多西他赛对前列腺癌细胞株C-jun与雄激素受体相互作用的影响
摘要
研究背景和目的
前列腺癌(Prostate carcinoma, PCa)是西方国家男性最常见的恶性肿瘤,美国男性因肿瘤导致的死亡率中,前列腺癌排第2位。流行病学统计,我国的前列腺癌发病率也在逐年上升,以上海市为例,1973-2000年,上海市男性前列腺癌的发病率增加了4.8倍,跃居男性泌尿生殖系肿瘤的第1位。目前在我国前列腺癌已位居泌尿系肿瘤的第二位,仅次于膀胱癌。尽管近年来前列腺癌的诊断和治疗取得了比较人的进展,如PSA筛查、前列腺活检穿刺、根治性前列腺切除、内分泌剥夺、放疗、化疗等综合诊疗手段有效的降低了前列腺癌的死亡率
但由于前列腺癌发病机制不清、病情隐匿、临床症状不典型,大约有1/3的病人在诊断时已经存在局部浸润或远处转移,失去了手术治疗的机会。目前50-70%晚期前列腺癌早期采用的标准治疗方法是雄激素消除治疗(Androgen ablation therapy)。
不幸的是,雄激素阻断仅能暂时缓解疾病,中位缓解时间12-20个月,最终所有病人都将进展为雄激素非依赖性前列腺癌(androgen-independe nt prostate cancer, AIPC),雄激素阻断疗法因此失效。如何对雄激素非依赖性前列腺癌进行进一步治疗,足临床上一个非常棘手的问题。
雄激素依赖型前列腺癌在进行雄激素阻断的内分泌治疗后,逐渐将转变成雄激素非依赖型前列腺癌,从而导致内分泌治疗失败。虽然目前的研究还远远不能阐明其中的机理,但许多学者们都认为雄激素受体始终在转变中发挥着重要的作用。
紫杉醇类药物被临床证实对前列腺癌有一定的疗效,研究表明,紫杉醇化疗可以提高雄激素非依赖性前列腺癌的生存率,降低患者血浆前列腺特异抗原(PSA, prostate special antibody)水平。多西他赛是紫杉醇类的代表药物,以多西他赛为中心的化疗方案,是晚期前列腺癌的一线化疗方案的金标准。
多西他赛的抗癌作用机理尚未被完全阐明。一般认为,多西他赛抗肿瘤的机理在于其优先结合细胞内的β-微管蛋白,阻碍了GTP及一些辅助蛋白因子与β-微管蛋白的结合为位点。β-微管蛋白在缺少GTP和辅助因子时发生静态聚合,破坏了细胞正常的有丝分裂,使细胞分裂停滞在G2/M期,最终导致细胞的凋亡。
但近年来的一些研究纷纷发现,多西他赛还在下调转录、细胞增殖、有丝分裂和肿瘤发生中起作用的某些基因,起到抗肿瘤作用。另有研究发现,多西他赛可以通过抑制雄激素受体来发挥对前列腺癌的治疗作用。这些研究提示我们,对微管系统的作用及对G2/M期的阻滞并不是多西他赛诱导细胞凋亡的唯一机制,可能有许多信号传导通路和机制参与紫杉醇诱导肿瘤细胞的凋亡。其中一个重要的信号传导通路就是JNK信号转导通路,研究发现,C-jun-氨基末端激酶(C-jun-N-terminal-kinase, JNK)被激活之后,可以通过激活内源性通路,激活BAX(Bcl-2家族的前凋亡蛋白)和Caspase-3(细胞凋亡过程中最主要的终末剪切酶),并磷酸化Bcl-2(抗凋亡蛋白)使其失活,使得促凋亡分子释放,从而导致细胞凋亡。
原癌蛋白C-jun己被大量实验证实与许多肿瘤进展有关,其作为JNK的下游分子,可以被JNK磷酸化,其磷酸化位点位于C-jun氨基末端活性区Set63和Ser73,磷酸化C-jun可以增强其转录活性。C-jun是被认为是转录因子AP-1的组成部分,被认为是AP-1中起主要功能的重要组分。AP-1作为雄激素受体的转录因子,其活性又与雄激素受体基因调控能力紧密相关。在前列腺癌的发生发展中,雄激素受体发挥着至关重要的作用。
本实验通过采用多西他赛处理体外培养的前列腺癌细胞株,通过western blotting、DNA转染等方式,试图了解C-jun与AR在多西他赛作用下的相互作用,为探索多西他赛抗前列腺癌的机理和寻找前列腺癌多西他赛抵抗现象的原因提供线索。
在之前的研究中,我们通过雄激素依赖型前列腺癌细胞株LNCaP和雄激素非依赖型前列腺癌细胞株PC-3在紫杉醇药物多西他赛处理下雄激素受体和C-jun蛋白表达等的对比,发现这雄激素依赖型和雄激素非依赖型前列腺癌细胞在多西他赛处理下,都表现出C-jun磷酸化增强的现象。转染了雄激素受体后的PC-3细胞获得了一定的抵抗多西他赛的能力。但由于两种细胞来源不同(LNCaP细胞来源于人前列腺癌淋巴转移灶,PC-3细胞来源于人前列腺癌骨转移灶),所表达的前列腺癌标志物也不尽相同,因此用两者分别模拟雄激素依赖型和雄激素非依赖型前列腺癌细胞,并在多西他赛处理下进行比较时,可比性欠佳。
近年来国内外均有学者采用激素递减法或激素阻断法培养LNCaP细胞,从而获得稳定传代的非雄激素依赖的LNCaP细胞亚系,尽管LNCaP细胞表达的AR基因并非野生型,但LNCaP与其非雄激素依赖的细胞亚系仍是较理想的前列腺癌细胞研究模型。
我们采用多西紫杉醇处理雄激素依赖型LNCaP前列腺癌细胞及其亚型比卡鲁胺抵抗型LNCaP-bic细胞(非雄激素依赖),采用荧光素酶分析检测AR及AP-1基因的表达,利用Western blotting,免疫沉淀方法分析C-jun以及AR的蛋白表达和相互作用。试图进一步分析AR和C-jun之间的相互作用对前列腺癌多西他赛化疗结果的影响。
二、研究目的
本实验拟采用多西他赛处理雄激素非依赖型细胞株PC-3、雄激素依赖型细胞株LNCaP及其雄激素非依赖亚型细胞株LNCaP-bic,探索C-jun与AR在多西他赛处理的前列腺癌细胞中的相互关系。
三、实验方法
1、将PC-3细胞及LNCaP细胞,分别分为实验组(加入2.5nm多西他赛)和对照组(相应浓度的DMSO),0.25%胰蛋白酶常规消化后以1.5×105/瓶接种于25cm2细胞培养瓶,在37℃、5%CO2、饱和湿度条件下培养24小时后,吸除原培养液,按分组加入相应药物处理,传代培养30天。用倒置显微镜观察并照相记录用药组和对照组的细胞形态变化。Western blotting方法分析两组细胞中p-C-jun的表达。
2、将PC-3细胞、LNCaP细胞消化收集,按5×103个细胞/孔接种于96孔培养板,在37℃,5%C02、饱和湿度条件下培养。待细胞贴壁后,每孔进行换液。吸除原培养液后,分别加入含多西他赛lOnmol(对照组)、lOnmol的RPMI培养液180u1,另设只加培养液不加细胞和药物的为空白组。每组四个复孔。将96孔板移入C02孵箱静置培养。MTT法测量细胞存活率。
4、用脂质法将C-jun以及C-jun与AR共转染PC-3细胞株,western blotting方法检测转染后C-jun和AR的蛋白表达。设立空质粒转染为对照组,用多西他赛10nm处理后,MTT法测量细胞存活率
5、Western blotting分析多西他赛处理的PC-3和LNCaP细胞在各个时刻p-C-jun和p-JNK的表达。
6、培养传代LNCaP细胞非雄激素依赖亚型LNCaP-bic细胞,用含萤火虫和海’肾荧光酶素的ARE-luc报告基因质粒、AP-1-luc报告基因质粒转染细胞。转染24h后,细胞分为(1)对照组；(2)DOC组,多西他赛10nm处理48小时；(3)DHT组,双氢睾酮100nm处理48小时。裂解细胞,收集细胞提取物用双荧光素酶报告基因检测试剂盒检测(Promega公司),海肾荧光酶素活性作为内对照。分别以ARE和AP-1作为AR和C-jun的指示,荧光素酶方法分析LNCaP细胞和LNCaP-bic细胞在多西他赛处理下AR和C-jun基因的表达。
7、Western blotting方法检测多西他赛处理的LNCaP细胞和LNCaP-bic细胞PSA蛋白的表达情况,免疫沉淀法检测二者在多西他赛处耻下C-jun以及AR的蛋白表达和相关关系。
四、实验结果
1、PC-3细胞和LNCaP细胞10nm多西他赛处理后,C-jun蛋白表达与未处理组无明显差别,但磷酸化C-jun (p-C-jun)蛋白表达明显增强。
2、在同样浓度的多西他赛处理下,PC-3细胞的存活率与LNCaP细胞相比有显著性差异(P<0.05),转染C-jun基因后的PC-3细胞提高了对多西他赛的耐受性,而共转染C-jun/AR基因的PC-3细胞恢复了部分对多西他赛的敏感性。
3、western blotting分析提示在多西他赛处理的前列腺癌细胞中,p-JNK蛋白表达并没有显著性变化,体现出和p-C-jun蛋白表达增强不一致的特点。
4、荧光素酶分析提示在多西他赛处理下,LNCaP细胞和LNCaP-bic细胞的AR和C-jun在基因表达水平上均增强。
5、LNCaP-bic细胞在多西他赛处理下PSA表达强于其父代LNCaP细胞。免疫沉淀提示多西他赛处理后的LNCaP-bic细胞比起LNCaP细胞,C-jun与AR有着更强的相关性。
五、结论
在体外实验中,多西他赛可以采用JNK通路以外的方式磷酸化前列腺癌细胞中的C-jun, AR和C-jun可以提高前列腺癌细胞对多西他赛的耐受程度,多西他赛可以激活AR非配体依赖的转录功能,AR和C-jun之间的相互作用可能影响多西他赛对前列腺癌的化疗结果。
Background and Objection:
Prostate cancer (Prostate carcinoma, PCa) is the one of the most common malignant tumor in the Western countries, Prostate cancer is second as the leading cause of cancer death, respectively, in US men. Epidemiological data show the incidence of prostate cancer in China is also increasing year by year, for example, from1973to2000, the incidence rate of prostate cancer in Shanghai men increased by4.8folds, making it to be the first leading cause of male genitourinary tumors. At present, prostate cancer incidence rate is second only to bladder cancer in Chinese men's genitourinary system. The diagnosis and treatment of prostate cancer in recent years has made progress, some techniques, such as PSA screening, prostate biopsy and radical prostatectomy, androgen deprivation, radiotherapy, chemotherapy and other treatment, effectively reduce prostate cancer mortality. However, the pathogenesis of prostate cancer is still unclear, many prostate cancer grow concealed and asymptomatic, almost1/3patients lost opportunity to receive surgical treatment, because of the existence of local invasion or distant metastasis when they are diagnosed prostate cancer. The standard treatment for advanced prostate cancer is androgen elimination of treatment (Androgen ablation therapy).
