新型人工合成钠离子通道阻断剂抑制前列腺癌生长的体内体外研究

发布时间：2018-01-06 08:15

本文关键词：新型人工合成钠离子通道阻断剂抑制前列腺癌生长的体内体外研究　出处：《第三军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景和目的:前列腺癌(Prostate cancer)在全球男性中是发病率第二的肿瘤,2012年有1,100,000例新发病例,占男性全部癌症患者的15%。发达国家男性人口占全球男性的17%,但其前列腺癌发病率最高,占全部前列腺癌患者的2/3。虽然亚洲地区前列腺癌发病率较低,但呈现逐年增加的趋势。早期前列腺癌通常没有症状,但发展到晚期常会引起一系列症状,包括排尿困难(尿量减少,排尿次数增多)、血尿、勃起功能障碍等;前列腺癌进一步进展易发生骨转移,癌症转移到骨后会引起臀部、背部、胸部或其他地方的疼痛;当肿瘤压迫脊髓还可能引起腿或足部虚弱麻木、膀胱或者肠失控等。由于前列腺癌的危险因素包括年龄、种族、地理、家族史和基因改变等多不可改变,且具有复杂的分子通路,化疗被认为是治疗前列腺癌的主要治疗方法。因此,找到高效低毒的放化疗药物对于前列腺癌的治疗显得至关重要。电压门控-钠离子通道(Voltage-gated sodium channels,VGSCs)与人类多种恶性肿瘤有关,VGSCs在癌症进程中的各个阶段都发挥着重要的作用,与肿瘤细胞黏附、增殖、迁移、侵袭和转移等相关。研究显示,VGSCs的激动剂能促进癌症细胞的转移,VGSCs的阻断剂能抑制细胞增殖、迁移和侵袭等。VGSCs已经成为癌症治疗的新靶标。因此,重庆医科大学药学院实验室以VGSCs通道蛋白为靶标,运用3D定量构效关系结构模拟软件建立药物结合模型,设计并人工合成了一系列具有VGSCs通道蛋白结合活性的小分子配体-新型钠离子通道胺类配体(Sodium channels amine ligands,SCALs)共15种,分别命名为S0103,S0132,S0135,S0142,S0143,S0152,S0154,S0156,S0158,S0160,S0161,S0163,S0165,S0205,S0307。这一系列小分子配体试图通过与VGSCs的结合,封阻该通道,从而发挥钠离子通道阻断剂的作用抑制前列腺癌转移。本研究以15种SCALs作为筛选化合物,以前列腺癌作为研究对象,评估人工合成的SCALs对前列腺癌生长的影响。VGSCs的通道蛋白Nav1.6和Nav1.7在前列腺癌PC3和LNCa P细胞中大量表达,其m RNA表达水平比正常或增生的前列腺组织高6-27倍(p0.05),它们可能是潜在的前列腺癌诊断指标和治疗靶点。本研究运用S0154和S0161对PC3细胞进行干预,通过Western blotting实验检测细胞中Nav1.6和Nav1.7蛋白的表达,从而观察SCALs对钠离子通道蛋白的影响。肿瘤细胞发生远处转移是一个复杂的生理过程,需要减少细胞黏附、增加细胞能动性和侵袭能力、蛋白酶解、以及抵抗凋亡等。VGSCs阻断剂多通过抑制肿瘤细胞的侵袭和转移发挥抗肿瘤效应。基质金属蛋白酶类(Matrix metalloproteinases,MMPs)是与癌症转移密切相关的细胞内信号分子,肿瘤细胞分泌MMPs,通过降解细胞基底膜进而迁移和侵袭到其他组织,实现癌症的转移。在一大类MMPs中关于MMP-2和MMP-9的研究较为深入,MMP-2或MMP-9过表达时转移的发生率增加,抑制MMP-2或者MMP-9的表达转移发生率减少。本研究运用S0154和S0161对PC3细胞进行干预,通过检测细胞中MMP-2和MMP-9蛋白的表达,从分子水平上观察SCALs对PC3细胞转移能力的影响。同时,我们还运用BALB/c,nu/nu裸鼠建立了PC3细胞移植瘤模型,用S0154或S0161(以DMSO为对照)干预裸鼠移植瘤小鼠,通过监测其体重变化以及移植瘤大小,进而评估S0154和S0161在体内对PC3移植瘤生长的影响。实验方法:1.运用MTT实验初筛15种SCALs对3株前列腺癌细胞(PC3、LNCa P、DU145)生长能力的影响;2.运用钠离子探针实验检测经S0154和S0161处理的PC3细胞内Na+的表达情况,进行Western blotting技术检测干预后PC3细胞中Nav1.6和Nav1.7蛋白的表达;3.运用流式细胞术检测经化合物干预后PC3细胞周期,运用Western blotting技术检测周期相关蛋白的表达情况;4.运用流式细胞术检测经S0154和S0161干预后PC3细胞凋亡情况;5.进行Transwell小室实验,检测S0154和S0161对PC3细胞侵袭能力的影响,Western blotting技术检测MMP-2和MMP-9蛋白的表达;6.采用划痕实验检测S0154和S0161对PC3细胞迁移能力的影响;7.建立PC3前列腺癌移植瘤模型,研究S0154和S0161对裸鼠移植瘤生长情况的影响。实验结果:1.15种人工合成的SCALs中,S0154和S0161抑制前列腺癌细胞株PC3、DU145和LNCa P生长的作用最为显著,IC50值分别在10.51-26.60μM(S0154)、5.07-11.92μM(S0161)之间;2.S0154和S0161增加了PC3细胞内Na+的浓度,抑制PC3细胞中钠离子通道蛋白Nav1.6和/或Nav1.7的表达;3.S0154和S0161通过下调G2/M期的关键蛋白CDK1和Cyclin D1的表达,从而诱导PC3细胞周期阻滞于G2/M期;4.20μM的S0154能促进PC3细胞发生凋亡,但S0161对PC3细胞凋亡作用不明显;5.S0154和S0161下调PC3细胞基质金属蛋白酶类中MMP-2和/或MMP-9蛋白的表达;同时显著抑制PC3细胞的侵袭能力(Transwell小室实验),与对照组相比,2.5μM时即存在显著性差异,10μM的S0154能抑制95%的细胞侵袭,10μM的S0161能完全抑制PC3细胞的侵袭能力;6.划痕实验结果显示,S0154和S0161能抑制PC3细胞的迁移能力,但作用效果不如抑制侵袭明显;7.S0154和S0161能显著抑制PC3移植瘤裸鼠中肿瘤的生长,其中S0161干预2周,实验结束时移植瘤的生长被抑制了50.4%,但其安全性不如S0154。实验结论:在15种SCALs中,S0154和S0161主要通过抑制CDK1和Cyclin D1蛋白表达诱导G2/M期周期阻滞,从而发挥抑制前列腺癌在体内外生长的作用;通过下调MMP-2和/或MMP-9表达抑制肿瘤细胞侵袭进而抑制其转移活性。S0154和S0161可抑制肿瘤细胞生长和侵袭,可抑制前列腺癌移植瘤的生长,是潜在的前列腺癌化疗药。
[Abstract]:Background and objective: prostate cancer (Prostate cancer) in the world of men is second incidence of tumors, 1100000 new cases in 2012, developed countries accounted for 15%. of all cancers in male male population accounted for 17% of the male world, but the incidence of prostate cancer has the highest total prostate cancer patients with 2/3. while in Asia the incidence of prostate cancer is low, but increased year by year. Early prostate cancer often has no symptoms, but to the development of advanced and often cause a series of symptoms, including dysuria (urine volume