【作者】 王建松；

【导师】 齐范；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 背景：前列腺癌是男性泌尿生殖系统最常见的恶性肿瘤之一。早期局限性前列腺癌可行根治性手术或放疗,治愈率高。然而对于转移性前列腺癌,内分泌治疗虽然初期有一定作用,可是,几乎所有病变会最终发展为激素非依赖性前列腺癌,尚无有效的治疗方法。近期,以多西紫杉醇为基础的化疗方案经临床试验证明可以提高激素非依赖性前列腺癌患者的生存率,改善生活质量。不过,副作用较大,且药物作用有限,需要寻找新型的治疗激素非依赖性前列腺癌的策略和药物。Hedgehog (Hh)信号通路在哺乳动物中广泛存在,参与胚胎的生长发育和器官形成。该通路同样在前列腺的发育中起作用,而且,有证据显示,前列腺癌可表达该通路的靶基因PTCH1和GLI1；在体外细胞实验及体内动物实验中发现,该通路抑制剂环耙明能抑制前列腺癌肿瘤生长、改变侵袭能力,表明该通路不适当的激活与前列腺癌的发生、进展、转移密切相关。然而,Hh通路在前列腺癌中的作用方式不能明确,存在自分泌和旁分泌之争。此外,有关研究也提示了环耙明作用特异性的可疑,表明环耙明对前列腺癌细胞的抑制可能是不依赖Hh通路的,具有脱靶效应。和化学合成的小分子药物相比,抗体对相应抗原具有高特异性和高亲和力的靶向结合能力。SMO蛋白是Hh信号通路中关键的受体蛋白。本实验拟制备出抗SMO抗体,将其应用于激素非依赖性前列腺癌细胞DU145,观察其变化,一方面为自分泌或旁分泌方式提供证据,另一方面,判断该抗体能否应用于激素非依赖性前列腺癌的治疗。方法和结果：首先,对SMO蛋白进行抗原表位预测,然后合成多肽,与钥孔血蓝蛋白偶联后对新西兰兔进行免疫。第四次免疫后取血清,ELISA检测抗血清效价为1：240,000。抗血清经抗原亲和纯化后进行Western blot免疫印迹检测,结果显示,抗血清可特异性结合DU145细胞中SMO蛋白,表明成功制备了抗SMO多克隆抗体。其次,用MTT实验判断抗SMO抗体对DU145细胞增殖的影响,用流式细胞仪观察其诱导DU145凋亡情况,用小室试验检测抗体对DU145细胞侵袭力的改变状况,并行RT-PCR,依据PTCH1和GLI1mRNA表达水平判断Hh信号通路在DU145细胞中的状态,同时设置空白对照和正常兔IgG对照。结果表明,在DU145细胞存在自分泌的Hh信号通路,抗SMO抗体可特异性地阻断Hh信号通路,抑制DU145细胞的增殖,并诱导其凋亡,降低侵袭力。最后,联合应用抗SMO抗体和多西紫杉醇于DU145细胞,并使用同样的实验技术路线。结果表明,二药联用具有协同作用,较单药应用效应更强,包括对DU145细胞的抑制增殖能力,诱导凋亡以及降低侵袭力等方面。结论：在激素非依赖性前列腺癌存在自分泌方式的Hh信号通路。抗SMO抗体可单独应用或与多西紫杉醇联合应用治疗激素非依赖性前列腺癌。更多还原

【Abstract】 Background:Prostate cancer is currently the most frequent malignancy in men and represents the second leading cause of cancer-related deaths. While early-stage prostate cancer can be cured with surgery or radiation therapy, this is not the case for metastatic disease. The mainstay of treatment for metastatic prostate cancer is androgen deprivation.Unfortunately, most of men ultimately develop androgen-independent prostate cancers (AIPC), unresponsive to hormonal manipulations. Recently, docetaxel has replaced mitoxantrone as the standard of care for patients with nonlocalized AIPC.The docetaxel-based chemtherapies has been reported to improve the quality of life for patients, offering pain relief. However,the side-effects are significant.In addition, it is clear that, while docetaxel is effective, this efficacy is modest and better therapies are definitely needed.Therefore, novel therapeutic strategies that target the molecular basis of androgen resistance are required.Hedgehog(Hh) is a developmental signaling pathway that regulates embryonic cell growth, body pattern formation and organogenesis in certain vertebrate tissues. Hh signaling is involved in normal prostate development and there is mounting evidence that dysregulated Hh signaling plays some role in prostate cancer. This evidence includes reports that human prostate tumors express Hh ligand and Hh-target genes and other reports citing experimental evidence that a chemical inhibitor of Hh signaling, cyclopamine, suppresses the growth of cultured and xenografted human prostate cancer cell lines. Collectively, this evidence implies that autocrine Hh signaling has some role in prostate cancer tumor growth or progression. The validity of this hypothesis is contradicted, however, by more recent experimental studies that failed to find a canonical signaling effect of Hh-activators or inhibitors in the most commonly utilized prostate cancer cell lines. Moreover, there are increasing concerns that cyclopamine might have off-target effects in cancer cells that belie its specificity as an Hh signaling inhibitor.Antibody is a favorable molecule for drug development due to its high binding specificity and affinity to target. Smoothened (SMO) is an important member of the Hedgehog signaling pathway. Antibodies which recognize an oligopeptide of SMO on the cancer cell surface and have the ability to silence Shh stimuli were raised in the present study.The aim was to find a new strategy for controling prostate cancer and other Hh dependent carcinomas by precisely controling the Hh activity to avoid the side effects.Methods and Results:To prepare and characterize the polyclonal antibody against SMO,a polypeptide