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减少。几种天然和合成的他汀类药物可增强人淋巴母细胞、骨髓瘤和乳腺癌细胞的凋亡。这种作用与它们抑制HMG CoA还原酶的能力直接相关，HMG CoA还原酶阻断异戊二烯化小GTP酶的合成，而不是通过角鲨烯（胆固醇的直接前体）。该提议是基于以下发现：他汀类药物通过诱导型一氧化氮合酶（iNOS）和一氧化氮（NO）的表达增加和/或抑制型一氧化氮合酶（NOS）的表达减少而引起对乳腺癌细胞的细胞毒性增加。他汀介导的细胞死亡可被1400 W（一种更特异性的iNOS（NOS II）抑制剂）和甲羟戊酸（乙酰CoA/HMG-CoA还原酶反应的直接代谢产物）部分逆转。甲羟戊酸补充抑制他汀类诱导的iNOS和NO，并恢复β-内酰胺酶的表达。在体内动物模型中，氟伐他汀剂量依赖性地抑制乳腺肿瘤的发展。待检验的假设为：（i）他汀类药物刺激乳腺癌细胞中的NO，这是其促凋亡、杀肿瘤和抗增殖作用的原因，（ii）他汀类药物通过抑制乳腺癌细胞中的RhoA信号传导来抑制乳腺癌酶的表达和活性，和（iii）补充sepiapterin（iNOS辅因子）和生育三烯酚类药物增强了乳腺癌细胞和大鼠模型中他汀类药物诱导的杀肿瘤作用。具体目标：（i）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细胞凋亡的影响，（ii）确定在单独用他汀类药物以及用sepiapterin和Nf酶抑制剂处理的细胞中iNOS和NO形成的诱导，（iii）确定RhoA在他汀类药物介导的NO产生、Nf酶表达、Nf：乳腺癌细胞中的B抑制和抗增殖作用。（iv）建立化学预防大鼠模型，并评估他汀类药物单独和与3-生育三烯酚或sepiapterin组合的有效性。方法：采用MCF- 7和MDA-MB-231细胞及化学诱导的大鼠乳腺癌模型。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。将使用磁共振成像（MRI）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各种类型的癌症，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仍然未知。这一建议将促进我们对他汀类药物单独使用和与天然生育三烯酚联合使用的化学预防和化学治疗能力的理解。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/促凋亡作用的分子机制。使用生育三烯酚协同增强他汀类药物在乳腺癌细胞和乳腺癌动物模型中的化学预防功效是创新的。MRI将用于监测大鼠模型中乳腺癌的化学预防作用。公共卫生相关性：他汀类药物是最广泛的处方药之一。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，他汀类药物，当与其他营养素结合，成为更有效的抗癌药物。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.