Unfortunately, the effectiveness of androgen deprivation therapy is temporary, Androgen ablation therapy will be failure when patients develop to androgen-independent prostate cancer (AIPC), mean time is12-20months. Improving and optimizing therapeutic strategies for men with AIPC is one of the most challenging aspects of prostate cancer management today.
Androgen-dependent prostate cancer gradually progress to androgen-independent prostate cancer leading to the resistant of androgen ablation therapy. Although the current study is far from being able to clarify the mechanisms, many researchers believe that the androgen receptor plays an important role during the whole procedure.
Taxane mediated chemotherapy has been proven to have certain effect on prostate cancer, studies have shown that docetaxel can improve the survival rate of androgen-independent prostate cancer patients, and reduce the serum level of prostate-specific antigen (PSA), docetaxel chemotherapy is the gold standard of first-line chemotherapy in advanced prostate cancer.
The anticancer mechanism of docetaxel has not yet been fully elucidated. One of the general opinions believed that docetaxel combined with intracellular β-tubulin, prevented the combination of GTP and other protein factors with β-tubulin. The absence of GTP-and microtubule-associated proteins will stabilize microtubules, which are usually essential for cells mitosis, make cells division be arrested in the G2/M phase and lead to apoptosis.
Recent studies have also found that docetaxel antitumor effect as a result of down-regulating certain genes which related to transcription, cell proliferation, mitosis and tumorigenesis. Another study showed that docetaxel can play a role in the treatment of prostate cancer by inhibiting the androgen receptor. These studies suggest that the impact on microtubule system and the G2/M phase arrest is not the only mechanism of docetaxel induced apoptosis, there may be many signaling pathways and mechanisms involved in docetaxel antitumor effect. One of the important signaling pathways is JNK signal pathway.Activated C-jun N-terminal kinase (C-jun-N-the terminal-kinase, JNK) inducing activation of BAX (Bcl-2family) and Caspase-3, phosphorylation and inactivation Bcl-2, making The pro-apoptotic factor released, resulting in apoptosis.
C-jun is one of oncoprotein has been confirmed that related to the number of tumor progression by many experiments. C-jun is a transcription factor downstream of the JNK signaling pathway, it can enhance its transcriptional activity through the JNK-induced phosphorylation, the phosphorylation sites located in Ser63and Ser73. C-jun is considered to be an essential part of the transcription factor AP-1plays a major function. The activity of transcription factor AP-1is closely related to with the androgen receptor, which plays a crucial role in the development of prostate cancer.
In this study, we use docetaxel to treat prostate cancer cell lines in vitro, trying to understand the interaction of C-jun and AR by western blotting and DNA transfection, explore the mechanism of docetaxel against prostate cancer and look for clues of the docetaxel-resistant phenomenon. We also used docetaxel to treat androgen-dependent LNCaP prostate cancer cells and its subtypes LNCaP-bic cell (non-androgen dependent), using luciferase assays detect AR and AP-1gene expression, analyze C-jun, and AR protein expression by Western blotting, immunoprecipitation analysis. Attempt to further investigate the interaction between AR and C-jun in prostate cancer cell lines treated with docetaxel.
Objective Treat androgen-independent cell lines PC-3, androgen-dependent cell line LNCaP and its androgen-independent subtype cell lines LNCaP-bic with docetaxel, investigated the complex interaction of c-jun with AR in these cell lines after docetaxel treatment.
Material s and Methods
1, PC-3cells and LNCaP cells were divided into experimental group (treat with2.5nm docetaxel) and control group (treat with the corresponding concentration of DMSO), after0.25%trypsin digestion, inoculating cells to25cm2cell culture flasks(1.5×105/bottle, at37℃,5%CO2and saturated humidity) for24hours, then abandoned of the original medium, joined the appropriate drug to treat cells according their group. Cells were subcultured for30days. Observed and photographed the changes of cell morphology in treatment group and control group by inverted microscope, western blotting analysis p-C-jun expression of two groups of cells.
2, After digestion, PC-3cells, LNCaP cells were collected and seeded in a96-well plates,5×103cells/well, incubated in37℃,5%CO2and saturated humidity conditions. After the adherent cells growth, medium was changed. Abandon the original culture medium, add10nmol RPMI medium180μl, which contained10mol docetaxel. Use only medium without cells and drug as contrast. Each group has four wells. Incubating cells in a C02incubator, then measuring viability of cells by MTT assay.
3, Transfect PC-3cell with C-jun and C-jun/AR gene (liposome transfection), after detect C-jun and AR protein expression in PC-3cell after transfection by western blotting. Use empty plasmid transfected cells as the control group, MTT assay measuring cell viability in each group after1Onm docetaxel treatment.
4, Western blotting analysis the expression of p-C-jun and p-JNK in PC-3and LNCaP cells deal with docetaxel in different moments.
5, Continuously passaging LNCaP cells and its androgen-dependent subtypes, LNCaP-bic cell, were transfected with Luciferase reporter constructs ARE-luc and AP-1-luc using Lipofectamine2000reagent (Invitrogen), which containing the firefly and Renilla enzyme.The cells were divided into (1) control group;(2) DOC group (docetaxel10nm treated for48hours);(3) DHT group(DHT100nm treated for48hours).
After24h the cells were collected and analyzed with Luciferase assay according to manufacturer's instructions (Promega). Use Renilla enzyme activity as internal control.The activity of AP-1reporter was normalized to the protein concentration. Use ARE as indication for AR and AP-1for C-jun, luciferase analyzied AR and C-jun gene expression in LNCaP cells and LNCaP-bic cells after treatment of docetaxel.
6、To evaluate AR and AP-1gene activity using luciferase assay. To analyze the effects of docetaxel treatment on the expression/interaction of C-jun, and AR by Western blotting and immunoprecipitation assay.
Results
1, C-jun protein expression was no significant difference with the untreated group, but the phosphorylation of C-jun (pC and-jun) protein expression was significantly enhanced in PC-3cells and LNCaP cells after lOnm docetaxel treatment,
2, The survival rate of PC-3cells and LNCaP cells has a significant difference (P<0.05) after the treatment of same concentration docetaxel, PC-3cells improved docetaxel tolerability by transfected with the C-jun gene, and partially restored the sensitivity to docetaxel after co-transfected C-jun/AR gene.
3, western blotting analysis indicated that in docetaxel treated prostate cancer cells, p-JNK protein expression had no significant change and was not in correspondence with the expression of p-C-jun.
4, Luciferase assay showed LNCaP and LNCaP-bic cells expressed an increased activity of AR and C-jun in response to docetaxel.
5, Western blotting showed docetaxel induced strong PSA protein expression in LNCaP-bic. Immunoprecipitation assay demonstrated that docetaxel induced strong inreactions of AR with C-jun in LNCaP-bic.
