天地散干预乳腺癌侵袭转移及其机制研究

发布时间：2018-04-12 19:23

本文选题：As_4O_6 + EGFR信号通路　；参考：《北京中医药大学》2017年硕士论文

【摘要】：乳腺癌是全球女性患者高发性的癌症,这种高转移率的癌症有着较高的死亡率。虽然乳腺不是维持人类生命活动的主要器官,而且原位乳腺癌并不是致命的,但是由于乳腺癌组织的细胞失去了正常细胞的特性,这些癌细胞的粘附能力较低,并且很容易通过血液循环或者淋巴循环进行扩散转移。到目前为止,尽管在临床许多治疗乳腺癌的方法被应用于治疗当中,但是治疗效果并不是令人十分满意,尤其对HER-2阴性的乳腺癌患者几乎没有作用。曲妥珠单抗是一个近年来开发得比较成功的治疗乳腺癌的抗体药物,临床上用其治疗HER-2阳性的乳腺癌患者取得了较好疗效,但对于HER-2阴性的乳腺癌患者无效。所以研究和开发一个针对HER-2阴性患者的抗肿瘤药物非常必要。EGFR和HER-2是同一家族膜蛋白受体,并且它们是乳腺癌临床治疗的主要靶点。虽然近年来有许多靶向EGFR和HER-2的药物在开发,但是最终在临床上获得卓越疗效的药物却少之又少。另外,随着不断的研究发现(EGF)/EGFR参与了细胞转移,并在这个过程起到促进作用,可以诱导上皮-间质转化过程。EGFR的下游信号通路,EGFR-Akt-mTOR信号轴,在多种迁移的癌细胞中被发现激活,因此以EGFR和HER-2为靶点的有效治疗药物很有必要被开发。天地散是从砒霜开发出来的一种传统医学药物,其主要成分As_4O_6是一种砷制剂,其在韩国被广泛用于恶性肿瘤的治疗。自从上个世纪八十年代,As_4O_6就在韩国当做民间偏方来使用。在近十几年的研究中,As_4O_6在体外实验中对不同的肿瘤细胞(子宫颈癌、结肠癌、脑胶质瘤和白血病)表现出了显著的抑制作用,并在抗肿瘤新生血管、提高化疗药物敏感性和联合用药方面有着一定的作用。尽管对As406抗癌症的研究报道颇多,但是其对乳腺癌方面的研究报道仅有一篇,该篇文章证明了 As_4O_6可以通过NF-κB信号通路对MCF-7乳腺癌细胞起到抑制作用,但是As_4O_6对乳腺癌侵袭转移的影响未见报道,所以本研究在前期研究的基础上,查阅相关的参考文献,选取MCF-7、MDA-MB-231、MDA-MB-453和SKBR3四种不同类型的乳腺癌细胞为研究对象,采用MTT法、划痕实验、Transwell小室实验、粘附实验、RT-PCR和Western blotting分子生物学实验方法,探究As406对乳腺癌细胞侵袭转移作用的影响并阐明其分子机制,并建立BABL/C小鼠乳腺癌移植瘤模型,进一步探讨As406对动物体内移植瘤的影响。本文的主要研究结果如下:(1)As_4O_6可以抑制MCF-7细胞的增殖。As_4O_6可以降低MCF-7细胞的迁移和侵袭能力。经EGF诱导后,细胞的EGFR的磷酸化明显增加,但是经As_4O_6干预后被抑制。现有的数据表明,As_4O_6可以有效地通过抑制EGFR介导的信号通路,抑制相关基因(EGFR)的表达,降低MCF-7细胞的迁移和侵袭能力。(2)As_4O_6可以抑制MDA-MB-231细胞的增殖呈时间和剂量依赖性。As_4O_6可以降低MDA-MB-231细胞的迁移和侵袭能力,并且可以降低MDA-MB-231细胞的MMP-2和MMP-9的表达,特别是对EGFR磷酸化的抑制作用。同时,经EGF诱导后,EGFR的磷酸化明显增加,但是经As_4O_6干预后被抑制。现有的数据表明,As_4O_6可以有效地通过抑制EGFR介导的信号通路,抑制相关基因(EGFR)的表达,降低MDA-MB-231细胞的迁移和侵袭能力。(3)As_4O_6可以抑制MDA-MB-453细胞的增殖,并呈时间和剂量依赖性。As_4O_6可以降低MDA-MB-453细胞的迁移和侵袭能力。经EGF诱导后,MDA-MB-453细胞的侵袭和迁移能力明显增强,As_4O_6干预后明显被抑制。与Lapatinib对比,发现As_4O_6抑制MDA-MB-453细胞迁移和侵袭能力的效果要优于Lapatinib,两药进行等剂量的联合使用后,对MDA-MB-453细胞迁移和侵袭能力的抑制作用要优于两药单独使用。(4)As_4O_6可以抑制SKBR3细胞的增殖,并呈时间和剂量依赖性。As_4O_6能有效抑制HER-2阳性乳腺癌SKBR3细胞的侵袭和迁移,并且SKBR3细胞的粘附能力在As_4O_6的干预下也有所减弱。实验结果表明,As_4O_6抗肿瘤效果与HER-2/EGFR信号通路有关,通过调节在HER-2/EGFR信号通路中的细胞因子(EGFR,HER-2,Akt,MMP-9)和其他关键分子,实现对HER-2阳性乳腺癌细胞迁移和侵袭的抑制作用。总之,As_4O_6抑制HER-2阳性乳腺癌SKBR3细胞的侵袭和迁移能力是通过HER-2/EGFR信号通路的负调节实现的。综上,As406对人三阴性乳腺细胞MDA-MB-231作用较明显,人三阴性乳腺细胞MDA-MB-231 对 As_4O_6 较敏感。(5)天地散可以抑制BABL/C裸鼠乳腺癌肿瘤生长,在本实验剂量下其抑瘤率可达40%以上,并且裸鼠给药前后的体重没有显著性差异(P0.05),说明天地散比较安全,未见明显的毒性作用。
[Abstract]:Breast cancer is the high incidence of cancer in women worldwide, the high metastasis rate of cancer has a high mortality. Although the main organs of breast not sustain human life activities, and in situ breast cancer is not fatal, but because breast cancer cells lose the characteristics of normal cells, the adhesion ability of these cancer cells low and easily through blood circulation or lymph circulation by diffusion transfer. So far, although many methods in clinical treatment of breast cancer was used in the treatment, but