前列腺癌内分泌治疗抵抗机制的初步研究

发布时间：2018-05-21 10:03

本文选题：激素依赖性前列腺癌 + 激素非依赖性前列腺癌　；参考：《吉林大学》2015年博士论文

【摘要】：前列腺癌是欧美国家男性最常见的恶性肿瘤，死亡率居男性肿瘤的第二位。我国前列腺癌的发病率虽然低于西方国家，但近年来呈显著增长且年轻化趋势。前列腺癌的持续和扩散依赖于雄激素受体传导信号，因此雄激素去势治疗是除外科手术与放射治疗外的标准前列腺癌治疗方法，多数前列腺癌患者在首次接受雄激素去势治疗后都有显著疗效。但经过短暂的缓解期后，几乎所有患者都会复发并由雄激素依赖性前列腺癌发展成高度恶化且广泛转移的雄激素非依赖性前列腺癌，，使常规的雄激素去势治疗失去效果。转变后的前列腺癌治疗非常棘手，死亡率极高，因此成为危害老年男性人群健康的重大疾病。对AIPC的转变机制的研究，是目前国内外前列腺癌研究领域的热点，主要包括：雄激素受体基因的扩增、过表达以及突变，AR信号通路异常等。基因表达的改变及信号通路异常，原癌基因、抑癌基因以及生长因子调控失调被认为是ADPC发展为AIPC的主要原因，研究人员运用各种技术发现了大量前列腺癌的相关基因及信号通路。但是前列腺癌的发生发展过程十分复杂，呈现多步骤、多基因复合的特点。另外，多数AIPC研究都是以激素非依赖性细胞PC3、DU145作为模型，因其不表达内源性雄激素受体而产生雄激素抵抗。但前列腺癌的发生发展，以及从激素依赖到非依赖的转变，都不可避免的经历激素与受体的结合，经雄激素-雄激素受体信号通路，将信号传递至细胞内特异靶基因而发挥作用。因此，以上理论都不能合理地阐述AIPC的发生机制，雄激素非依赖性前列腺癌发病仍需进一步深入研究。本研究选择保留了人前列腺癌功能分化特征，同时具有很强雄激素依赖性的LNCaP细胞系作为对象，建立能模拟临床前列腺癌由雄激素依赖转变为雄激素非依赖过程的细胞模型，采用现代细胞生物学、分子生物学、生物信息学技术探索人前列腺癌基因表达改变，进一步揭示雄激素依赖性前列腺癌向雄激素非依赖性前列腺癌转化过程中可能的发生机制，有望为雄激素非依赖性前列腺癌的治疗提供新的理论依据。 1.雄激素非依赖性前列腺癌亚细胞模型的建立选择保留了人前列腺癌功能分化特征的LNCaP细胞为基础，以激素递减法诱导建立雄激素非依赖性前列腺癌细胞模型。LNCaP细胞分别经过去雄激素培养液和去雄激素+氟他胺环境连续传代培养后，观察细胞形态学改变并检测细胞增殖能力、侵袭和迁移能力、PSA分泌等生物学活性变化。结果可见，经去雄激素诱导并连续培养30代的LNCaP-A细胞形态发生明显变化：细胞胞体变小，细胞形态由三角形、长梭型、圆形变为不规则；细胞不再均匀分布，多见单个细胞独立生长，同时出现聚集生长现象。细胞增殖能力增高，可以在去雄激素环境快速生长，并对雄激素竞争性抑制剂氟他胺产生抵抗。细胞的侵袭性及迁移能力也大大增强，在无激素环境下仍能恢复部分PSA的分泌，且随时间的延长而增加。说明该细胞模型从形态学以及生物学行为上都很接近临床内分泌治疗形成的AIPC过程，可以用来进行前列腺癌在细胞学以及分子生物学上的发生机制的初探。 2.雄激素非依赖性前列腺癌基因表达变化以基因芯片技术对雄激素依赖性LNCaP细胞和雄激素非依赖亚细胞模型LNCaP-A进行全基因表达谱扫描，筛选AIPC形成过程中差异表达基因，同时整合KEGG和PANTHER的在线分析以及MAS等生物信息学工具，对AIPC相关基因进行生物信息通路、基因调控网络及生物学功能等多方面分析，以了解ADPC转变成AIPC后的基因表达变化，并在此基础上筛选ADPC转变成AIPC的关键基因及可能信号通路。对基因芯片测得的原始数据进行分析处理，共比对表达基因12207个。以差异倍数＞2.0为标准，得到雄激素非依赖LNCaP-A细胞与对照组LNCaP细胞差异表达基因347个，其中表达上调基因156个，表达下调基因191个，表明激素非依赖性的前列腺癌细胞在基因表达上发生了明显变化。而对比雄激素非依赖LNCaP-A细胞与去雄激素后再以氟他胺处理的LNCaP-A+F细胞，两组细胞基因表达则无明显差异，这也说明经去雄激素诱导产生的激素非依赖性的前列腺癌细胞生长不再受到雄激素抑制剂的影响，对雄激素抑制剂产生抵抗。应用ToppGene工具对差异表达基因进行归类分析，结果显示下调的基因主要涉及膜蛋白、跨膜蛋白、离子结合、细胞间信号传导、基因表达调控等功能，上调基因的基因功能主要涉及细胞内部基因调控、表达、代谢、可变剪切等。参与细胞内部基因调控、表达、新陈代谢等功能的基因表达水平升高，参与外源性物质进入细胞内部的膜蛋白、信号传导，以及细胞间识别粘附等作用的基因表达水平降低，这也与非依赖性前列腺癌细胞不再受外部去激素调控的实验现象吻合。结合差异表达基因的功能、生物学过程和已发表文献，对该过程中的关键基因及可能涉及的信号通路进行初步分析，表达上调基因中主要有4个基因涉及了4种类型的信号通路，而表达下调基因中有7个基因涉及了多种类型的信号通路。这些基因及相关信号通路的提出为进一步研究AIPC发生机制提供了方向。 3.雄激素非依赖性前列腺癌机制的初步探讨采用RT-PCR及western blot方法对TLR2介导的凋亡作用途径、PI3K/AKT途径、MAPK途径等信号通路关键蛋白进行验证，探讨其在激素依赖性前列腺癌向激素非依赖性转化过程中的可能作用。 Western结果可见，caspase3、FADD蛋白表达水平均无明显变化，且均未见激活caspase3条带，提示toll-like信号通路中的凋亡作用途径没有参与非激素依赖性前列腺癌的转变。AKT、p-AKT表达均无明显增高，但NF-κB蛋白表达上调，IκBα蛋白表达下降。提示NF-κB可能为信号通路中一个关键位点，但在LNCaP-A细胞系中不是通过PI3K/AKT通路激活，可能存在着其他激活通路，抑制蛋白IκBα表达的下调可能影响了NF-κB表达变化。对MAPK信号通路相关蛋白进行检测，ERK、MEK、JNK蛋白表达均无显著变化，而p38、HSP27、IL-8表达显著升高，提示在LNCaP细胞转变为LNCaP-A细胞的过程中，ERK途径、JNK途径没有参与，但MAPK/p38信号通路发挥了重要作用，IL-8、HSP70都可能是该途径中重要的信息传递分子。有研究表明MAPK/p38在乳腺癌、卵巢癌、子宫内膜癌耐药机制中发挥着重要作用，但在前列腺癌激素耐药中尚无报道。这为AIPC机制的研究以及AIPC药物作用靶点的发掘提供了新的理论基础。
[Abstract]:Prostate cancer is the most common malignant tumor in the European and American countries, with the death rate of the second male tumor. Although the incidence of prostate cancer in China is lower than that of the western countries, it has been increasing and young in recent years.
