Mechanisms of tumor suppressor gene reactivation in colon cancer by berries

描述（由申请人提供）：尽管在过去的二十年中，结直肠癌的死亡率在男性和女性中都有所下降，但这种疾病的5年生存率为64%，并在诊断后10年继续下降至57%。对于确诊时远处转移的患者，5年生存率仅为10%。因此，预防结肠癌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途径。我们的实验室一直在评估冷冻干燥浆果，主要是黑覆盆子（BRBs）的使用，以预防动物和人类的结肠癌。饲粮中添加BRB粉可抑制偶氮氧甲烷（AOM）处理大鼠结肠腺瘤和癌的发生，以及ApcMin/+小鼠肠道自发腺瘤的发生。已经在人类中进行了两项先导干预试验：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。我们的研究结果表明，短期内口服BRB散治疗散发性结直肠癌患者（20g/3x/day）可显著降低细胞增殖率，并对结直肠癌肿瘤细胞凋亡、血管生成和Wnt通路相关基因的变化产生积极的趋势。我们的FAP试验表明，对FAP患者口服BRB粉（20g/3次/天）和直肠栓剂形式治疗9个月后，直肠息肉的消退率为53%。这些结果表明，浆果有预防人类结直肠癌的潜力。通过生物定向分离，我们和我们的合作者发现brb中的花青素对它们的化学预防作用很重要。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类结肠肿瘤细胞中DNA甲基转移酶1 （DNMT1）蛋白表达和总DNMT活性，从而导致高甲基化沉默的肿瘤抑制基因（如p16）的去甲基化和再激活。结直肠癌患者的短期BRB干预导致p16去甲基化，并在结肠肿瘤组织的异常隐窝中重新表达该基因。因此，我们建议确定BRB花青素在人结肠癌细胞和ApcMin/+小鼠中对高甲基化沉默基因的去甲基化和再激活是否与肿瘤发生的抑制有关（Specific Aim 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（Specific Aim 2）。我们还将确定在两项人类BRB干预试验中获得的标本中，BRB是否会影响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消退的关系（Specific Aim 3）。最后，我们将进行一项药代动力学研究，以确定在ApcMin/+小鼠饮食中以不同浓度给予花青素及其代谢物时，花青素在人类结肠细胞、肠组织和血清中的摄取情况，以及尿中花青素及其代谢物的水平（Specific Aim 4）。最终，我们计划评估花青素对预防人类结直肠癌的作用。

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. PUBLIC HEALTH RELEVANCE: Colon cancer is the third most prevalent cancer, and the third leading cause of cancer death in the United States. Our laboratory is developing strategies for the prevention of colon cancer using freeze-dried berries. We now propose to evaluate the anthocyanins in berries for their ability to demethylate and reactivate tumor suppressor genes in colon cancer.