Carotenoid/Retinoid Modulation of Cellular Redox/Cancer

最近的预防和干预试验的结果表明，在疾病状态早期食用富含抗氧化剂的食物提供了保护优势，而晚期干预饮食中的类胡萝卜素/维甲酸可能会加剧癌症。我们正在进行一系列基于机制的假设驱动研究，旨在解释在人类干预试验中观察到的自相矛盾的结果。我们假设，在化学抗氧化剂N-乙酰-L-半胱氨酸(NAC)或类胡萝卜素/维甲酸的高水平饮食补充下，增加抗氧化细胞环境将改变细胞的氧化还原状态，并促进致癌物质启动的小鼠的癌症发生。正在进行以下具体目标：1)通过使用基因改变的小鼠癌症模型定量评估癌症潜伏期和发病率，确定高水平补充抗氧化剂N-乙酰-L-半胱氨酸(NAC)与苯并[a]芘的肿瘤进展变化之间的联系。2)通过原位研究关键信号转导通路中基因表达的变化模式，确定抗氧化剂N-乙酰-L-半胱氨酸(NAC)通过细胞增殖和凋亡调节失调而改变细胞氧化还原状态，从而改变肿瘤进展的机制和/或作用模式。*确定抗氧化剂N-乙酰-L-半胱氨酸在体外通过调节细胞增殖和凋亡改变了脾细胞的氧化还原状态。细胞凋亡，以及关键信号转导通路中的基因表达。到目前为止，我们观察到饮食中的NAC提高了存活率，减少了皮肤恶性肿瘤的多样性，但增加了局部给予B[a]P诱发的恶性肿瘤的比例。在FVB/N-P53基因缺失的Tg.AC小鼠中诱导的大多数恶性肿瘤(角化棘皮瘤、鳞癌和梭形细胞肿瘤)显示出突变的转基因ras表达，并且P53核阳性(从0到+4区域可变强度)并且不依赖于治疗。内源性c-Ha-ras原癌基因第12位密码子和61位密码子的突变在转基因表达阴性和阳性的恶性肿瘤中均为阴性。NAC单独对脾细胞有丝分裂作用。目前，我们正在进行一项生活研究(0、2、4或6Gy伽玛射线照射的FVB/N杂合子p53+/-小鼠)，在饮食中补充和不补充NAC。我们观察到对照组在24周后胸腺淋巴瘤潜伏期缩短，胸腺淋巴瘤发生率增加(0/15比10/15)，而对照组仅为4Gy加NAC。这些结果可能至少部分是由于体外研究表明，脂多糖(一种B淋巴细胞有丝分裂原)可显著促进NAC诱导的脾细胞增殖(T/B细胞比例为3：1)，但显著抑制细胞凋亡。用B淋巴细胞和T淋巴细胞特异性单抗进行流式细胞仪检测，以确定B淋巴细胞的特异性。当目前的生后和活体组织病理学研究完成后，我们将重点研究抗氧化剂加剧淋巴肿大的分子机制。这些研究将包括在mRNA和蛋白质水平上的基因表达的变化，以及对增殖和凋亡至关重要的信号通路的分子遗传变化。此外，体外研究将集中在1)NAC通过基于微阵列的基因表达来抑制细胞凋亡的机制，随后将使用蛋白质2D凝胶电泳法和Western分析来证实(如果需要确认失调的通路)。

Results from recent prevention and intervention trials imply that consumption of antioxidant-rich foods early in disease states provides a protective advantage, while late intervention with dietary carotenoids/retinoids may exacerbates cancer. We are conducting a series of mechanism-based hypothesis driven studies designed to explain the paradoxical results observed in human intervention trials. We hypothesize that increased antioxidant cellular environment (provided by high levels of dietary supplementation with the chemical antioxidant N-acetyl-L-cysteine (NAC), or carotenoids/retinoids, will alter the cellular redox state and promote cancer in carcinogen initiated mice. The following specific aims are being carried out:1) Determination of the association between high levels of dietary supplementation with the antioxidant N-acetyl-L-cysteine (NAC) and the alteration of tumor progression of benzo[a]pyrene by quantitatively assessing cancer latency and incidence using genetically-altered mouse models of cancer.2)Determination of the mechanism an/or mode of action of the antioxidant N-acetyl-L-cysteine (NAC) altered cellular redox state on tumor progression via dysregulation of cell proliferation and apoptosis by investigation of altered patterns of gene expression in critical signal transduction pathways in situ.* Determination of the mechanism an/or mode of action of the antioxidant N-acetyl-L-cysteine (NAC) altered cellular redox state in vitro in splenocytes via dysregulation of cell proliferation and apoptosis by investigation of altered patterns of proliferation, apoptosis, and gene expression in critical signal transduction pathways. Thus far, we have observed that dietary NAC increases survival and reduces multiplicity of skin malignancies but increases the fraction of malignancies induced by topically administered B[a]P. The majority of the malignancies (keratoacanthomas, squamous carcinomas, and spindle cell tumors) induced in FVB/N-p53 deficient Tg.AC mice demonstrated mutant transgene ras expression and were nucleus positive for p53 (variable intensity from 0 to +4 areas) and treatment independent. Malignancies, both negative and positive for transgene expression were also negative for mutations in codon 12 and codon 61 of endogenous c-Ha-ras protooncogene. NAC alone is mitogenic to splenocytes. Currently, we are conducting an in life study (gamma irradiated FVB/N-heterozygous p53+/- mice at 0, 2, 4, or 6 Gy) with and without NAC supplementation in the diet. We have observed a decreased latency for thymic lymphoma and an increased incidence of thymic lymphoma after 24 wks (0/15 vs 10/15) in the controls versus 4 Gy plus NAC. These results may be explained, at least in part, by in vitro studies that show that lipopolysaccharide (LPS, a B-lymphocyte mitogen) induced splenic cell proliferation (3:1 T- to B-cell ratio) is significantly increased by NAC, but apoptosis was significantly suppressedSuppression of apoptosis was confirmed by independent assays (TUNEL and Annexin V). B-lymphocyte specificity was confirmed by flow cytometry using B- and T-lymphocyte specific monoclonal antibodies. When current post- and in-life studies are completed for histopathology, we will focus on investigation of the molecular mechanisms involved in antioxidant exacerbation of lymphomagenesis. These studies will include changes in gene expression at the mRNA and protein level as well as molecular genetic changes in signaling pathways critical to proliferation and apoptosis. In addition, in vitro studies will be focused on 1) mechanism of NAC associated suppression of apoptosis using microarray based gene expression, followed by confirmation using protein 2D gel electrophoresis and western analysis (where required for confirming dysregulated pathways).