decreased, urination, hematuria), erectile dysfunction; prostate cancer prone to further progression of bone metastasis, cancer metastasis to bone may cause the hips, back, chest or other parts of the pain; when the tumor compression of the spinal cord may cause leg or foot numbness weakness, bladder or bowel control due to the risk of prostate cancer. Factors including age, ethnicity, geography, family history and genetic changes can not change, and has a complex molecular pathways, chemotherapy is considered to be the main method for the treatment of prostate cancer. Therefore, to find efficient and low toxicity of chemotherapy in the treatment of prostate cancer is very important. The voltage-gated sodium channel (Voltage-gated - sodium channels, VGSCs) associated with a variety of human malignant tumors, VGSCs at each stage in the process of cancer plays an important role, and tumor cell adhesion, proliferation, migration, invasion and metastasis. Research shows that VGSCs agonist can promote the metastasis of cancer cells, VGSCs blockers can inhibit cell proliferation. The migration and invasion of.VGSCs has become a new target for cancer therapy. Therefore, the laboratory Medical University Of Chongqing School of medicine in VGSCs channel protein as the target, the use of 3D quantitative structure-activity relationship model structure The software to set up the drug binding model, designed and synthesized a series of small molecules with VGSCs channel protein ligand binding activity of novel sodium channel amine ligand (Sodium channels amine ligands, SCALs) a total of 15 species, which were named S0103, S0132, S0135, S0142, S0143, S0152, S0154, S0156, S0158. S0160, S0161, S0163, S0165, S0205, S0307. series of small molecule ligands by combining with VGSCs, blocking the channel, so as to play the role of sodium ion channel blockers inhibit the metastasis of prostate cancer. In this study, 15 kinds of SCALs as screening compounds, with prostate cancer as the research object, a lot of artificial expression evaluation the synthesis of SCALs on the growth of prostate cancer.VGSCs channel protein Nav1.6 and Nav1.7 PC3 and LNCa P in prostate cancer cells, the expression level of the m RNA 6-27 times higher than normal or hyperplastic prostate tissue (P0.05), which May be a potential prostate cancer diagnostic markers and therapeutic targets. This study uses S0154 and S0161 intervention in PC3 cells, the expression of Nav1.6 and Nav1.7 protein by Western blotting assay in cells, to observe the effect of SCALs on sodium channel protein. The tumor cells had distant metastasis is a complex physiological process. The need to reduce cell adhesion, invasion and increase of cell activity ability, proteolysis, and resistance to apoptosis by inhibiting.VGSCs blocker multiple tumor cell invasion and metastasis play an anti-tumor effect. Matrix metalloproteinases (Matrix, metalloproteinases, MMPs) is closely related to the intracellular signaling molecules and cancer metastasis, tumor cell MMPs secretion. Through the degradation of basement membrane and cell migration and invasion to other organizations, realize the transfer of cancer. About MMP-2 and MMP-9's research is in a large class of MMPs Further, the transfer of over expression of MMP-2 or MMP-9 increased incidence, inhibit the expression of MMP-2 or MMP-9 reduced the incidence of metastasis. This study uses S0154 and S0161 to intervene by