还原酶反应的代谢产物。甲羟戊酸补充剂抑制他汀类药物- 诱导iNOS和NO的表达，并恢复iNOS的表达。氟伐他汀剂量依赖性抑制乳腺癌 在体内动物模型中的肿瘤发展。待检验的假设是：（i）他汀类药物刺激乳腺中的NO 负责其促凋亡、杀肿瘤和抗增殖作用的癌细胞，（ii）他汀类抑制 通过抑制乳腺癌细胞中的RhoA信号传导来抑制RhoA酶表达和活性，以及（iii） 补充Sepiapterin（iNOS辅因子）和生育三烯酚增强他汀类药物诱导的杀肿瘤作用 在乳腺癌细胞和大鼠模型中的作用。具体目标：（一）评估各种他汀类药物的作用 （亲脂性和亲水性）和生育三烯酚对乳腺癌细胞增殖和凋亡的影响， 单独用他汀类药物以及用sepiapterin和pepiapase处理的细胞中诱导iNOS和<$NO形成 抑制剂，（iii）定义RhoA在他汀类药物介导的NO生成、Nf B抑制、Nf抑制中的作用 （iv）建立乳腺癌化学预防大鼠模型，并评价其对乳腺癌细胞增殖的抑制作用。 他汀类药物单独使用和与生育三烯酚或sepiapterin联合使用的有效性。方法：采用MCF-7细胞培养技术， 7和MDA-MB-231细胞以及化学诱导的乳腺癌大鼠模型。HPLC技术将用于 检测和定量用他汀类药物处理的细胞中的NO形成。磁共振成像（MRI）将 用于评估大鼠模型对乳腺癌治疗的反应。重要性：最近的研究表明 他汀类药物可以预防包括乳腺癌在内的各种类型的癌症。然而，分子 他汀类药物诱导乳腺癌细胞死亡的机制仍不清楚。这项建议将促进我们的 了解他汀类药物单独使用和联合使用的化学预防和化疗能力 天然存在的生育三烯酚新奇：总体目标是阐明 他汀类药物在乳腺癌细胞中发挥抗增殖/促凋亡作用。生育三烯酚用于 协同增强他汀类药物在乳腺癌细胞和乳腺癌动物中化学预防功效 模式是创新的。MRI将用于监测大鼠模型中乳腺癌的化学预防作用。项目叙述/相关性。 他汀类药物是最广泛使用的处方药物之一。最近的研究表明， 他汀类药物可能对绝经后妇女有益。研究还表明，他汀类药物，当与 其他营养素，成为更有效的抗癌药物。乳腺癌是导致死亡的主要原因， 妇女因此，了解他汀类药物杀死乳腺癌的机制既及时又重要。 癌细胞，并探讨他汀类药物作为化学预防药物的临床实施的可能性。

PROJECT SUMMARY / ABSTRACT. Long-term goal: Statins selectively inhibit the enzyme hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase leading to decreased cholesterol biosynthesis. Several natural and synthetic statins enhanced apoptosis in human lymphoblastoid, myeloma and breast cancer cells. This effect was directly related to their ability to inhibit HMG CoA reductase, which blocks the synthesis of isoprenylated small GTPases, and not by squalene, an immediate precursor of cholesterol. This proposal is based on the discovery that statins cause increased cytotoxicity to breast cancer cells through either increased expression of inducible nitric oxide synthase (iNOS) and nitric oxide (¿NO) and/or decreased arginase expression. Statin-mediated cell death was partially reversed by 1400W, a more specific inhibitor of iNOS (NOS II), and by mevalonate, an immediate metabolic product of acetyl CoA/HMG-CoA reductase reaction. Mevalonate supplementation inhibited statin- induced iNOS and ¿NO and restored arginase expression. Fluvastatin dose-dependently inhibited mammary tumor development in an in vivo animal model. Hypotheses to be tested are: (i) statins stimulate ¿NO in breast cancer cells that is responsible for their proapoptotic, tumoricidal and antiproliferative effects, (ii) statins inhibit arginase expression and activity through inhibition of RhoA signaling in breast cancer cells, and (iii) supplementation with sepiapterin (iNOS co-factor) and tocotrienols potentiates statin-induced tumoricidal effects in breast cancer cells and in a rat model. Specific aims: (i) Assess the effects of various statins (lipophilic and hydrophilic) and tocotrienols on breast cancer cell proliferation, and apoptosis, (ii) Determine the induction of iNOS and ¿NO formation in cells treated with statins alone and with sepiapterin and arginase inhibitors, (iii) Define the role of RhoA in statin-mediated ¿NO generation, arginase expression, Nf¿B inhibition and antiproliferative effects in breast cancer cells, (iv) Establish a chemopreventive rat model, and evaluate the effectiveness of statins alone and in combination with ¿-tocotrienol or sepiapterin. Methods: We will use MCF- 7 and MDA-MB-231 cells and a chemically-induced breast cancer rat model. HPLC techniques will be used to detect and quantitate ¿NO formation in cells treated with statins. Magnetic resonance imaging (MRI) will be used to assess the response to breast cancer therapy in a rat model. Significance: Recent research suggests that statins may prevent various types of cancers including breast cancer. However, the molecular mechanisms by which statins induce breast cancer cell death remain unknown. This proposal will advance our understanding of the chemopreventive and chemotherapeutic ability of statins, alone and in combination with naturally-occurring tocotrienols. Novelty: The overall goal is to elucidate the molecular mechanism by which statins exert antiproliferative/proapoptotic effects in breast cancer cells. The use of tocotrienols to synergistically enhance chemopreventive efficacy of statin in breast cancer cells and breast cancer animal model is innovative. MRI will be used to monitor chemopreventive effects of breast cancer in a rat model. PROJECT NARRATIVE / RELEVANCE. Statins are one of the most widely prescribed group of drugs. Recent studies suggest that lipophilic statins may be beneficial for postmenopausal women. Studies also suggest that statins, when combined with other nutrients, become more potent as anticancer drugs. Breast cancer is the leading cause of death in women. Thus, it is both timely and important to understand the mechanism(s) by which statins kill breast cancer cells and to explore the possibility for clinical implementation of statins as chemopreventive drugs.