was synthesized based on the bioinformatics analysis of SMO protein and coupled with keyhole limpet hemocyanin(KLH) for immunization. The antiserum containing specific polyclonal antibodies were prepared by immunizing healthy male rabbits,about 4-month-old and 3 kilogram weight. Antibodies were collected after the fourth immunization injection. The titre of the antiserum was detected with enzyme-linked immunosorbent assay (ELISA). The antiserum was purified with immuno-affinity chromatography. Western blot was performed with the purified antiserum on the cell lysates of DU145 cells.The antiserum had titres of 1:240,000 in ELISA. The results obtained by Western blot analysis showed that the antiserum could react with SMO with highly specific and sensitive affinities. So,the specific antibody against human SMO (anti-SMO-antibody) was obtained,which will be useful for further investigation.The effect of anti-SMO-antibody on Hh signaling activity in AIPC cell line DU145 was analyzed by RT-PCR.Because GLI1 and PTCH1 are not only the components of Hh signaling but also target genes of GLI1 trans-activation, the mRNA levels of GLI1 and PTCH1 as markers of the activity of Hh signaling pathway were examined. Anti-SMO-antibody suppressed the expression levels of both GLI1 and PTCH1 when compared with a treatment with normal rabbit IgG. To evaluate the antiproliferative effect of drugs on AIPC cells, the growth-inhibitory effects of anti-SMO-antibody was evaluated on DU145 prostate cancer cells. The flowcytometric analyses were made to determinate the percentage of apoptotic cell death induced by anti-SMO-antibody. On the other hand,to estimate the inhibitory effect of the anti-SMO-antibody on the invasiveness of prostate cancer cells, the in vitro invasive potential of untreated or drug-treated DU145 cells was evaluated by their ability to penetrate a Matrigel invasion chamber.The results showed that anti-SMO-antibody inhibited the proliferation of DU145 cells, down-regulated cell invasiveness and induced apoptosis.To establish whether the anti-proliferative effect of docetaxel on AIPC cell proliferation may be improved by combined use with anti-SMO-antibody, the growth-inhibitory effects either of single agent or in combination, were evaluated on the DU145 prostate cancer cells.To estimate the beneficial effect of combining anti-SMO-antibody for improving the docetaxel-based therapeutic regimens, the percentage of apoptotic cell death induced by docetaxel, either alone or in drug combination, were estimated by the flow cytometric analyses. Importantly, to estimate the inhibitory effect of the drugs, alone or in combination, on the invasiveness of the AIPC cells, the invasive potential of untreated or drug-treated DU145 cells was evaluated by their ability to penetrate a Matrigel-invasion chamber. Synergistic effects were observed when anti-SMO-antibody and docetaxel were used together.The results revealed that the drugs, alone or in combination,at lower concentrations inhibited the growth of androgen-independent DU145 cells. Moreover, the combined docetaxel and anti-SMO-antibody also caused a higher rate of apoptotic death of prostate cancer cells compared with individual agents. Additionally,the combined agents were more effective at suppressing the invasiveness of DU145 cells through Matrigel in vitro than the single drugs. It appeared that combined treatment with docetaxel caused additive and/or synergistic cytostatic effects on prostate cancer cells.Conclusion:Androgen-independent prostate cancer cell proliferation was associated with activity of the Hedgehog signalling pathway. Autonomous Hedgehog signalling was detectable in DU145 prostate cancer cells.The other findings also indicate that the anti-SMO-antibody alone or in combination with docetaxel could represent two promising strategies for improving the treatment in patients with metastatic and androgen-independent prostate cancers.更多还原