【作者】 王平；

【导师】 陈昭典；

【作者基本信息】 浙江大学 ， 外科学， 2007， 博士

【摘要】 研究背景前列腺癌可从局灶性病变，向局部进展期和进展期发展。前列腺癌的治疗方案取决于疾病的分期，对早期和局部进展期患者可通过根治性前列腺切除或放射治疗方法治愈。对进展期和发生转移的前列腺癌最初可用手术去势或药物去势等雄激素剥夺治疗，或在以上基础上加用雄激素受体(Androgen receptor；AR)阻止剂达到最大雄激素剥夺。大多数患者对雄激素剥夺治疗起初反应良好，18～24月后，几乎所有患者不可避免地出现循环血前列腺特异抗原(Prostate specific antigen；PSA)升高、肿瘤重新生长为特征的激素抵抗前列腺癌(Hormone refractory prostate cancer；HRPC)。HRPC治疗是世界性难题，目前尚无理想治疗方法。通过对HRPC细胞生物学行为进行修饰的策略可能对HRPC治疗有潜在的临床意义。同源框基因是一类与发育相关的基因，该类基因都有一段共同的DNA序列，长约180bp，即同源框，该序列编码的蛋白质含60个氨基酸，这段氨基酸被称为同源域。具有同源域的蛋白通过对目标基因的表达调节来控制机体的发育程序，使机体在发育时按正确的时间、空间和组织类型发育。同时该类基因也能参与对细胞癌化过程的调节。但是该类基因并不足以直接造成癌化，而只是对癌化起促进或者抑制作用。由于同源框基因在发育和分化中具有的关键性调控作用以及在肿瘤时经常发生表达失常，目前认为，它们是研究肿瘤形成和胚胎发育间联系的理想靶点。在前列腺癌，也有同源框基因的表达异常。Nkx3.1是受雄激素调节的前列腺特异表达的同源框基因。人类Nkx3.1定位于染色体8p21，约85％前列腺癌患者该区域缺损。通过对前列腺癌样本的基因分析发现Nkx3.1蛋白编码区无肿瘤特异性突变。然而，在前列腺癌中Nkx3.1失表达与前列腺癌进展和激素抵抗疾病强烈相关(p＜0.0001)。在激素依懒前列腺癌LNCaP细胞中Nkx3.1表达且雄激素刺激下其表达水平增加，而在激素抵抗前列腺癌的PC3细胞株中Nkx3.1失表达。最近在复合的突变鼠模型中研究发现Nkx3.1失表达与脂质磷酸酶PTEN失表达一起协同激活Akt信号途径促进前列腺癌进展。p27主要与细胞周期素(cyclin)结合而发挥对cyclin-CDK的抑制作用。p27对周期素依赖激酶(Cyclin-dependent kinase；CDK)的抑制作用有两方面，一方面p27能抑制已结合到cyclin并被激活的CDK活性；另一方面p27也可以抑制CDK的激活过程，最终抑制细胞周期G1→S的转变，故目前认为其是一个肿瘤抑制因子。与Nkx3.1相似，在前列腺癌中大部分p27的表达丢失，而且p27的表达降低和丢失与差的临床预后相关。研究发现，外源性恢复前列腺癌细胞株的p27可引起细胞周期阻滞在G0／G1期，并引起细胞凋亡增加。在大鼠中p27缺损也可引起前列腺上皮增生，并可与PTEN缺损一起协同引起前列腺肿瘤的发展。尽管Nkx3.1与p27都与前列腺癌抑制有关，但是二者的肿瘤抑制均存在存在单体失能，亦即单个等位基因缺损不足以引起前列腺癌发生。Gary等在Nkx3.1与p27复合突变的等位基因大鼠模型中研究发现，Nkx3.1和p27协同抑制前列腺上皮细胞增殖和类似前列腺上皮内瘤的瘤前病变形成。这说明在前列腺癌的发动中，Nkx3.1与p27二者存在协同联系作用。随着前列腺癌的进展，向激素抵抗状态转化时Nkx3.1与p27的表达渐渐降低直至消失，如恢复激素抵抗前列腺癌PC3细胞株的p27表达可一定程度抑制其生长。如同时恢复二者在PC3细胞株中的表达获得协同的抗肿瘤效应吗?这方面研究目前国内外尚无报道，我们拟对此问题作进一步探讨。第一部分人类Nkx3.1和p27基因全长读码框(ORF)的克隆及真核表达载体的构建【研究目的】为了对Nkx3.1与p27的功能及其二者在激素抵抗前列腺细胞株中结合效应进行全面的研究，我们构建了Nkx3.1和p27真核表达载体。利用这几套载体我们可以恢复PC3细胞的Nkx3.1或p27的表达，或二者共同表达。【材料和方法】我们在人类正常前列腺组织中使用RT-PCR技术扩增了Nkx3.1与p27全长读码框(ORF)，并将二者克隆到T载体。并进一步以T载体为模板，把Nkx3.1克隆到真核细胞高效表达载体pcDNA3.1(+)(Invitrogen)，用同样的方法把p27克隆到真核细胞高效表达载体pcDNA3.1(-)(Invitrogen)。特别要说明的是在Nkx3.1 ORF和p27 ORF上分别加上HisTag和HAtag并构建在载体内，这样对表达产物可以用抗HisTag和抗HAtag单抗进行鉴定。此外，为了评估质粒的转染效率和基因核定位，我们还构建了报告基因表达载体。分别把Nkx3.1 ORF和p27 ORF构建到pEGFP-C1和pEGFP-N1(Clontech)。所有构建质粒得到测序(上海博亚生物公司)证实。【结果】我们成功地构建了Nkx3.1和p27真核系列表达载体，这二套载体可在真核细胞内高效表达、His或HA标签纯化等。我们还成功构建Nkx3.1和p27的报告基因质粒系统，可以鉴定质粒的转染效率，基因定位等。通过以上二套质粒系统，我们可对Nkx3.1或／和p27的功能进行较为全面的评价。【结论】1．本部分成功构建了Nkx3.1和p27真核表达载体和报告载体系列。利用这二套载体我们可对Nkx3.1、p27的功能进行全面的评估。2．本部分在PCR扩增技术上，发展了“富含GC-缓冲液”，能有效降低Tm值。与二甲基亚砜(DMSO)、甲酰胺等经典的PCR扩增添加试剂比较，该缓冲液具有更多的优势。第二部分质粒表达分析、报告基因分析和基因表达定位分析【研究目的】为了评估构建质粒在真核细胞中转染效率、表达水平，从而为进一步研究Nkx3.1和p27生物学功能打好基础。【材料和方法】我们用Lipofectamine2000瞬时转染PC3细胞，转染后24、48小时时间点用western blot法检测Nkx3.1，p27蛋白表达水平。在报告基因分析中，我们用荧光显微镜观察GFP融合蛋白表达状况，并用流式细胞术检测质粒转染效率。同时我们在用4％多聚甲醛固定细胞后，用DAPI染色对Nkx3.1与p27基因表达进行定位。【结果】Western blot检查发现转染后Nkx3.1和p27蛋白在PC3细胞中高表达，并且表达水平与HisTag和HAtag表达相一致。在荧光显微镜下Nkx3.1-GFP和p27-GFG融合蛋白大量表达，用流式细胞检测转染效率为50％-55％(24小时)和46％-50％(48小时)。用DAPI染色基因表达定位发现Nkx3.1与p27均定位于细胞核。【结论】1．在真核细胞中pcDNA3.1-Nkx3.1-His和pcDNA3.1-P27-HA高表达；2．