Conclusions
Docetaxel phosphorylates C-jun by unknown patheway bypassing JNK. AR and C-jun may affects the prostate cancer cell resistance to docetaxel in vitro.
Docetaxel activates ligand independent AR transcriptional activity. Balance between AR and C-jun may affects outcome of docetaxel chemotherapy.
前列腺癌(Prostate carcinoma, PCa)是西方国家男性最常见的恶性肿瘤,美国男性因肿瘤导致的死亡率中,前列腺癌排第2位。流行病学统计,我国的前列腺癌发病率也在逐年上升,以上海市为例,1973-2000年,上海市男性前列腺癌的发病率增加了4.8倍,跃居男性泌尿生殖系肿瘤的第1位。目前在我国前列腺癌已位居泌尿系肿瘤的第二位,仅次于膀胱癌。尽管近年来前列腺癌的诊断和治疗取得了比较人的进展,如PSA筛查、前列腺活检穿刺、根治性前列腺切除、内分泌剥夺、放疗、化疗等综合诊疗手段有效的降低了前列腺癌的死亡率
但由于前列腺癌发病机制不清、病情隐匿、临床症状不典型,大约有1/3的病人在诊断时已经存在局部浸润或远处转移,失去了手术治疗的机会。目前50-70%晚期前列腺癌早期采用的标准治疗方法是雄激素消除治疗(Androgen ablation therapy)。
不幸的是,雄激素阻断仅能暂时缓解疾病,中位缓解时间12-20个月,最终所有病人都将进展为雄激素非依赖性前列腺癌(androgen-independe nt prostate cancer, AIPC),雄激素阻断疗法因此失效。如何对雄激素非依赖性前列腺癌进行进一步治疗,足临床上一个非常棘手的问题。
雄激素依赖型前列腺癌在进行雄激素阻断的内分泌治疗后,逐渐将转变成雄激素非依赖型前列腺癌,从而导致内分泌治疗失败。虽然目前的研究还远远不能阐明其中的机理,但许多学者们都认为雄激素受体始终在转变中发挥着重要的作用。
紫杉醇类药物被临床证实对前列腺癌有一定的疗效,研究表明,紫杉醇化疗可以提高雄激素非依赖性前列腺癌的生存率,降低患者血浆前列腺特异抗原(PSA, prostate special antibody)水平。多西他赛是紫杉醇类的代表药物,以多西他赛为中心的化疗方案,是晚期前列腺癌的一线化疗方案的金标准。
多西他赛的抗癌作用机理尚未被完全阐明。一般认为,多西他赛抗肿瘤的机理在于其优先结合细胞内的β-微管蛋白,阻碍了GTP及一些辅助蛋白因子与β-微管蛋白的结合为位点。β-微管蛋白在缺少GTP和辅助因子时发生静态聚合,破坏了细胞正常的有丝分裂,使细胞分裂停滞在G2/M期,最终导致细胞的凋亡。
但近年来的一些研究纷纷发现,多西他赛还在下调转录、细胞增殖、有丝分裂和肿瘤发生中起作用的某些基因,起到抗肿瘤作用。另有研究发现,多西他赛可以通过抑制雄激素受体来发挥对前列腺癌的治疗作用。这些研究提示我们,对微管系统的作用及对G2/M期的阻滞并不是多西他赛诱导细胞凋亡的唯一机制,可能有许多信号传导通路和机制参与紫杉醇诱导肿瘤细胞的凋亡。其中一个重要的信号传导通路就是JNK信号转导通路,研究发现,C-jun-氨基末端激酶(C-jun-N-terminal-kinase, JNK)被激活之后,可以通过激活内源性通路,激活BAX(Bcl-2家族的前凋亡蛋白)和Caspase-3(细胞凋亡过程中最主要的终末剪切酶),并磷酸化Bcl-2(抗凋亡蛋白)使其失活,使得促凋亡分子释放,从而导致细胞凋亡。
原癌蛋白C-jun己被大量实验证实与许多肿瘤进展有关,其作为JNK的下游分子,可以被JNK磷酸化,其磷酸化位点位于C-jun氨基末端活性区Set63和Ser73,磷酸化C-jun可以增强其转录活性。C-jun是被认为是转录因子AP-1的组成部分,被认为是AP-1中起主要功能的重要组分。AP-1作为雄激素受体的转录因子,其活性又与雄激素受体基因调控能力紧密相关。在前列腺癌的发生发展中,雄激素受体发挥着至关重要的作用。
本实验通过采用多西他赛处理体外培养的前列腺癌细胞株,通过western blotting、DNA转染等方式,试图了解C-jun与AR在多西他赛作用下的相互作用,为探索多西他赛抗前列腺癌的机理和寻找前列腺癌多西他赛抵抗现象的原因提供线索。
在之前的研究中,我们通过雄激素依赖型前列腺癌细胞株LNCaP和雄激素非依赖型前列腺癌细胞株PC-3在紫杉醇药物多西他赛处理下雄激素受体和C-jun蛋白表达等的对比,发现这雄激素依赖型和雄激素非依赖型前列腺癌细胞在多西他赛处理下,都表现出C-jun磷酸化增强的现象。转染了雄激素受体后的PC-3细胞获得了一定的抵抗多西他赛的能力。但由于两种细胞来源不同(LNCaP细胞来源于人前列腺癌淋巴转移灶,PC-3细胞来源于人前列腺癌骨转移灶),所表达的前列腺癌标志物也不尽相同,因此用两者分别模拟雄激素依赖型和雄激素非依赖型前列腺癌细胞,并在多西他赛处理下进行比较时,可比性欠佳。
近年来国内外均有学者采用激素递减法或激素阻断法培养LNCaP细胞,从而获得稳定传代的非雄激素依赖的LNCaP细胞亚系,尽管LNCaP细胞表达的AR基因并非野生型,但LNCaP与其非雄激素依赖的细胞亚系仍是较理想的前列腺癌细胞研究模型。
我们采用多西紫杉醇处理雄激素依赖型LNCaP前列腺癌细胞及其亚型比卡鲁胺抵抗型LNCaP-bic细胞(非雄激素依赖),采用荧光素酶分析检测AR及AP-1基因的表达,利用Western blotting,免疫沉淀方法分析C-jun以及AR的蛋白表达和相互作用。试图进一步分析AR和C-jun之间的相互作用对前列腺癌多西他赛化疗结果的影响。
二、研究目的
本实验拟采用多西他赛处理雄激素非依赖型细胞株PC-3、雄激素依赖型细胞株LNCaP及其雄激素非依赖亚型细胞株LNCaP-bic,探索C-jun与AR在多西他赛处理的前列腺癌细胞中的相互关系。
三、实验方法
1、将PC-3细胞及LNCaP细胞,分别分为实验组(加入2.5nm多西他赛)和对照组(相应浓度的DMSO),0.25%胰蛋白酶常规消化后以1.5×105/瓶接种于25cm2细胞培养瓶,在37℃、5%CO2、饱和湿度条件下培养24小时后,吸除原培养液,按分组加入相应药物处理,传代培养30天。用倒置显微镜观察并照相记录用药组和对照组的细胞形态变化。Western blotting方法分析两组细胞中p-C-jun的表达。
2、将PC-3细胞、LNCaP细胞消化收集,按5×103个细胞/孔接种于96孔培养板,在37℃,5%C02、饱和湿度条件下培养。待细胞贴壁后,每孔进行换液。吸除原培养液后,分别加入含多西他赛lOnmol(对照组)、lOnmol的RPMI培养液180u1,另设只加培养液不加细胞和药物的为空白组。每组四个复孔。将96孔板移入C02孵箱静置培养。MTT法测量细胞存活率。
4、用脂质法将C-jun以及C-jun与AR共转染PC-3细胞株,western blotting方法检测转染后C-jun和AR的蛋白表达。设立空质粒转染为对照组,用多西他赛10nm处理后,MTT法测量细胞存活率
5、Western blotting分析多西他赛处理的PC-3和LNCaP细胞在各个时刻p-C-jun和p-JNK的表达。
6、培养传代LNCaP细胞非雄激素依赖亚型LNCaP-bic细胞,用含萤火虫和海’肾荧光酶素的ARE-luc报告基因质粒、AP-1-luc报告基因质粒转染细胞。转染24h后,细胞分为(1)对照组；(2)DOC组,多西他赛10nm处理48小时；(3)DHT组,双氢睾酮100nm处理48小时。裂解细胞,收集细胞提取物用双荧光素酶报告基因检测试剂盒检测(Promega公司),海肾荧光酶素活性作为内对照。分别以ARE和AP-1作为AR和C-jun的指示,荧光素酶方法分析LNCaP细胞和LNCaP-bic细胞在多西他赛处理下AR和C-jun基因的表达。
7、Western blotting方法检测多西他赛处理的LNCaP细胞和LNCaP-bic细胞PSA蛋白的表达情况,免疫沉淀法检测二者在多西他赛处耻下C-jun以及AR的蛋白表达和相关关系。
四、实验结果
1、PC-3细胞和LNCaP细胞10nm多西他赛处理后,C-jun蛋白表达与未处理组无明显差别,但磷酸化C-jun (p-C-jun)蛋白表达明显增强。
2、在同样浓度的多西他赛处理下,PC-3细胞的存活率与LNCaP细胞相比有显著性差异(P<0.05),转染C-jun基因后的PC-3细胞提高了对多西他赛的耐受性,而共转染C-jun/AR基因的PC-3细胞恢复了部分对多西他赛的敏感性。
3、western blotting分析提示在多西他赛处理的前列腺癌细胞中,p-JNK蛋白表达并没有显著性变化,体现出和p-C-jun蛋白表达增强不一致的特点。
4、荧光素酶分析提示在多西他赛处理下,LNCaP细胞和LNCaP-bic细胞的AR和C-jun在基因表达水平上均增强。
5、LNCaP-bic细胞在多西他赛处理下PSA表达强于其父代LNCaP细胞。免疫沉淀提示多西他赛处理后的LNCaP-bic细胞比起LNCaP细胞,C-jun与AR有着更强的相关性。
五、结论
在体外实验中,多西他赛可以采用JNK通路以外的方式磷酸化前列腺癌细胞中的C-jun, AR和C-jun可以提高前列腺癌细胞对多西他赛的耐受程度,多西他赛可以激活AR非配体依赖的转录功能,AR和C-jun之间的相互作用可能影响多西他赛对前列腺癌的化疗结果。
Background and Objection:
Prostate cancer (Prostate carcinoma, PCa) is the one of the most common malignant tumor in the Western countries, Prostate cancer is second as the leading cause of cancer death, respectively, in US men. Epidemiological data show the incidence of prostate cancer in China is also increasing year by year, for example, from1973to2000, the incidence rate of prostate cancer in Shanghai men increased by4.8folds, making it to be the first leading cause of male genitourinary tumors. At present, prostate cancer incidence rate is second only to bladder cancer in Chinese men's genitourinary system. The diagnosis and treatment of prostate cancer in recent years has made progress, some techniques, such as PSA screening, prostate biopsy and radical prostatectomy, androgen deprivation, radiotherapy, chemotherapy and other treatment, effectively reduce prostate cancer mortality. However, the pathogenesis of prostate cancer is still unclear, many prostate cancer grow concealed and asymptomatic, almost1/3patients lost opportunity to receive surgical treatment, because of the existence of local invasion or distant metastasis when they are diagnosed prostate cancer. The standard treatment for advanced prostate cancer is androgen elimination of treatment (Androgen ablation therapy).