The persistence and diffusion of prostate cancer is dependent on the androgen receptor conduction signal, so androgen castration is a standard treatment for prostate cancer except for surgical and radiological treatment. Most of the prostate cancer patients have a significant effect after the first androgenic treatment. After a short period of remission, almost all patients are treated. The recurrent androgen dependent prostate cancer is developed into a highly deteriorating and widely metastatic androgen independent prostate cancer, which makes the routine androgenic treatment of androgen deprivation treatment out of effect. The post transition prostate cancer is very difficult to treat and has a very high mortality rate, so it has become a major disease that endangers the health of old men.
The research on the transformation mechanism of AIPC is a hot spot in the field of prostate cancer at home and abroad. It mainly includes the amplification, overexpression and mutation of androgen receptor gene, abnormal AR signaling pathway, the change of gene expression and the abnormal signal pathway. The proto oncogene, tumor suppressor base and the regulation of growth factors are considered to be the development of ADPC. The main reason for AIPC is that the researchers have used various techniques to discover a large number of genes and signaling pathways in prostate cancer. However, the development of prostate cancer is very complex and has the characteristics of multistep and multi gene recombination. In addition, most of the AIPC studies are based on the hormone non dependent PC3, DU145 as a model, because of its non expression. Androgen resistance is produced by the androgenic androgen receptor. However, the development of prostate cancer, as well as the hormone dependent to non dependent transformation, inevitably experience the combination of hormones and receptors, and the signal passes through the androgen receptor signaling pathway to the specific target genes in the cell. The pathogenesis of AIPC is discussed, and the pathogenesis of androgen independent prostate cancer still needs further study.
In this study, we chose to retain the functional differentiation of human prostate cancer, with a strong androgen dependent LNCaP cell line as an object, to establish a cell model that can simulate the transformation of prostatic cancer from androgen dependence to androgens, and to explore people with modern cell biology, molecular biology and bioinformatics. The changes in the gene expression of prostate cancer further reveal the possible mechanism of androgen dependent prostate cancer in the process of androgen independent prostate cancer transformation. It is expected to provide a new theoretical basis for the treatment of androgen independent prostate cancer.