Lipofectamine法转染效率在24h时间点约50％-55％，48h时间点约为46％-50％；3．基因表达定位分析示Nkx3.1和p27均为核蛋白。第三部分Nkx3.1、p27及二者联合对PC3细胞增殖影响的研究【研究目的】通过对不同转染组别细胞增殖指标的检测，分析Nkx3.1，p27抗增殖效应及联合应用Nkx3.1与p27是否存在抗PC3细胞增殖结合效应。【材料和方法】用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，以空质粒组为对照组。分别在0h，24h，48h时间点用MTT方法检测细胞增殖。在相同时间点用台盼蓝染色行活细胞计数和死亡细胞计数。【结果】MTT分析发现，24h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)；在48h时间点p27组与对照组存在差异(p＜0.05)，而Nkx3.1+p27组与对照组存在显著差异(p＜0.01)。台盼蓝排斥试验活细胞计数，24h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)，48h时间点Nkx3.1+p27组与对照组存在显著差异(p＜0.01)。台盼蓝死亡细胞计数，在24h和48h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)。【结论】1．Nkx3.1对PC3细胞增殖无明显抑制作用；2．p27对PC3细胞增殖存在抑制作用；3．Nkx3.1与p27存在协同的抗PC3细胞增殖作用。第四部分Nkx3.1、p27及二者联合对PC3细胞周期影响的研究【研究目的】通过对不同转染组细胞周期检测，分析Nkx3.1、p27及Nkx3.1+p27对PC3细胞周期影响。【材料和方法】用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，在24h、48h时间点分别收集细胞，用流式细胞仪(PI染色法)检测各组细胞周期分布。【结果】Nkx3.1，p27，Nkx3.1+p27转染组，G0／G1期细胞比例渐渐升高，但S期和G2／M期比例相对下降。在24h时间点p27和Nkx3.1+p27组与对照组相比，G0／G1期存在差异(p＜0.05)。在48h时间点，p27组与对照组相比G0／G1期存在差异(p＜0.05)，而Nkx3.1+p27组与对照组相比G0／G1期存在显著差异。【结论】1．Nkx3.1虽然可使PC3细胞的G0／G1期细胞比例增加，但与对照组比较无统计意义；2．p27可使PC3细胞周期阻滞在G0／G1期；3．Nkx3.1与p27使PC3细胞阻滞在G0／G1期具有协同性。第五部分Nkx3.1、p27及二者联合对PC3细胞凋亡及其凋亡相关蛋白影响的研究【研究目的】通过对转染各组细胞的不同时间点细胞凋亡率及凋亡相关蛋白检测，分析Nkx3.1、p27及Nkx3.1+p27对PC3细胞凋亡影响及对凋亡机制作初步探讨。【材料和方法】用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，在24h，48h时间点收集细胞。用AV+PI双染法流式细胞术检测细胞凋亡率。用western blot方法检测各组不同时间点的凋亡相关蛋白(bcl-2，bax，caspase-3，PARP)表达变化。【结果】与对照组相比，Nkx3.1与p27引起PC3细胞凋亡率均增加。当Nkx3.1与p27结合处理后，引起明显细胞凋亡协同效应。与我们的调亡率检测结果一致的是，在Nkx3.1+p27组与p27及Nkx3.1组相比，bcl-2表达明显增加，bax表达上升，caspase-3表达被激活，PARP劈裂片断表达增加。【结论】1．Nkx3.1与p27均可引起PC3细胞凋亡率增加；2．Nkx3.1与p27引起PC3凋亡存在协同效应；3．凋亡协同性产生主要是原因是凋亡拮抗物bcl-2表达下降、凋亡促进物bax表达增加，最终引起caspase-3激活。产生协同凋亡效应主要通过线粒体途径来实现。更多还原

【Abstract】 Prostate cancer now represents a serious health problem worldwide. Despite intense investigations, it has remained a daunting task to elucidate the mechanisms underlying disease progression. Androgen ablation, however, remains the only effective therapy for patients with advanced disease. Approximately 80% of patients achieve symptomatic and /or objective response after androgen withdrawal, but progression to androgen independence ultimately occurs in almost all cases. Because androgen-independent prostate cancer cells eventually lead to death, successful strategies to modify the biological behavior of these cells may potentially have the most significant clinical impact.Nkx3.1 is an androgen-regulated homeobox gene that is largely specific to prostate for expression[1,2]. The human Nkx3.1 has been mapped to chromosome 8p21, and has been located in a region of the chromosome deleted in approximately 85%of prostate cancer[3,4]. However, no tumor-specific mutations of the Nkx3.1 protein-coding region have been identified by genetic analysis of human prostate cancer samples[5]. Nevertheless loss of Nkx3.1 expression is strongly