紫甘蓝花青素抑制三阴性乳腺癌和黑色素瘤的机理研究

发布时间：2018-02-24 02:51

  本文关键词： 紫甘蓝 花青素 矢车菊-3-O-葡萄糖苷 乳腺癌 黑色素瘤　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：花青素(anthocyanin)属于类黄酮,是植物中的一种水溶性天然色素,在蔬菜中分布极广,已有研究报道它能抑制癌症。本研究通过测定几种常见蔬菜中总花青素含量,发现紫甘蓝为总花青素含量最高的,并且测定了紫甘蓝不同部位花青素含量。而紫甘蓝中主要的花青素成分是矢车菊-3-O-葡萄糖苷(Cy-3-glu)。所以本研究利用人三阴性乳腺癌(TNBC)和小鼠黑色素瘤为模型,通过体外体内评价了紫甘蓝中花青素和Cy-3-glu对人TNBC和小鼠黑色素瘤的作用。TNBC是指雌激素受体(ER),孕激素受体(PR),人表皮生长因子受体(HER-2)表达均为阴性的乳腺癌,约占乳腺癌的10-15%,具有远处转移风险大,愈后差等特点。黑色素瘤是一种皮肤癌症,起源于皮肤中的黑素细胞,这两种癌症目前都没有良好的靶向治疗方法。本实验通过单因素实验考察不同浸提条件对花青素提取效果的影响,选取几种常见蔬菜用pH差示法测定其总花青素含量,发现花青素的含量与蔬菜的颜色密切相关：紫色蔬菜比红色蔬菜花青素含量高,红色蔬菜比黄色蔬菜花青素含量高。所测定的紫色蔬菜中以紫甘蓝花青素含量最高。而且同一棵紫甘蓝不同部位花青素含量也不同,由外向内呈递减趋势。本研究发现紫甘蓝花青素主要成分Cy-3-glu能特异的抑制雌激素受体的变体(ERa36)和表皮生长因子受体(EGFR)共表达的人TNBC细胞系(MDA-MB-231, MDA-MB-436和BT20)生长,并促进其凋亡,而对非TNBC细胞(MCF-7,SK-BR-3)和正常乳腺上皮细胞(MCF-10A)没有明显作用。进一步研究发现Cy-3-glu能直接与NBC细胞膜上的ERα36的配体结合域(LBD)结合抑制其下游EGFR/AKT信号通路并促进EGFR蛋白酶体降解。激活肿瘤坏死因子(TNF-α)诱导激活cleaved-caspase-8/3级联凋亡通路,最终不经过线粒体途径使细胞凋亡。同时,通过给裸鼠肿瘤模型饲喂主要花青素成分为Cy-3-glu的紫甘蓝提取物饲料,进一步在小鼠体内验证体外结果,发现紫甘蓝提取物饲料能显著抑制肿瘤细胞生长,这与体外结果一致。另外,32例TNBC病人的免疫组化结果表明TNBC病人ERa36(75.00%)与EGFR(68.75%)都高表达,且二者表达有相关性(r=0.536),暗示Cy-3-glu在临床治疗ERα36与EGFR共阳性的TNBC病人潜在的作用。对于黑色素瘤来说,我们发现Cy-3-glu也能促进小鼠黑色素瘤细胞系(B16-F10)凋亡。进一步研究发现Cy-3-glu是通过靶向重组酶RAD51使B16-F10细胞周期停滞在G2/M期,破坏同源重组,从而导致细胞凋亡。另外,通过给C57小鼠肿瘤模型饲喂主要花青素成分为Cy-3-glu的紫甘蓝提取物饲料,进一步在小鼠体内验证体外结果,发现紫甘蓝提取物饲料能显著抑制肿瘤细胞生长,这与体外结果一致。综上所述,我们发现紫甘蓝中花青素Cy-3-glu通过不同机理抑制癌细胞生长,促进其凋亡。因此我们提出紫甘蓝等富含Cy-3-glu的果蔬将有望成为临床治疗或辅助治疗TNBC和黑色素瘤的潜在食疗产品。
[Abstract]:Anthocyanin, a kind of flavonoid, is a kind of water-soluble natural pigment in plants, which is widely distributed in vegetables. It has been reported that anthocyanin can inhibit cancer. It was found that purple cabbage had the highest total anthocyanin content. The anthocyanin content in different parts of purple cabbage was determined, and the main anthocyanin component in purple cabbage was Cy-3-gluonine. So the study used human three-negative breast cancer TNBC) and mouse melanoma as the model. The effects of anthocyanin and Cy-3-glu on human TNBC and mouse melanoma were evaluated in vitro. About 10-15 percent of breast cancer is characterized by a high risk of distant metastasis and poor recovery. Melanoma is a skin cancer that originates from melanocytes in the skin. There is no good target therapy for these two kinds of cancer at present. The effects of different extraction conditions on anthocyanin extraction were investigated by single factor experiment, and some common vegetables were selected to determine the total anthocyanin content by pH difference method. It was found that the content of anthocyanin was closely related to the color of vegetables: purple vegetables had higher anthocyanin content than red vegetables. The anthocyanin content of red vegetables is higher than that of yellow vegetables, and the anthocyanin content of purple vegetables is the highest, and the anthocyanin content of different parts of the same purple cabbage is different. In this study, we found that Cy-3-glu, the main component of anthocyanin, can specifically inhibit the growth and apoptosis of human TNBC cell line MDA-MB-231, MDA-MB-436 and BT20, which can specifically inhibit the expression of estrogen receptor variant ERa36) and epidermal growth factor receptor (EGFR). There was no significant effect on MCF-7 SK-BR-3) and normal breast epithelial cells (MCF-10A). Further studies showed that Cy-3-glu could directly bind to the ligand binding domain of ER 伪 36 on NBC cell membrane and inhibit its downstream EGFR/AKT signaling pathway and promote EGFR proteasome. Degradation. Activation of tumor necrosis factor TNF- 伪 (TNF- 伪) induces activation of cleaved-caspase-8/3 cascade apoptosis pathway, At the same time, the tumor model of nude mice was fed with Violet Cabbage extract (Cy-3-glu), which was the main component of anthocyanin, to further verify the results in vivo and in vitro. In addition, the immunohistochemical results of 32 patients with TNBC showed that ERa3675.00 and EGFR 68.75) were highly expressed. There was a correlation between the two expressions, suggesting the potential role of Cy-3-glu in the clinical treatment of TNBC patients with co-positive ER 伪 36 and EGFR. We found that Cy-3-glu could also promote the apoptosis of mouse melanoma cell line B16-F10. Further studies showed that Cy-3-glu caused the cell cycle arrest in G _ 2 / M phase by targeting the recombinant enzyme RAD51, which destroyed homologous recombination and led to apoptosis. By feeding C57 mice tumor model with Violet cabbage extract feed with Cy-3-glu as the main anthocyanin component, the results were further verified in vivo and in vitro. It was found that the extract feed could significantly inhibit the growth of tumor cells. In conclusion, we found that anthocyanin Cy-3-glu in purple cabbage inhibits the growth of cancer cells through different mechanisms. Therefore, we suggest that fruits and vegetables rich in Cy-3-glu, such as purple cabbage, may become potential therapeutic products for TNBC and melanoma in clinical treatment or adjuvant therapy.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R151
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本文编号：1528605