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，而膳食类胡萝卜素/类维生素A的后期干预可能会加剧癌症。我们正在进行一系列基于机制的假设驱动的研究，旨在解释在人类干预试验中观察到的矛盾结果。我们假设，增加抗氧化剂细胞环境（由高水平的化学抗氧化剂N-乙酰-L-半胱氨酸（NAC）或类胡萝卜素/类维生素A的膳食补充剂提供）将改变细胞氧化还原状态，并促进致癌物引发的小鼠的癌症。 正在进行以下具体目标：1）通过使用基因改变的癌症小鼠模型定量评估癌症潜伏期和发病率，确定高水平膳食补充抗氧化剂N-乙酰-L-半胱氨酸（NAC）与苯并[a]芘改变肿瘤进展之间的关联。2）通过原位研究关键信号转导途径中基因表达模式的改变，确定抗氧化剂N-乙酰-L-半胱氨酸（NAC）通过细胞增殖和凋亡的失调改变细胞氧化还原状态对肿瘤进展的作用机制和/或模式。确定抗氧化剂N-乙酰-L-半胱氨酸（NAC）的作用机制和/或模式，通过研究关键信号转导途径中增殖、凋亡和基因表达的改变模式，通过细胞增殖和凋亡的失调在体外改变脾细胞的细胞氧化还原状态。到目前为止，我们已经观察到饮食NAC增加了存活率并减少了皮肤恶性肿瘤的多样性，但增加了由局部施用的B[a] P诱导的恶性肿瘤的分数。（角化棘皮瘤，鳞状细胞癌，在FVB/N-p53缺陷的Tg.AC小鼠中诱导的梭形细胞瘤）表现出突变的转基因ras表达，并且对p53呈核阳性（强度从0到+4区域变化）和治疗无关。 转基因表达阴性和阳性的恶性肿瘤，内源性c-Ha-ras原癌基因的密码子12和密码子61突变也是阴性的。 NAC单独对脾细胞具有促有丝分裂作用。 目前，我们正在进行一项在饮食中添加和不添加NAC的活体研究（0、2、4或6戈伊γ辐照FVB/N杂合p53+/-小鼠）。 我们观察到，与4戈伊加NAC相比，对照组胸腺淋巴瘤潜伏期缩短，24周后胸腺淋巴瘤发病率增加（0/15 vs 10/15）。 这些结果可以解释，至少部分地，在体外研究表明，脂多糖（LPS，B淋巴细胞有丝分裂原）诱导的脾细胞增殖（3：1 T-B-细胞比）显着增加NAC，但细胞凋亡显着抑制细胞凋亡的抑制证实了独立的测定（TUNEL和Annexin V）。 使用B-和T-淋巴细胞特异性单克隆抗体通过流式细胞术确认B-淋巴细胞特异性。 当目前的后期和活体研究完成组织病理学，我们将重点调查参与抗氧化剂加重淋巴瘤的分子机制。 这些研究将包括mRNA和蛋白质水平的基因表达变化，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相关的细胞凋亡抑制机制，使用基于基因表达的微阵列，然后使用蛋白质2D凝胶电泳和蛋白质印迹分析（需要确认失调途径）进行确认。

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).