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

描述（由申请人提供）：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降低。几种天然的和合成的他汀类药物促进人淋巴母细胞样细胞、骨髓瘤和乳腺癌细胞的凋亡。这种效果与它们抑制HMG辅酶a还原酶的能力直接相关，而不是由角鲨烯（胆固醇的直接前体）抑制，而HMG辅酶a还原酶会阻止异丙烯化小gtpase的合成。这一建议是基于他汀类药物通过增加诱导型一氧化氮合酶（iNOS）和一氧化氮（NO）的表达和/或降低精氨酸酶的表达来增加乳腺癌细胞毒性的发现。1400W（一种特异性更强的iNOS抑制剂）和甲羟戊酸（乙酰辅酶a / hmg -辅酶a还原酶反应的直接代谢产物）可部分逆转他汀类药物介导的细胞死亡。补充甲羟戊酸抑制他汀类药物诱导的iNOS和NO，恢复精氨酸酶的表达。氟伐他汀在体内动物模型中剂量依赖性抑制乳腺肿瘤的发展。有待验证的假设有：(1)他汀类药物刺激乳腺癌细胞中的一氧化氮，这是其促凋亡、杀瘤和抗增殖作用的原因；(2)他汀类药物通过抑制乳腺癌细胞中的RhoA信号传导抑制精氨酸酶的表达和活性；(3)在乳腺癌细胞和大鼠模型中，补充七叶蒿素（iNOS辅助因子）和生育三烯醇增强了他汀类药物诱导的杀瘤作用。具体目标:(i)评估各种他汀类药物（亲脂性和亲水性）和生育三烯醇对乳腺癌细胞增殖和凋亡的影响，（ii）确定他汀类药物单独治疗和用海蒿素和精氨酸酶抑制剂治疗的细胞中iNOS和NO形成的诱导作用，（iii）确定RhoA在他汀类药物介导的乳腺癌细胞中NO生成、精氨酸酶表达、Nf:B抑制和抗增殖作用中的作用，（iv）建立化学预防大鼠模型。并评价他汀类药物单用及与3-生育三烯醇或皮蝶呤合用的疗效。方法：采用MCF- 7、MDA-MB-231细胞和化学诱导的乳腺癌大鼠模型。HPLC技术将用于检测和定量他汀类药物处理细胞中NO的形成。磁共振成像（MRI）将用于评估大鼠模型对乳腺癌治疗的反应。意义：最近的研究表明，他汀类药物可以预防多种类型的癌症，包括乳腺癌。然而，他汀类药物诱导乳腺癌细胞死亡的分子机制尚不清楚。这一建议将促进我们对他汀类药物的化学预防和化疗能力的理解，无论是单独使用还是与天然存在的生育三烯醇联合使用。新颖性：总体目标是阐明他汀类药物在乳腺癌细胞中发挥抗增殖/促凋亡作用的分子机制。利用生育三烯醇协同增强他汀类药物对乳腺癌细胞和乳腺癌动物模型的化学预防作用是一种创新。MRI将用于监测大鼠模型中乳腺癌的化学预防作用。公共卫生相关性：他汀类药物是最广泛使用的药物之一。最近的研究表明，亲脂性他汀类药物可能对绝经后妇女有益。研究还表明，当他汀类药物与其他营养物质结合使用时，作为抗癌药物会变得更有效。乳腺癌是妇女死亡的主要原因。因此，了解他汀类药物杀死乳腺癌细胞的机制并探索他汀类药物作为化学预防药物在临床应用的可能性是及时而重要的。

DESCRIPTION (provided by applicant): 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 3-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. PUBLIC HEARLTH 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.