associated with hormone-refractory disease and advanced tumor stage in prostate cancer （p<0.0001） [6]. In the LNCaP androgen-dependent prostate cancer cell line, Nkx3.1 is expressed at a basal level that was increased upon androgen stimulation. In contrast, there was no Nkx3.1 expression in androgen-independent PC3 cells[7]. More recently it was shown that in compound mutant mice the loss of Nkx3.1 along with the loss of the lipid phosphatase Pten cooperates in prostate cancer progression through synergistic activation of the Akt pathway[8].Similar to Nkx3.1, expression of the cyclin-dependent kinase inhibitor p27^KIP1 is lost in a large fraction of human prostate cancer, and reduced or absent expression of p27^KIP1 correlates with poor clinical outcome[9]. Exogenous p27^KIP1 overexpression results in cell cycle regulation and an increase in cell apoptosis in the human prostate carcinoma cell lines[10]. Deletion of p27^KIP1 in mice also results in prostatic epithelial hyperplasia and cooperates with loss of Pten to promote prostate tumor development[11]. Gary et al found that Nkx3.1 and p27^KIP1 cooperate to suppress the proliferation of prostatic epithelial cells and the formation of preneoplastic lesions resembling prostatic intraepithelial neoplasia[12], whereas there was no literature report about combined effect of Nkx3.1 and p27^KIP1 in androgen independent prostate cancer.Consequently, we hypothesized that the anticancer effect by combination therapy of Nkx3.1 and p27^KIP1 would be superior to single gene in PC3 prostate cancer cells. The objectives of this study were to test whether induction of apoptotic cell death by p27^KIP1 or Nkx3.1 gene transfer is enhanced by Nkx3.1 or p27^KIP1, and to determine whether combined use of Nkx3.1 and p27^KIP1 exist synergistic antitumor effect in the androgen-independent human prostate PC3 cells.We use two kinds of prostate cell line LNCaP and PC3 to demonstrate our hypothesis. LNCaP is an androgen-depedent prostate cacer cell line and expresses NKX3.1 and p27, and PC3 is an androgen-independent prostate cancer cell line without expressing NKX3.1 and p27. These cell were maintained at 37℃ in a humidified atmosphere of 5% CO₂/95% air and serially passaged in RPMI-1640 medium （Hyclone）, supplemented with 10% fetal bovine serum.RT-PCR method was used to obtain the amplified Nkx3.1-HisTag and p27-HAtag DNA, Nkx3.1-HisTag fragment was directionally cloned into the EcoR I and BamH I restriction sites of plasmid pcDNA3.1（+） （Invitrogen） and p27-HAtag was cloned into the same restriction sites of pcDNA3.1（-） （Invitrogen）.In addition, Nkx3.1 was cloned into pEGFP-C1（Clontech） at Bgl II and EcoR I sites, p27 was cloned into pEGFP-N1 （Clontech） at BamH I and EcoR I sites. The successful cloning was confirmed by sequencing the plasmid.Plasmid DNAs were delivered into cells with LipofectamineTM 2000 （Invitrogen） following