Mechanisms of tumor suppressor gene reactivation in colon cancer by berries

描述(申请人提供)：尽管在过去20年中，男性和女性的结直肠癌死亡率都有所下降，但这种疾病的5年生存率为%，并在确诊后10年继续下降至57%。对于确诊时有远处转移的患者，5年存活率仅为10%。因此，结肠癌的预防仍然是一个重要的目标，而化学预防是实现这一目标的可行方法。我们的实验室一直在评估冷冻干燥浆果(主要是黑树莓)在动物和人类身上预防结肠癌的使用。饮食中添加BRB粉可抑制偶氮甲烷(AOM)处理的大鼠结肠腺瘤和肿瘤的发展，以及ApcMin/+小鼠肠道自发性腺瘤的发展。已经在人类身上进行了两项试验性干预试验：一项是在散发性结直肠癌患者中进行的，另一项是在家族性腺瘤性息肉病(FAP)患者中进行的。我们的结果表明，口服BRB粉(20g/3x/天)短期治疗散发性结直肠癌患者显著降低了细胞增殖率，并使结直肠癌中细胞凋亡、血管生成和Wnt途径相关基因的变化呈积极趋势。我们的FAP试验表明，口服BRB粉剂(20g/3x/天)和直肠栓剂治疗FAP患者9个月后，直肠息肉消退率为53%。这些结果表明，浆果具有预防人类结直肠癌的潜力。使用生物定向分级，我们和我们的合作者发现，BRBs中的花青素对其化学预防作用非常重要。最近的初步研究表明，BRBs的结直肠癌抑制作用可能至少部分是由于它们能够抑制人结肠肿瘤细胞中DNA甲基转移酶1(DNMT1)的蛋白表达和总DNMT活性，从而导致p16等高甲基化沉默的抑癌基因去甲基化和重新激活。对结直肠癌患者进行短期BRB干预后，p16基因去甲基化，并在结肠肿瘤组织的异常隐窝中重新表达。因此，我们建议确定在人类结肠癌细胞和ApcMin/+小鼠中，BRB花青素对高甲基化沉默基因的去甲基化和重新激活是否与抑制肿瘤形成有关(特异性目标1)，以及DNMT1在BRB花青素诱导的肿瘤抑制基因去甲基化中的作用(特异性目标2)。我们还将确定BRBs是否影响从两个人类BRB干预试验获得的样本中高甲基化沉默的肿瘤抑制基因的甲基化状态，以及它与细胞增殖减少和息肉退化的关系(特定目标3)。最后，我们将进行药代动力学研究，以确定花青素在人体结肠细胞、肠道组织和血清中的摄取，以及在ApcMin/+小鼠饮食中以不同浓度给药时花青素及其代谢物的尿液水平(特定目标4)。最终，我们计划评估花青素在预防人类结直肠癌方面的作用。

DESCRIPTION (provided by applicant): Although mortality rates from colorectal cancer have declined in both men and women over the past two decades, the 5-year survival from this disease is 64% and continues to decline to 57% at 10 years after diagnosis. For persons with distant metastases at diagnosis, the 5-year survival is only 10%. Therefore, the prevention of colon cancer remains an important goal and chemoprevention is a viable approach to achieve this goal. Our laboratory has been evaluating the use of freeze-dried berries, mainly black raspberries (BRBs), for the prevention of colon cancer in animals and in humans. The addition of BRB powder to the diet inhibited the development of colonic adenomas and carcinomas in azoxymethane (AOM)-treated rats, and of spontaneous adenomas in the intestine of ApcMin/+ mice. Two pilot intervention trials have been conducted in humans: one in patients with sporadic colorectal cancer and the other in patients with familial adenomatous polyposis (FAP). Our results suggest that short-term treatment of sporadic colorectal cancer patients with oral BRB powder (20g/3x/day) significantly reduced cell proliferation rates and produced a positive trend for changes in apoptosis, angiogenesis, and in genes associated with the Wnt pathway in colorectal tumors. Our FAP trial indicated that nine month treatment of FAP patients with BRB powder administered both orally (20g/3x/day) and in the form of rectal suppositories caused a 53% regression rate of rectal polyps. These results suggest that berries have potential for prevention of colorectal cancer in humans. Using bio-directed fractionation, we and our collaborators found that the anthocyanins in BRBs are important for their chemopreventive effects. Recent preliminary studies suggest that the colorectal cancer inhibitory effect of BRBs may be due, at least in part, to their ability to inhibit DNA methyltransferase 1 (DNMT1) protein expression and total DNMT activity in human colon tumor cells, which led to demethylation and reactivation of hypermethylation-silenced tumor suppressor genes such as p16. Short-term BRB intervention in colorectal cancer patients resulted in the demethylation of p16 and re-expression of the gene in aberrant crypts of colonic tumor tissues. We propose therefore to determine if demethylation and reactivation of hypermethylation- silenced genes by BRB anthocyanins in human colon cancer cells and in ApcMin/+ mice is associated with inhibition of tumorigenesis (Specific Aim 1) and the role of DNMT1 in BRB anthocyanin-induced demethylation of tumor suppressor genes (Specific Aim 2). We will also determine if BRBs influence the methylation state of hypermethylation-silenced tumor suppressor genes in specimens obtained from the two human BRB intervention trials, and its association with reduced cell proliferation and polyp regression (Specific Aim 3). Finally, we will conduct a pharmacokinetic study to determine the uptake of anthocyanins into human colon cells, and intestinal tissue and serum, as well as urinary levels of anthocyanins and their metabolites when administered at different concentrations in the diet to ApcMin/+ mice (Specific Aim 4). Ultimately, we plan to evaluate anthocyanins for prevention of colorectal cancer in humans.