Unfortunately, the effectiveness of androgen deprivation therapy is temporary, Androgen ablation therapy will be failure when patients develop to androgen-independent prostate cancer (AIPC), mean time is12-20months. Improving and optimizing therapeutic strategies for men with AIPC is one of the most challenging aspects of prostate cancer management today.
Androgen-dependent prostate cancer gradually progress to androgen-independent prostate cancer leading to the resistant of androgen ablation therapy. Although the current study is far from being able to clarify the mechanisms, many researchers believe that the androgen receptor plays an important role during the whole procedure.
Taxane mediated chemotherapy has been proven to have certain effect on prostate cancer, studies have shown that docetaxel can improve the survival rate of androgen-independent prostate cancer patients, and reduce the serum level of prostate-specific antigen (PSA), docetaxel chemotherapy is the gold standard of first-line chemotherapy in advanced prostate cancer.
The anticancer mechanism of docetaxel has not yet been fully elucidated. One of the general opinions believed that docetaxel combined with intracellular β-tubulin, prevented the combination of GTP and other protein factors with β-tubulin. The absence of GTP-and microtubule-associated proteins will stabilize microtubules, which are usually essential for cells mitosis, make cells division be arrested in the G2/M phase and lead to apoptosis.
Recent studies have also found that docetaxel antitumor effect as a result of down-regulating certain genes which related to transcription, cell proliferation, mitosis and tumorigenesis. Another study showed that docetaxel can play a role in the treatment of prostate cancer by inhibiting the androgen receptor. These studies suggest that the impact on microtubule system and the G2/M phase arrest is not the only mechanism of docetaxel induced apoptosis, there may be many signaling pathways and mechanisms involved in docetaxel antitumor effect. One of the important signaling pathways is JNK signal pathway.Activated C-jun N-terminal kinase (C-jun-N-the terminal-kinase, JNK) inducing activation of BAX (Bcl-2family) and Caspase-3, phosphorylation and inactivation Bcl-2, making The pro-apoptotic factor released, resulting in apoptosis.
C-jun is one of oncoprotein has been confirmed that related to the number of tumor progression by many experiments. C-jun is a transcription factor downstream of the JNK signaling pathway, it can enhance its transcriptional activity through the JNK-induced phosphorylation, the phosphorylation sites located in Ser63and Ser73. C-jun is considered to be an essential part of the transcription factor AP-1plays a major function. The activity of transcription factor AP-1is closely related to with the androgen receptor, which plays a crucial role in the development of prostate cancer.
In this study, we use docetaxel to treat prostate cancer cell lines in vitro, trying to understand the interaction of C-jun and AR by western blotting and DNA transfection, explore the mechanism of docetaxel against prostate cancer and look for clues of the docetaxel-resistant phenomenon. We also used docetaxel to treat androgen-dependent LNCaP prostate cancer cells and its subtypes LNCaP-bic cell (non-androgen dependent), using luciferase assays detect AR and AP-1gene expression, analyze C-jun, and AR protein expression by Western blotting, immunoprecipitation analysis. Attempt to further investigate the interaction between AR and C-jun in prostate cancer cell lines treated with docetaxel.
Objective Treat androgen-independent cell lines PC-3, androgen-dependent cell line LNCaP and its androgen-independent subtype cell lines LNCaP-bic with docetaxel, investigated the complex interaction of c-jun with AR in these cell lines after docetaxel treatment.
Material s and Methods
1, PC-3cells and LNCaP cells were divided into experimental group (treat with2.5nm docetaxel) and control group (treat with the corresponding concentration of DMSO), after0.25%trypsin digestion, inoculating cells to25cm2cell culture flasks(1.5×105/bottle, at37℃,5%CO2and saturated humidity) for24hours, then abandoned of the original medium, joined the appropriate drug to treat cells according their group. Cells were subcultured for30days. Observed and photographed the changes of cell morphology in treatment group and control group by inverted microscope, western blotting analysis p-C-jun expression of two groups of cells.
2, After digestion, PC-3cells, LNCaP cells were collected and seeded in a96-well plates,5×103cells/well, incubated in37℃,5%CO2and saturated humidity conditions. After the adherent cells growth, medium was changed. Abandon the original culture medium, add10nmol RPMI medium180μl, which contained10mol docetaxel. Use only medium without cells and drug as contrast. Each group has four wells. Incubating cells in a C02incubator, then measuring viability of cells by MTT assay.
3, Transfect PC-3cell with C-jun and C-jun/AR gene (liposome transfection), after detect C-jun and AR protein expression in PC-3cell after transfection by western blotting. Use empty plasmid transfected cells as the control group, MTT assay measuring cell viability in each group after1Onm docetaxel treatment.
4, Western blotting analysis the expression of p-C-jun and p-JNK in PC-3and LNCaP cells deal with docetaxel in different moments.
5, Continuously passaging LNCaP cells and its androgen-dependent subtypes, LNCaP-bic cell, were transfected with Luciferase reporter constructs ARE-luc and AP-1-luc using Lipofectamine2000reagent (Invitrogen), which containing the firefly and Renilla enzyme.The cells were divided into (1) control group;(2) DOC group (docetaxel10nm treated for48hours);(3) DHT group(DHT100nm treated for48hours).
After24h the cells were collected and analyzed with Luciferase assay according to manufacturer's instructions (Promega). Use Renilla enzyme activity as internal control.The activity of AP-1reporter was normalized to the protein concentration. Use ARE as indication for AR and AP-1for C-jun, luciferase analyzied AR and C-jun gene expression in LNCaP cells and LNCaP-bic cells after treatment of docetaxel.
6、To evaluate AR and AP-1gene activity using luciferase assay. To analyze the effects of docetaxel treatment on the expression/interaction of C-jun, and AR by Western blotting and immunoprecipitation assay.
Results
1, C-jun protein expression was no significant difference with the untreated group, but the phosphorylation of C-jun (pC and-jun) protein expression was significantly enhanced in PC-3cells and LNCaP cells after lOnm docetaxel treatment,
2, The survival rate of PC-3cells and LNCaP cells has a significant difference (P<0.05) after the treatment of same concentration docetaxel, PC-3cells improved docetaxel tolerability by transfected with the C-jun gene, and partially restored the sensitivity to docetaxel after co-transfected C-jun/AR gene.
3, western blotting analysis indicated that in docetaxel treated prostate cancer cells, p-JNK protein expression had no significant change and was not in correspondence with the expression of p-C-jun.