the manufacturer’s protocol. Reporter gene expression was monitored by fluorescence microscopy and quantification of GFP fusion protein by flow cytometryCell viability was evaluated by MTT assay. The viable cell number was monitored with absorbance at 570nm. At the same time, We used a trypan blue dye exclusion assay to determinecell viability after the treatments. After the cells were collected by trypsinization, they were stained with trypan blue, and the total number cells and the number of viable cells in each well were counted.We used flow cytometric to analysis the cell cycle and apoptosis. The cells transfected with various plasmid DNA for 24 h or 48 h were assayed for cell cycle progression by the propidium iodide （PI） staining method. However, Annexin V （Annexin V-FITC） and PI double staining was used to determine apoptosis.To prodive further confirmation that the synergistic effect of combined use Nkx3.1 and p27 induces apoptosis in PC3 cells, we also analyzed apoptotic-related proteins using western blot method.In the present study, significant anti-proliferative effect of p27^KIP1 was shown on PC3 human prostate cancer cells in vitro. The combination of Nkx3.1 and p27^KIP1 exhibited synergistic inhibitory effects on growth of PC3 cells: increase in cellular apoptosis and enhancement in G0/G1 phase arrest of cell cycle. These result suggest that the combining use of Nkx3.1 and p27^KIP1 possesses novel anti-tumor action on PC3 human prostate cancer cells.Proliferation status was assessed by MTT assay. Compared with control-plasmid group, cells in Nkx3.1plus p27 group experienced more greater growth inhibition than Nkx3.1 group and p27 group. Transfected PC3 cells fail to produce a measurable difference between control and Nkx3.1 group at any of the time point. However, transfection of PC3 cells with p27 did produce a significantly lower absorbance at the 48 h time point when compare to the control （p < 0.05）. When Nkx3.1 and p27 were combined, the PC3 cell line exhibited a significant decrease in cell growth at 24 （p < 0.05） and 48 （p < 0.01） hours, respectively, compared with the control. These data were also supported by the results of the trypan blue assay for studying cell viability.There were substantial increase in the fraction of cells in the G0/G1 phases and a corresponding decrease in the number of cells in S and G2/M phases after transfected with Nkx3.1 alone, p27 alone, or their combination. P27 group did produce a higher percentage of cells in G0/G1 at 24 （p < 0.05） and 48 （p < 0.05 ） hours when compare to control. We found that the combination of both Nkx3.1 and p27 synergistically induced increase in the G0/G1 phase at 48 h （p < 0.01） but not at 24 h time point （p < 0.05）.The results from an annexin-V FITC binding assay showed that the higher proportion ofannexinV-positive cells was observed in co-transfected group at the 24 and 