Carotenoid/retinoid Modulation Of Cellular Redox Status

最近的预防和干预试验结果表明，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。我们进行了一系列基于机制的假设驱动的研究，旨在调查在人类干预试验中观察到的自相矛盾的结果。我们假设，增加抗氧化细胞环境(通过高水平的饮食补充化学抗氧化剂N-乙酰-L半胱氨酸或NAC)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。我们观察到，高水平的饮食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)和治疗无关。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。此外，我们进行了一项生活研究(0、2、4或6Gy伽玛射线照射的FVB/N杂合子P53+/-小鼠)，在饮食中添加和不添加NAC。在这项研究中，我们观察到对照组的胸腺淋巴瘤潜伏期显著缩短，而胸腺淋巴瘤的发病率在24周后(0/15比10/15)比4Gy加NAC组显著增加。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)诱导的脾细胞增殖(T/B细胞比例为3：1)在NAC作用下显著增加，但明显抑制了细胞凋亡。细胞凋亡的抑制通过独立的检测(TUNEL和Annexin V)得到证实。用B和T淋巴细胞特异性单抗进行流式细胞仪检测，以确定淋巴细胞来源的特异性。目前，我们正在研究促氧化加重淋巴肿大的分子机制。这些研究包括基因在mRNA和蛋白质水平的表达变化，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，更多的体外研究集中在1)NAC通过基于微阵列的基因表达来抑制细胞凋亡的机制，然后使用蛋白质2D凝胶电泳法和Western分析来证实(在需要的情况下确认异常的通路)。

Recent prevention and intervention trial results 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 conducted a series of mechanism-based hypothesis driven studies designed to investigate the paradoxical results observed in human intervention trials. We hypothesized that increased antioxidant cellular environment (provided by high levels of dietary supplementation with the chemical antioxidant N-acetyl-L-cysteine or NAC), would alter the cellular redox state and promote cancer in carcinogen initiated mice. We have observed that high levels of 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. In addition, we conducted 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. In this study, we observed a significant decrease in 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 suppressed. Suppression of apoptosis was confirmed by independent assays (TUNEL and Annexin V). Lymphocyte origin specificity was confirmed by flow cytometry using B- and T-lymphocyte specific monoclonal antibodies. We are currently investigating the molecular mechanisms involved in pro-oxidant exacerbation of lymphomagenesis. These studies 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, additional in vitro studies have 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).