Establishment of a subcellular model for androgen independent prostate cancer 1.
On the basis of LNCaP cells, which have retained the characteristics of human prostate cancer function differentiation, the androgen independent prostate cancer cell model.LNCaP cells were induced by hormone decreasing method, and the cell morphology changes were observed and the cell proliferation ability was detected by the continuous subculture of androgen and androgen plus flutamide. Invasion and migration, PSA secretion and other biological activities.
The results showed that the morphology of LNCaP-A cells, which were induced by androgens and continuously cultured for 30 generations, showed that the cell body became smaller and the cell morphology changed from triangle, long shuttle type and round to irregular, and the cells were no longer uniformly distributed. The androgen environment is fast growing and resists the androgen competitive inhibitor fluroamine. The cell invasiveness and migration ability is also greatly enhanced, and the secretion of partial PSA can be restored in the hormone free environment, and increases with the time. It shows that the cell model is close to the clinical endocrinology from the morphological and biological behavior. The AIPC process can be used to explore the mechanism of prostate cancer in cytology and molecular biology.
2. gene expression changes in androgen independent prostate cancer
Using gene chip technology to scan the full gene expression spectrum of androgen dependent LNCaP cells and androgen independent subcellular model LNCaP-A, screening the differentially expressed genes in the process of AIPC formation, and integrating the online analysis of KEGG and PANTHER and MAS and other bioinformatics tools to carry out the biological information pathway and gene modulation of the AIPC related genes. In order to understand the changes in gene expression after the transformation of ADPC into AIPC, the changes of gene expression after the transformation of ADPC into AIPC were analyzed, and on this basis, the key genes and possible signaling pathways of AIPC were screened.
The original data measured by gene chip were analyzed and compared with 12207 expression genes. The difference multiplier > 2 was used as the standard to obtain 347 differentially expressed genes of androgen independent LNCaP-A cells and control group LNCaP cells, including 156 up-regulated genes and 191 down-regulation groups, indicating that hormone non dependent prostate cancer is fine. There was a significant change in the cell gene expression, but there was no significant difference in gene expression between the two groups of androgen independent LNCaP-A cells and the LNCaP-A+F cells treated with androgens and flutamide, which also indicated that the growth of the hormone non dependent prostatic adenocarcinoma cells induced by androgens was no longer inhibited by androgens. The effect of the agent is resistant to androgen inhibitors.
The ToppGene tool is used to classify the differentially expressed genes. The results show that the down regulated genes are mainly involved in membrane protein, transmembrane protein, ion binding, intercellular signal transduction, gene expression regulation and so on. The regulation of gene regulation, expression, metabolism and variable shear are involved in the gene function of the gene. The gene expression level of gene regulation, expression, metabolism and other functions increased, and the level of gene expression in the membrane proteins involved in exogenous substances entering the cell, signal transduction, and intercellular recognition and adhesion were reduced, which also coincided with the experimental phenomenon that non dependent prostate cancer cells were no longer regulated by external hormone.
In combination with the function of differentially expressed genes, biological processes and published literature, the key genes and possible signaling pathways involved in the process are preliminarily analyzed. The main 4 genes in the up regulation genes involve 4 types of signaling pathways, while 7 genes in the down regulated genes involve a variety of signaling pathways. These genes and related signaling pathways provide a direction for further study of the pathogenesis of AIPC.
Preliminary study on the mechanism of 3. androgen independent prostate cancer
The possible role of TLR2 mediated apoptosis pathway, PI3K/AKT pathway, MAPK pathway and other key signaling pathway proteins in the process of hormone dependent prostate cancer to hormone dependent transformation was examined by RT-PCR and Western blot.
Western results showed that there was no obvious change in the expression level of Caspase3 and FADD protein, and no Caspase3 bands were activated, suggesting that the apoptotic pathway in the Toll-like signaling pathway did not participate in the transformation of.AKT in non hormone dependent prostate cancer, but the expression of p-AKT was not significantly increased, but the expression of NF- kappa B protein was up and I kappa B alpha protein expression declined. NF- kappa B may be a key site in the signal pathway, but it is not activated by the PI3K/AKT pathway in the LNCaP-A cell line, and there may be other activation pathways. The inhibition of the down regulation of the expression of I kappa B alpha may affect the expression of NF- kappa B.
There was no significant change in the expression of ERK, MEK, and JNK protein in the MAPK signaling pathway related proteins, and the expression of p38, HSP27 and IL-8 significantly increased, suggesting that ERK pathway and JNK pathway were not involved in the transformation of LNCaP cells into LNCaP-A cells, but the MAPK/p38 signaling pathway was important. Information transfer molecules.
Studies have shown that MAPK/p38 plays an important role in the mechanism of breast cancer, ovarian cancer, and endometrial cancer, but it has not been reported in the drug resistance of the prostate cancer. This provides a new theoretical basis for the study of the AIPC mechanism and the discovery of the target of the AIPC drug.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25

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