4, Luciferase assay showed LNCaP and LNCaP-bic cells expressed an increased activity of AR and C-jun in response to docetaxel.
5, Western blotting showed docetaxel induced strong PSA protein expression in LNCaP-bic. Immunoprecipitation assay demonstrated that docetaxel induced strong inreactions of AR with C-jun in LNCaP-bic.
Conclusions
Docetaxel phosphorylates C-jun by unknown patheway bypassing JNK. AR and C-jun may affects the prostate cancer cell resistance to docetaxel in vitro.
Docetaxel activates ligand independent AR transcriptional activity. Balance between AR and C-jun may affects outcome of docetaxel chemotherapy.
引文
1.Zenz R.Cjun regulates eyelid closure and skin tumor development through EGFR signaling. Dev Cell,2003,879-889.
2.Djavan, B., Waldert, M., Seitz, C., Marberger, M.,2001. Insulin-like growth factorsand prostate cancer. World J. Urol.19,225-233.
3.Waldum H, Brenna E, Sandvik A, Syversen U, Falkmer S.Hormones and carcinogenesis. Endocr Relat Cancer 1998;5:45-48.
4.叶定伟.前列腺癌的流行病学和中国的发病趋势[J].中华外科杂志,2006,44:362-364.
5.TANNOCK IF, DEW IT R, BERRY WJ, et al. Docetaxel plus prednisone or mit oxantrone plus prednisone for advanced prostate cancer[J]. N Engl J Med,2004, 351:1502-1512.
6.Wani MC, Taylor HL, Wall ME, et al. Plant antiumor agents. VI. The isolation and structure of taxol. a novel antileukemia and antitumor agen from Taxus brevifolia[J]. J Am Chem Soc,1971,93(3):2325.
7.Harada T, Ogura S, Yamazaki K, et al. Predictive value of expression of p53, Bcl-2 and lung resistance-related protein for respone to chemotherapy in non-small cell cancers [J]. Cancer Sci,2003,94(4):394.
8.Syrahruddin E, Oguri T, Takahashi T, et al. Differential expression of DNA topoisomerase Ⅱ alpha ang Ⅱ beta genes between small cell and non-small cell lung cancer[J]. Jpn J Cancer Res,1998,89(8):885.
9.Khan MA, Carducci MA, Partin AW, et al. The evolving role of docetaxel in the management of androgen independent prostate cancer[J]. Urol,2003,170(5): 1709-1716
10.Digemans AM, Wiflox MA, Stallaert RA, et al. Expression of DNA topoisomerase Ⅱ alpha and topoisomerase Ⅱ beta genes predicts survival and response to chemotherapy in patients with small cell lung cancer [J]. Clin Cancer Res,1999,5(8):2048
11.Tannock IF, de Wit R, Berry WR, et al. Docetaxel and estramustine compared plus prednisone or mitoxantrone plus prednisone for advanced independent prostate cancer[J]. N EnsI J Med,2004,351(15):1502-1512.
12.Miyake H, Nelson C, Rennie PS. Et al. Acquisition of chemoresistant phenotype by overexpression of the antiapoptotic gene testosterone-repressed prostate message-2 in prostate cancer xenograft models. Cancer Res,2000,60:2547-2554.
13.Zellweger T, Kiyama S. Chi K, et al. Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model. BJU Int.2003.92:463-469
14.Makarovskiy AN, Siryaporn E, Hixson DC. et al.Survival of docetaxel-resistant prostate cancer cells in vitro depends on phenotype alterations and continuity of drug exposure.Cell Mol life Sci,2002,59:1198-1211.
15.Hara T, Ushio K, Nishiwaki M, et al. A mutation in beta-tubulin and a sustained dependence on androgen receptor signalling in a newly established docetaxel-resistant prostate cancer cell line. Cell Biol Int,2010,34:177-184.
16.Chatterjee B. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer [J]. Mol Cell Biochem,2003,253(1):89-101.
17.Androgen receptor drives cellular senescence. Mirochnik Y, Veliceasa D, Williams L, et al. PLoS One.2012;7(3):e31052. Epub 2012 Mar 5.
18.Heinlein C A, Chang C. Androgen receptor in prostate cancer [J]. Endocr Rev, 2004,25(2):276-308
19.Feldman BJ. Feldman D. The development of androgen-independent prostate cancer. [J] Nat Rev Cancer.2001 Oct;1(1):34-45.
20.Surh YJ, Na HK, Lee SS. Transcription factors and mitogenactivated protein kinases as molecular target for chemoprevention with anti-inflammatory phytochemicals. Biofactors,2004,21:103-108.
21.Bubulya, A., Chen, S-Y., Fisher, C.J., Zheng, Z., Shen, X-Q., and Shemshedini, L. (2001) c-Jun potentiates the functional interaction between the amino and carboxy termini of the androgen receptor. J. Biol. Chem.276,44704-44711.
22.Bubulya A, Wise SC, Shen X-Q et al:c-Jun can mediate androgen receptor-induced transactivation. J Biol Chem,1996; 271:24583-24589
23.Chen SY, Cai C, Fisher CJ, Zheng Z, Omwancha J, Hsieh CL, Shemshedini L c-Jun enhancement of androgen receptor transactivation is associated with prostate cancer cell proliferation. Oncogene.2006 Nov 16;25(54):7212-23.
24.Fan M, Chambers TC Role of mitogen-activated protein kinases in the response of tumor cells to chemotherapy. Drug Resis Update (2001) 4,253-267.
25.Brantley-Finley C, Lyle CS, Du L, et alThe JNK, ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine, doxorubicin, and etoposide. Biochem Pharmacol (2003) 66,459-469.
26.Kolomeichuk SN, Terrano DT, Lyle CS, et al.Distinct signaling pathways of microtubule inhibitors vinblastine and Taxol induce JNK-dependent cell death but through AP-1-dependent and AP-1-independent mechanisms, respectively. FEBS J. 2008 Apr;275(8):1889-99. Epub 2008 Mar 13.
1.Denmeade SR, Lin XS, Lsaacs J T. Rote of programmed cell death during the progression and therapy for prostate cancer, Prostate,1996,28:251-265.
2.Raffo AJ. Perlman H, Chen MW:et al. Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res,1995,55:4438-4445.
3.Colombel M, Symmans F, Gil S, et al, Detection of the apoptosis-suppressing oncoprotein bcl-2 in hormone refractory human prostate cancers. Am J Pathol,1993,143:390-400.
4.Veldscholte J, Berrevoets CA, Ris-stalpers C, et al.The androgen receptor in LNCaP cells contains a mutation in ligand binding domain which affects steroid binding characteristics and response to antiandrogen. J Steroid Biochem Mol Biol 1992,41:665-669.
5.Culig Z, Hobisch A, Hittmair A. et al. Expression, structure, and function of androgen receptor in advanced prostatic carcinoma. Prostate.1998 Apr 1; 35(1):63-70.
6.Mitterberger M, Neuwirt H, Cavarretta IT, et al. Increased resistance to trail-induced apoptosis in prostate cancer cells selected in the presence of bicalutamide [J]. Prostate.2007 Aug 1;67(11):1194-201.
7.叶华茂,孙颖浩,许传亮,等.雄激素非依赖性前列腺癌细胞亚系模型LNCaP-AI的建立[J].第二军医大学学报,2008,29：59-62.
8.Onishi T, Yamakawa K, Franco OE, et al. Mitogen-activated protein kinase pathway is involved in alpha6 integrin gene expression in androgen-independent prostate cancer cells:role of proximal Sp1 consensus sequence. Biochim Biophys Acta.2001 Apr 23; 1538(2-3):218-27.
9.Kokontis JM, Hay N, Liao S.Progression of LNCaP prostate tumor cells during androgen deprivation:hormone-independent growth, repression of proliferation by androgen, and role for p27Kipl in androgen-induced cell cycle arrest. Mol Endocrinol.1998 Jul;12(7):941-53.
10.Hara T, Miyazaki J, Araki H, et al. Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome. Cancer Res.2003 Jan 1;63(1):149-53.
11.Tyrrell CJ, Iversen P, Tammela T, et al. Tolerability, efficacy and pharmacokinetics of bicalutamide 300 mg,450 mg or 600 mg as monotherapy for patients with locally advanced or metastatic prostate cancer, compared with castration. BJU Int.2006 Sep;98(3):563-72. Epub 2006 Jun 8.
12.Chatterjee B. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer [J]. Mol Cell Biochem,2003,253(1):89-101.
13.Tannock IF, de Wit R, Berry WR, et al. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer [J]. N Engl J Med 2004;351:1502-12.
14. Petrylak DP, Tangen CM, Hussain MH, et al. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer [J]. N Engl J Med 2004; 351:1513-20.
15. Gleave M, Miyake H, Chi K. Beyond simple castration:targeting the molecular basis of treatment resistance in advanced prostate cancer [J]. Cancer Chemother Pharmacol.2005; 56:s47-57.