the treatment effect is not very satisfactory, especially for patients with HER-2 negative breast cancer almost no role. Trastuzumab is an antibody drug in recent years the development of more successful treatment of breast cancer, the clinical use in the treatment of HER-2 positive breast cancer patients has good curative effect, but for HER-2 negative breast Invalid patients with adenocarcinoma. So the research and development of an HER-2 for patients with negative anti tumor drugs necessary to.EGFR and HER-2 are the same membrane protein receptor family, and they are the main target for the clinical treatment of breast cancer. In recent years there are many drugs targeting EGFR and HER-2 in the development of drugs, but ultimately excellent clinical efficacy is less and less. In addition, with the continuous study found that (EGF) /EGFR is involved in cell migration, and to promote the role in this process, can induce epithelial mesenchymal transition process downstream of.EGFR signaling pathway, EGFR-Akt-mTOR signaling axis, in a variety of cancer cell migration was found with EGFR and HER-2 activation, so it is necessary for the effective treatment of drug targets is developed. The world is scattered from a traditional medicine developed arsenic, the main component of As_4O_6 is a kind of arsenic, which was in South Korea Widely used in the treatment of malignant tumors. Since the last century in 80s, As_4O_6 in South Korea as folk remedies to use. In the recent decade, As_4O_6 in vitro experiments on different tumor cells (Zi Gongjing cancer, colon cancer, glioma and leukemia) showed significant inhibitory effect, and in tumor angiogenesis, has a certain role to improve the sensitivity of chemotherapy drugs and combination therapy. Although the research reports on As406 cancer a lot, but the breast cancer study reported only one article, the article proves that As_4O_6 can through the NF- B pathway of MCF-7 breast cancer cells to inhibit effect of As_4O_6 on invasion and metastasis, but no reports of breast cancer, so the study on the basis of previous research, access to relevant references, including MCF-7, MDA-MB-231, MDA-MB-453 and SKBR3 four Breast cancer cells of different types as the research object, using the method of MTT, scratch test, Transwell assay, adhesion assay, RT-PCR blotting and Western experimental method of molecular biology, to explore the impact of As406 on invasion and metastasis of breast cancer cells and to elucidate its molecular mechanism, and the establishment of BABL/C mice xenograft model of breast cancer, to further explore the effect of As406 the animal transplanted tumor. The main results are as follows: (1) As_4O_6 can inhibit the proliferation of MCF-7 cells.As_4O_6 can