PC3 cells, the expression of MMP-2 and MMP-9 protein was detected in cells, observe the effect of SCALs on metastasis of PC3 cells at the molecular level. At the same time. We also use BALB/c, nu/nu established PC3 cell xenografts in nude mice model with S0154 or S0161 (DMSO control) intervention xenograft in nude mice, by monitoring the changes of body weight and tumor size, S0154 and S0161 and evaluate the in vivo effects of PC3 on the growth of transplanted tumor. Methods: 1. using MTT at the beginning of experiment screen 15 SCALs of 3 strains of prostate cancer cells (PC3, LNCa, P, DU145) affected the growth ability; 2. using the expression of sodium ion probe assay by S0154 and S0161 PC3 cells in Na+, Western blottin G technique to detect the expression of Nav1.6 and Nav1.7 stem cells PC3 protein prognosis; 3. using flow cytometry to detect the compound after the intervention of PC3 cell cycle, the expression of Western blotting detected by cycle related proteins; 4. using flow cytometry to detect the S0154 and S0161 intervention PC3 cell apoptosis; 5. Transwell cell the effect of detection of S0154 and S0161 on the invasion ability of PC3 cells, the expression of Western blotting in detecting MMP-2 and MMP-9 protein; 6. by scratch assay and S0161 S0154 on the migration of PC3 cells; 7. to establish the xenograft model of prostate cancer PC3, effects of S0154 and S0161 on the growth of transplanted tumor in nude mice experiments. Results: 1.15 kinds of synthetic SCALs, S0154 and S0161 inhibit prostate cancer cell lines PC3, DU145 and LNCa P growth is the most significant, IC50 value in 10.51-26.60 M (S0 154), 5.07-11.92 M (S0161); 2.S0154 and S0161 increased the intracellular PC3 concentration of Na+, inhibiting the expression of sodium channel in PC3 cell protein Nav1.6 and / or Nav1.7; 3.S0154 and S0161 through the down-regulation of G2/M protein CDK1 and Cyclin in the key period of D1, thereby inducing PC3 cell cycle arrest in G2/M period; 4.20 M S0154 can promote the apoptosis of PC3 cells, but S0161 has no obvious effect on the apoptosis of PC3 cells; 5.S0154 and S0161 downregulated the expression of matrix metalloproteinases PC3 cells in MMP-2 and / or MMP-9 protein; also significantly inhibited PC3 cell invasion (Transwell assay), compared with the control group. That there is a significant difference between the 2.5 M, 10 M S0154 can inhibit the cell invasion of 95%, 10 M S0161 can completely inhibit the invasion of PC3 cells; 6. scratch experiments showed that S0154 and S0161 can inhibit PC3 cell migration, but the effect of As 7.S0154 and S0161 significantly inhibited the invasion; PC3 can significantly inhibit tumor growth in nude mice, the S0161 intervention for 2 weeks, at the end of the experiment, the tumor growth was inhibited by 50.4%, but its safety is better than S0154. in the experimental conclusion: 15 kinds of SCALs, S0154 and S0161 mainly through inhibition of CDK1 and Cyclin D1 protein the expression of G2/M induced by cycle arrest, inhibit the growth of prostate cancer in vitro and in vivo effect; through down-regulation of MMP-2 and / or MMP-9 expression inhibits tumor cell invasion and metastasis inhibited the activity of.S0154 and S0161 can inhibit the growth and invasion of tumor cells, inhibit the growth of transplanted tumor of prostate cancer, prostate cancer chemotherapy drug potential.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.25

【参考文献】