16. Mathew P, DiPaola R. Taxane refractory prostate cancer. J Urol.2007;178:S36-41.
17. Zhu ML, Horbinski CM, Garzotto M, et al. Tubulin-targeting chemotherapy impairs androgen receptor activity in prostate cancer[J]. Cancer Res.2010 Oct 15;70(20):7992-8002.
18.Hussain M, Tangen CM, Higano C, et al. Absolute prostate-specific antigen value after androgen deprivation is a strong independent predictor of survival in new metastatic prostate cancer; data from Southwest Oncology Group trial 9346 (INT-0162)[J]. J Clin Oncol.2006;24:3984-90.
19. Hara T, Ushio K, Nishiwaki M, et al. A mutation in beta-tubulin and a sustained dependence on androgen receptor signalling in a newly established docetaxel-resistant prostate cancer cell line[J]. Cell Biol Int.2010 Jan 25;34(2):177-84.
20.Gan L, Chen S, Wang Y, et al. Inhibition of the androgen receptor as a novel mechanism of taxol chemotherapy in prostate cancer. Cancer research 2009;69:8386-94.
21.Chetram MA, Odero-Marah V, Hinton CV.Loss of PTEN permits CXCR4-mediated tumori genesis through ERK1/2 in prostate cancer cells. Mol Cancer Res.2011 Jan;9(1):90-102. Epub 2010 Nov 12.
22.Bertram J, Peacock JW, Fazli L, et al. Loss of PTEN is associated with progression to androgen independence. Prostate 2006;66:895-902.
23.Chen SY, Cai C, Fisher CJ, et al.c-Jun enhancement of androgen receptor transactivation is associated with prostate cancer cell proliferation [J] Oncogene.2006 Nov 16;25(54):7212-23.
24. Wang Q,Li W, Zhang Y, et al. Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer. [J] Cell.2009 Jul 23;138(2):245-56.
1. Jemal A, Siegel R, Ward E, et al. Cancer statistics. CA Cancer J Clin. 2009;59:225-49.
2. Risk M, Corman JM. The role of immunotherapy in prostate cancer: an overview of current approaches in development. Rev Urol.2009; 11:16-27.
3. Scher HI, Halabi S, Tannock I, et al. Prostate Cancer Clinical TrialsWorking Group. Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone:recommendations of the Prostate Cancer Clinical Trials Working Group. J Clin Oncol. 2008;26:1148-59.
4. G. Attard, D. Sarker, A. Reid, et al. Improving the outcome of patients with castration resistant prostate cancer through rational drug development, British Journal of Cancer, vol.95, no.7, pp.767-774,2006.
5. O. Kucuk, E. Fisher, C. M. Moinpour, et al. Phase II trial of bicalutamide in patients with advanced prostate cancer in whom conventional hormonal therapy failed:a Southwest Oncology Group study (SWOG 9235), Urology, vol.58, no.1, pp.53-58,2001.
6. M. Lodde, L. Lacombe, and Y. Fradet, Salvage therapy with bicalutamide 150 mg in nonmetastatic castration-resistant prostate cancer, Urology, vol.76, no.5, pp.1189-1193,2010.
7. D. Bianchini, A. Zivi, S. Sandhu, et al. Horizon scanning for novel therapeutics for the treatment of prostate cancer, Annals of Oncology, vol.21, supplement 7, pp. vii43-vii55,2010.
8. L. Zarour and J. Alumkal, Emerging therapies in castrate resistant prostate cancer, through rational drug development,British Journal of Cancer, vol.95, no.7, pp.767-774,2006.
9. G. Attard, A. S. Belldegrun, and J. S. De Bono, Selective blockade of androgenic steroid synthesis by novel lyase inhibitors as a therapeutic strategy for treating metastatic prostate cancer,British Journal of Urology International, vol.96, no.9, pp.1241-1246,2005.
10. J. S. De Bono, C. J. Logothetis, A. Molina,et al.Abiraterone and increased survival in metastatic prostate cancer, New England Journal of Medicine, vol. 364, no.21, pp.995-2005,2011.
11. Wani MC,Taylor HL,Wall ME,et al.Plant antiumor agents.VI. The isolation and structure of taxol, a novel antileukemia and antitumor agent from Taxus brevifolia [J]. J Am Chem Soc,1971,93(3):2325.
12. Tannock IF, de Wit R, Berry WR, et al. Docetaxel and estramustine compared plus prednisone or mitoxantrone plus prednisone for advanced independent prostate cancer[J]. N Engl J Med,2004,351(15):1502-1512.
13. Beer TM, Eilers KM, GARzotto M,et al. Quality of life and pain relief during treatment with calcitriol and decetaxel in symptomatic metastatic androgen-independent prostate eancinoma[J]. Cancer,2004,100(4):758-763.
14. Safarinejad MR. Combination chemotherapy with docetaxel, estramustine and suramin for hormone refractory prostate cancer[J]. Urol Oncol,2005,23(2): 93-101.
15. Pili R, Haggman M, Stadler WM, et al. Phase Ⅱ randomized, double-blind, placebo-controlled study of tasquinimod in men with minimally symptomatic metastatic castrate-resistant prostate cancer. J Clin Oncol.2011 Oct 20;29(30):4022-8. Epub 2011 Sep 19.
16. Saylor PJ, Lee RJ, Smith MR. Emerging therapies to prevent skeletal morbidity in men with prostate cancer. J Clin Oncol.2011 Sep 20;29(27):3705-14. Epub 2011 Aug22.
17. Picus J, Halabi S, Rini B, et al. The use of bevacizumab (B) with docetaxel (D) and estramustine (E) in hormone refractory prostate cancer (HRPC):initial results of CALGB 90006 [Abstract]. Proc Am Soc Clin Oncol 2005; 22:393.
18. Kelly WK, Halabi S, Carducci M, et al Randomized, Double-Blind, Placebo-Controlled Phase III Trial Comparing Docetaxel and Prednisone With or Without Bevacizumab in Men With Metastatic Castration-Resistant Prostate Cancer:CALGB 90401. J Clin Oncol.2012 May 1;30(13):1534-40. Epub 2012 Mar 26.
19. Dahut WL, Gulley JL, Arlen PM, et al. Randomized phase II trial of docetaxel plus thalidomide in androgen-independent prostate cancer. J Clin Oncol.2004 Jul 1;22(13):2532-9.
20. Ning YM, Gulley JL, Arlen PM, et al. Phase II trial of bevacizumab, thalidomide, docetaxel, and prednisone in patients with metastatic castration-resistant prostate cancer. J Clin Oncol.2010 Apr 20;28(12):2070-6. Epub 2010 Mar 22.
21. Saad F, Gleason DM, Murray R, et al. Long-term efficacy of zoledronic acid for the prevention of skeletal complications in patients with metastatic hormone-refractory prostate cancer. J Natl Cancer Inst.2004 Jun 2;96(11):879-82.
22. Fizazi K, Carducci M, Smith M, et al. Denosumab vs. zoledronic acid for treatment of bone metastases in men with castration-resistant prostate cancer: A randomized, double-blind study. Lancet 2011;377:813-22.
23. Stopeck AT, Lipton A, Body JJ, et al. Denosumab compared with zoledronic acid for the treatment of bone metastases in patients with advanced breast cancer: A randomized, double-blind study. J Clin Oncol 2010;28:5132-9.
24. Henry DH, Costa L, Goldwasser F, et al. Randomized, double-blind study of denosumab vs. zoledronic Acid in the treatment of bone metastases in patients with advanced cancer (excluding breast and prostate cancer) or multiple myeloma. J Clin Oncol 2011;29:1125-32.
25. Bruland ΦS, Nilsson S, Fisher DR, et al. High-linear energy transfer irradiation targeted to skeletal metastases by the alpha-emitter 223Ra: adjuvant or alternative to conventional modalities? Clin Cancer Res.2006 Oct 15; 12(20 Pt 2):6250s-6257s.
26. P. W. Kantoff, C. S. Higano, N. D. Shore, et al.Sipuleucel-T immunotherapy for castration-resistant prostate cancer, New England Journal of Medicine, vol. 363, no.5, pp.411-422,2010.
27. D. R. Berthold, C. N. Sternberg, and I. F. Tannock, Management of advanced prostate cancer after first-line chemotherapy,Journal of Clinical Oncology, vol. 23, no.32, pp.8247-8252,2005.
28. S. J. Kim and S. I. Kim, "Current treatment strategies for castration-resistant prostate cancer,Korean Journal of Urology, vol.52, no.3, pp.157-165,2011.
29. E. J. Small, N. Sacks, J. Nemunaitis,et al. Granulocyte macrophage colony-stimulating factor-secreting allogeneic cellular immunotherapy for hormone-refractory prostate cancer, Clinical Cancer Research, vol.13, no.13, pp.3883-3891,2007.
30. M. Hussain, M. R. Smith, C. Sweeney et al. Cabozantinib (XL184) in metastatic castration-resistant prostate cancer (mCRPC):results from a phase Ⅱ randomized discontinuation trial, Journal of Clinical Oncology, vol.29, supplement, abstract 4516,2011.