48 hours time point, with values of 21.87% and 26.76%, respectively. Moreover, the combinatory effects of Nkx3.1 and p27 on cellular apoptosis correspond to the decrease in pro-caspase-3 protein expression （caspase-3 activation is presented by the loss of its pro-form）, concurrent with decrease Bcl-2 protein and increased Bax protein expression in transfected PC3 cells. Consistent with the caspase-3 activation results.the combination of Nkx3.1 and P27 markedly increased the level of cleaved PARP.In summary, this report describes a potentially useful approach, the combination of Nkx3.1 plus p27^KIP1 exhibited significant anti-proliferative effects on PC3 cells. Especially, the combination of Nkx3.1 and p27^KIP1 demonstrated synergistic multiple anticancer effects of activation of caspase-3 and PARP, down-regulating Bcl-2 protein and up-regulation Bax protein expression, triggering cellular apoptosis and inducing G0/G1 phase arrest. Our in vitro study suggests the potential combined use Nkx3.1 and p27^KIP1 in gene therapy of androgen-independent prostate cancer. References1. Bieberich CJ, Fujita K, He WW, Jay G. Prostate-specific and androgen-dependent expression of a novel homeobox gene. J Biol Chem 1996; 271: 31779-317822. Prescott JL, Blok L, Tindall DJ. Isolation and androgen regulation of the human homeobox cDNA,Nkx3.1. Prostate 1998; 35: 71-803. Vocke CD, Pozzatti RO, Linehan WM, et al. Analysis of 99 microdissected prostate carcinoma reveals a high frequency of allelic loss on chromosome 8p21-22. Cancer res 1996; 56: 2411-24164. Swalwell JI, Vocke CD, Yang Y,et al. Determination of a minimal deletion interval on chromosome band 8p21 in sporatic prostate cancer. Genes Chromosomes Cancer 2002; 33: 201-2055. Voeller HJ, Augustus M, Madlike V, et al. Coding region of Nkx3.1,prostate-specific homeo -box gene on 8P21,is not mutuated in human prostate cancers. Cancer Res 1997; 57: 4455-44596. Bowen C, Bubendorf L, Voeller HJ, et al. Loss of Nkx3.1 expression in human prostate cancers correlates with tumor progression. Cancer Res 2000; 60: 6111-61157. Korkmaz CG, Korkmaz KS, Manola J, et al. Analysis of androgen regulated homeobox gene Nkx3.1 during prostate carcinogenesis. J urol 2004; 172: 1134-11398. Kim MJ, Cardiff RD, Desai N, et al. Cooperativity of Nkx3.1 and Pten loss of function in a mouse modle of prostate carcinogenesis. Proc Natl Acad Sci USA 2002; 99:2884-28899. Eder IE, Bektic J, Haag P, Bartsch G, Klocker H. Genes differentially expressed in prostate cancer. BJU Int 2004;93:1151-115510. Katner AL, Hoang QB, Rayford W, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1. Prostate 2002; 53: 77-8711. Di Cristofano A, De Acetis M, Koff A, et al. Pten and p27KIPl cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-22412. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27KIP1 in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614更多还原