reduce the migration and invasion of MCF-7 cells. After induced by EGF cells, the phosphorylation of EGFR increased significantly, but the intervention of As_4O_6 was inhibited. The existing data show that As_4O_6 can effectively through the signal pathway of EGFR mediated inhibition, inhibition of gene expression (EGFR), reduced the migration and invasion of MCF-7 cells. (2) As_4O_6 can inhibit MDA-MB- 231 cell proliferation in a time and dose dependent.As_4O_6 can reduce the migration and invasion of MDA-MB-231 cells, and MDA-MB-231 cells can reduce the expression of MMP-2 and MMP-9, especially the inhibition of the phosphorylation of EGFR. At the same time, after induced by EGF, the phosphorylation of EGFR increased significantly, but the intervention of As_4O_6 was inhibited. The existing data show that As_4O_6 can effectively inhibit EGFR mediated by signaling pathways, inhibition of gene expression (EGFR), reduced the migration and invasion of MDA-MB-231 cells. (3) As_4O_6 can inhibit the proliferation of MDA-MB-453 cells in dose and time dependent.As_4O_6 can reduce the migration and invasion of MDA-MB-453 cells. After EGF induction, the migration and invasion of MDA-MB-453 cells was significantly enhanced, the intervention of As_4O_6 was significantly inhibited. Compared with Lapatinib, As_4O_6 was found to inhibit MDA-MB-453 cell Cell migration and invasion ability is better than the Lapatinib, the two drugs used in combination group, inhibited the invasion and migration of MDA-MB-453 cells is superior to the two drugs used alone. (4) As_4O_6 can inhibit the proliferation of SKBR3 cells in dose and time dependent.As_4O_6 can effectively inhibit the cell invasion and migration HER-2 positive breast cancer SKBR3, and the adhesion ability of SKBR3 cells was also reduced in the intervention of As_4O_6. Experimental results show that the antitumor effect of As_4O_6 HER-2/EGFR pathway, through the regulation of cytokines in HER-2/EGFR signal pathway in (EGFR, HER-2, Akt, MMP-9) and other key molecules that inhibit the migration and invasion of HER-2 positive breast cancer cells. In conclusion, As_4O_6 inhibited the invasion and migration of HER-2 positive breast cancer cell SKBR3 is a negative regulator of HER-2/EGFR signal through the path The summary, As406 on three breast MDA-MB-231 cells obviously, MDA-MB-231 three negative breast cells are sensitive to As_4O_6. (5) and San BABL/C can inhibit the growth of breast carcinoma in nude mice, the dose of the experiment the inhibition rate can reach more than 40%, and nude mice before and after administration of no significant weight the difference (P0.05), and that scattered relatively safe, no toxicity.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

【参考文献】