31. P. Vishnu and W. W. Tan, Update on options for treatment of metastatic castration-resistant prostate cancer, Onco Targets and Therapy, vol.3, pp. 39-51,2010.
32. D. Bianchini, A. Zivi, S. Sandhu, et al. Horizon scanning for novel therapeutics for the treatment of prostate cancer, Annals of Oncology, vol.21, supplement 7, pp. vii43-vii55,2010.
2.Djavan, B., Waldert, M., Seitz, C., Marberger, M.,2001. Insulin-like growth factorsand prostate cancer. World J. Urol.19,225-233.
3.Waldum H, Brenna E, Sandvik A, Syversen U, Falkmer S.Hormones and carcinogenesis. Endocr Relat Cancer 1998;5:45-48.
4.叶定伟.前列腺癌的流行病学和中国的发病趋势[J].中华外科杂志,2006,44:362-364.
5.TANNOCK IF, DEW IT R, BERRY WJ, et al. Docetaxel plus prednisone or mit oxantrone plus prednisone for advanced prostate cancer[J]. N Engl J Med,2004, 351:1502-1512.
6.Wani MC, Taylor HL, Wall ME, et al. Plant antiumor agents. VI. The isolation and structure of taxol. a novel antileukemia and antitumor agen from Taxus brevifolia[J]. J Am Chem Soc,1971,93(3):2325.
7.Harada T, Ogura S, Yamazaki K, et al. Predictive value of expression of p53, Bcl-2 and lung resistance-related protein for respone to chemotherapy in non-small cell cancers [J]. Cancer Sci,2003,94(4):394.
8.Syrahruddin E, Oguri T, Takahashi T, et al. Differential expression of DNA topoisomerase Ⅱ alpha ang Ⅱ beta genes between small cell and non-small cell lung cancer[J]. Jpn J Cancer Res,1998,89(8):885.
9.Khan MA, Carducci MA, Partin AW, et al. The evolving role of docetaxel in the management of androgen independent prostate cancer[J]. Urol,2003,170(5): 1709-1716
10.Digemans AM, Wiflox MA, Stallaert RA, et al. Expression of DNA topoisomerase Ⅱ alpha and topoisomerase Ⅱ beta genes predicts survival and response to chemotherapy in patients with small cell lung cancer [J]. Clin Cancer Res,1999,5(8):2048
11.Tannock IF, de Wit R, Berry WR, et al. Docetaxel and estramustine compared plus prednisone or mitoxantrone plus prednisone for advanced independent prostate cancer[J]. N EnsI J Med,2004,351(15):1502-1512.
12.Miyake H, Nelson C, Rennie PS. Et al. Acquisition of chemoresistant phenotype by overexpression of the antiapoptotic gene testosterone-repressed prostate message-2 in prostate cancer xenograft models. Cancer Res,2000,60:2547-2554.
13.Zellweger T, Kiyama S. Chi K, et al. Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model. BJU Int.2003.92:463-469
14.Makarovskiy AN, Siryaporn E, Hixson DC. et al.Survival of docetaxel-resistant prostate cancer cells in vitro depends on phenotype alterations and continuity of drug exposure.Cell Mol life Sci,2002,59:1198-1211.
15.Hara T, Ushio K, Nishiwaki M, et al. A mutation in beta-tubulin and a sustained dependence on androgen receptor signalling in a newly established docetaxel-resistant prostate cancer cell line. Cell Biol Int,2010,34:177-184.
16.Chatterjee B. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer [J]. Mol Cell Biochem,2003,253(1):89-101.
17.Androgen receptor drives cellular senescence. Mirochnik Y, Veliceasa D, Williams L, et al. PLoS One.2012;7(3):e31052. Epub 2012 Mar 5.
18.Heinlein C A, Chang C. Androgen receptor in prostate cancer [J]. Endocr Rev, 2004,25(2):276-308
19.Feldman BJ. Feldman D. The development of androgen-independent prostate cancer. [J] Nat Rev Cancer.2001 Oct;1(1):34-45.
20.Surh YJ, Na HK, Lee SS. Transcription factors and mitogenactivated protein kinases as molecular target for chemoprevention with anti-inflammatory phytochemicals. Biofactors,2004,21:103-108.
21.Bubulya, A., Chen, S-Y., Fisher, C.J., Zheng, Z., Shen, X-Q., and Shemshedini, L. (2001) c-Jun potentiates the functional interaction between the amino and carboxy termini of the androgen receptor. J. Biol. Chem.276,44704-44711.
22.Bubulya A, Wise SC, Shen X-Q et al:c-Jun can mediate androgen receptor-induced transactivation. J Biol Chem,1996; 271:24583-24589
23.Chen SY, Cai C, Fisher CJ, Zheng Z, Omwancha J, Hsieh CL, Shemshedini L c-Jun enhancement of androgen receptor transactivation is associated with prostate cancer cell proliferation. Oncogene.2006 Nov 16;25(54):7212-23.
24.Fan M, Chambers TC Role of mitogen-activated protein kinases in the response of tumor cells to chemotherapy. Drug Resis Update (2001) 4,253-267.
25.Brantley-Finley C, Lyle CS, Du L, et alThe JNK, ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine, doxorubicin, and etoposide. Biochem Pharmacol (2003) 66,459-469.
26.Kolomeichuk SN, Terrano DT, Lyle CS, et al.Distinct signaling pathways of microtubule inhibitors vinblastine and Taxol induce JNK-dependent cell death but through AP-1-dependent and AP-1-independent mechanisms, respectively. FEBS J. 2008 Apr;275(8):1889-99. Epub 2008 Mar 13.
1.Denmeade SR, Lin XS, Lsaacs J T. Rote of programmed cell death during the progression and therapy for prostate cancer, Prostate,1996,28:251-265.
2.Raffo AJ. Perlman H, Chen MW:et al. Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res,1995,55:4438-4445.
3.Colombel M, Symmans F, Gil S, et al, Detection of the apoptosis-suppressing oncoprotein bcl-2 in hormone refractory human prostate cancers. Am J Pathol,1993,143:390-400.
4.Veldscholte J, Berrevoets CA, Ris-stalpers C, et al.The androgen receptor in LNCaP cells contains a mutation in ligand binding domain which affects steroid binding characteristics and response to antiandrogen. J Steroid Biochem Mol Biol 1992,41:665-669.
5.Culig Z, Hobisch A, Hittmair A. et al. Expression, structure, and function of androgen receptor in advanced prostatic carcinoma. Prostate.1998 Apr 1; 35(1):63-70.
6.Mitterberger M, Neuwirt H, Cavarretta IT, et al. Increased resistance to trail-induced apoptosis in prostate cancer cells selected in the presence of bicalutamide [J]. Prostate.2007 Aug 1;67(11):1194-201.
7.叶华茂,孙颖浩,许传亮,等.雄激素非依赖性前列腺癌细胞亚系模型LNCaP-AI的建立[J].第二军医大学学报,2008,29：59-62.
8.Onishi T, Yamakawa K, Franco OE, et al. Mitogen-activated protein kinase pathway is involved in alpha6 integrin gene expression in androgen-independent prostate cancer cells:role of proximal Sp1 consensus sequence. Biochim Biophys Acta.2001 Apr 23; 1538(2-3):218-27.
9.Kokontis JM, Hay N, Liao S.Progression of LNCaP prostate tumor cells during androgen deprivation:hormone-independent growth, repression of proliferation by androgen, and role for p27Kipl in androgen-induced cell cycle arrest. Mol Endocrinol.1998 Jul;12(7):941-53.
10.Hara T, Miyazaki J, Araki H, et al. Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome. Cancer Res.2003 Jan 1;63(1):149-53.
11.Tyrrell CJ, Iversen P, Tammela T, et al. Tolerability, efficacy and pharmacokinetics of bicalutamide 300 mg,450 mg or 600 mg as monotherapy for patients with locally advanced or metastatic prostate cancer, compared with castration. BJU Int.2006 Sep;98(3):563-72. Epub 2006 Jun 8.
12.Chatterjee B. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer [J]. Mol Cell Biochem,2003,253(1):89-101.
13.Tannock IF, de Wit R, Berry WR, et al. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer [J]. N Engl J Med 2004;351:1502-12.
14. Petrylak DP, Tangen CM, Hussain MH, et al. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer [J]. N Engl J Med 2004; 351:1513-20.
15. Gleave M, Miyake H, Chi K. Beyond simple castration:targeting the molecular basis of treatment resistance in advanced prostate cancer [J]. Cancer Chemother Pharmacol.2005; 56:s47-57.
16. Mathew P, DiPaola R. Taxane refractory prostate cancer. J Urol.2007;178:S36-41.
17. Zhu ML, Horbinski CM, Garzotto M, et al. Tubulin-targeting chemotherapy impairs androgen receptor activity in prostate cancer[J]. Cancer Res.2010 Oct 15;70(20):7992-8002.
18.Hussain M, Tangen CM, Higano C, et al. Absolute prostate-specific antigen value after androgen deprivation is a strong independent predictor of survival in new metastatic prostate cancer; data from Southwest Oncology Group trial 9346 (INT-0162)[J]. J Clin Oncol.2006;24:3984-90.
19. Hara T, Ushio K, Nishiwaki M, et al. A mutation in beta-tubulin and a sustained dependence on androgen receptor signalling in a newly established docetaxel-resistant prostate cancer cell line[J]. Cell Biol Int.2010 Jan 25;34(2):177-84.
20.Gan L, Chen S, Wang Y, et al. Inhibition of the androgen receptor as a novel mechanism of taxol chemotherapy in prostate cancer. Cancer research 2009;69:8386-94.
21.Chetram MA, Odero-Marah V, Hinton CV.Loss of PTEN permits CXCR4-mediated tumori genesis through ERK1/2 in prostate cancer cells. Mol Cancer Res.2011 Jan;9(1):90-102. Epub 2010 Nov 12.
22.Bertram J, Peacock JW, Fazli L, et al. Loss of PTEN is associated with progression to androgen independence. Prostate 2006;66:895-902.
23.Chen SY, Cai C, Fisher CJ, et al.c-Jun enhancement of androgen receptor transactivation is associated with prostate cancer cell proliferation [J] Oncogene.2006 Nov 16;25(54):7212-23.
24. Wang Q,Li W, Zhang Y, et al. Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer. [J] Cell.2009 Jul 23;138(2):245-56.
1. Jemal A, Siegel R, Ward E, et al. Cancer statistics. CA Cancer J Clin. 2009;59:225-49.
2. Risk M, Corman JM. The role of immunotherapy in prostate cancer: an overview of current approaches in development. Rev Urol.2009; 11:16-27.
3. Scher HI, Halabi S, Tannock I, et al. Prostate Cancer Clinical TrialsWorking Group. Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone:recommendations of the Prostate Cancer Clinical Trials Working Group. J Clin Oncol. 2008;26:1148-59.
4. G. Attard, D. Sarker, A. Reid, et al. Improving the outcome of patients with castration resistant prostate cancer through rational drug development, British Journal of Cancer, vol.95, no.7, pp.767-774,2006.
5. O. Kucuk, E. Fisher, C. M. Moinpour, et al. Phase II trial of bicalutamide in patients with advanced prostate cancer in whom conventional hormonal therapy failed:a Southwest Oncology Group study (SWOG 9235), Urology, vol.58, no.1, pp.53-58,2001.
6. M. Lodde, L. Lacombe, and Y. Fradet, Salvage therapy with bicalutamide 150 mg in nonmetastatic castration-resistant prostate cancer, Urology, vol.76, no.5, pp.1189-1193,2010.
7. D. Bianchini, A. Zivi, S. Sandhu, et al. Horizon scanning for novel therapeutics for the treatment of prostate cancer, Annals of Oncology, vol.21, supplement 7, pp. vii43-vii55,2010.
8. L. Zarour and J. Alumkal, Emerging therapies in castrate resistant prostate cancer, through rational drug development,British Journal of Cancer, vol.95, no.7, pp.767-774,2006.
9. G. Attard, A. S. Belldegrun, and J. S. De Bono, Selective blockade of androgenic steroid synthesis by novel lyase inhibitors as a therapeutic strategy for treating metastatic prostate cancer,British Journal of Urology International, vol.96, no.9, pp.1241-1246,2005.
10. J. S. De Bono, C. J. Logothetis, A. Molina,et al.Abiraterone and increased survival in metastatic prostate cancer, New England Journal of Medicine, vol. 364, no.21, pp.995-2005,2011.
11. Wani MC,Taylor HL,Wall ME,et al.Plant antiumor agents.VI. The isolation and structure of taxol, a novel antileukemia and antitumor agent from Taxus brevifolia [J]. J Am Chem Soc,1971,93(3):2325.
12. Tannock IF, de Wit R, Berry WR, et al. Docetaxel and estramustine compared plus prednisone or mitoxantrone plus prednisone for advanced independent prostate cancer[J]. N Engl J Med,2004,351(15):1502-1512.
13. Beer TM, Eilers KM, GARzotto M,et al. Quality of life and pain relief during treatment with calcitriol and decetaxel in symptomatic metastatic androgen-independent prostate eancinoma[J]. Cancer,2004,100(4):758-763.
14. Safarinejad MR. Combination chemotherapy with docetaxel, estramustine and suramin for hormone refractory prostate cancer[J]. Urol Oncol,2005,23(2): 93-101.
15. Pili R, Haggman M, Stadler WM, et al. Phase Ⅱ randomized, double-blind, placebo-controlled study of tasquinimod in men with minimally symptomatic metastatic castrate-resistant prostate cancer. J Clin Oncol.2011 Oct 20;29(30):4022-8. Epub 2011 Sep 19.
16. Saylor PJ, Lee RJ, Smith MR. Emerging therapies to prevent skeletal morbidity in men with prostate cancer. J Clin Oncol.2011 Sep 20;29(27):3705-14. Epub 2011 Aug22.
17. Picus J, Halabi S, Rini B, et al. The use of bevacizumab (B) with docetaxel (D) and estramustine (E) in hormone refractory prostate cancer (HRPC):initial results of CALGB 90006 [Abstract]. Proc Am Soc Clin Oncol 2005; 22:393.
18. Kelly WK, Halabi S, Carducci M, et al Randomized, Double-Blind, Placebo-Controlled Phase III Trial Comparing Docetaxel and Prednisone With or Without Bevacizumab in Men With Metastatic Castration-Resistant Prostate Cancer:CALGB 90401. J Clin Oncol.2012 May 1;30(13):1534-40. Epub 2012 Mar 26.
19. Dahut WL, Gulley JL, Arlen PM, et al. Randomized phase II trial of docetaxel plus thalidomide in androgen-independent prostate cancer. J Clin Oncol.2004 Jul 1;22(13):2532-9.
20. Ning YM, Gulley JL, Arlen PM, et al. Phase II trial of bevacizumab, thalidomide, docetaxel, and prednisone in patients with metastatic castration-resistant prostate cancer. J Clin Oncol.2010 Apr 20;28(12):2070-6. Epub 2010 Mar 22.
21. Saad F, Gleason DM, Murray R, et al. Long-term efficacy of zoledronic acid for the prevention of skeletal complications in patients with metastatic hormone-refractory prostate cancer. J Natl Cancer Inst.2004 Jun 2;96(11):879-82.
22. Fizazi K, Carducci M, Smith M, et al. Denosumab vs. zoledronic acid for treatment of bone metastases in men with castration-resistant prostate cancer: A randomized, double-blind study. Lancet 2011;377:813-22.
23. Stopeck AT, Lipton A, Body JJ, et al. Denosumab compared with zoledronic acid for the treatment of bone metastases in patients with advanced breast cancer: A randomized, double-blind study. J Clin Oncol 2010;28:5132-9.
24. Henry DH, Costa L, Goldwasser F, et al. Randomized, double-blind study of denosumab vs. zoledronic Acid in the treatment of bone metastases in patients with advanced cancer (excluding breast and prostate cancer) or multiple myeloma. J Clin Oncol 2011;29:1125-32.
25. Bruland ΦS, Nilsson S, Fisher DR, et al. High-linear energy transfer irradiation targeted to skeletal metastases by the alpha-emitter 223Ra: adjuvant or alternative to conventional modalities? Clin Cancer Res.2006 Oct 15; 12(20 Pt 2):6250s-6257s.
26. P. W. Kantoff, C. S. Higano, N. D. Shore, et al.Sipuleucel-T immunotherapy for castration-resistant prostate cancer, New England Journal of Medicine, vol. 363, no.5, pp.411-422,2010.
27. D. R. Berthold, C. N. Sternberg, and I. F. Tannock, Management of advanced prostate cancer after first-line chemotherapy,Journal of Clinical Oncology, vol. 23, no.32, pp.8247-8252,2005.
28. S. J. Kim and S. I. Kim, "Current treatment strategies for castration-resistant prostate cancer,Korean Journal of Urology, vol.52, no.3, pp.157-165,2011.
29. E. J. Small, N. Sacks, J. Nemunaitis,et al. Granulocyte macrophage colony-stimulating factor-secreting allogeneic cellular immunotherapy for hormone-refractory prostate cancer, Clinical Cancer Research, vol.13, no.13, pp.3883-3891,2007.
30. M. Hussain, M. R. Smith, C. Sweeney et al. Cabozantinib (XL184) in metastatic castration-resistant prostate cancer (mCRPC):results from a phase Ⅱ randomized discontinuation trial, Journal of Clinical Oncology, vol.29, supplement, abstract 4516,2011.
31. P. Vishnu and W. W. Tan, Update on options for treatment of metastatic castration-resistant prostate cancer, Onco Targets and Therapy, vol.3, pp. 39-51,2010.
32. D. Bianchini, A. Zivi, S. Sandhu, et al. Horizon scanning for novel therapeutics for the treatment of prostate cancer, Annals of Oncology, vol.21, supplement 7, pp. vii43-vii55,2010.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |