多西紫杉醇诱导前列腺癌PC-3细胞凋亡及耐药前后的蛋白质组学研究
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摘要
背景
前列腺癌在美国和欧洲成年男性的恶性肿瘤中发病率最高,也是发达国家男性中因为癌症死亡的十分重要的原因之一。而随着人们生活条件逐渐提高,人均寿命不断延长以及饮食结构发生的巨大变化,前列腺癌的发病率和死亡率在我国均有逐渐升高的趋势。临床上通常将前列腺癌分为4期,其中早期前列腺癌(T2以下)的治疗多以根治性前列腺切除手术为主,而超过3期的前列腺癌,因癌细胞已经侵出前列腺包膜或外侵及转移,患者多失去手术机会。由于前列腺癌早期发病隐蔽,且在症状上表现为排尿困难,与良性前列腺增生症的主要症状类似,因此多被人们忽略,多数患者发现时肿瘤已有外侵及转移,失去了根治手术的时机。研究表明,人体内雄激素的分泌与前列腺癌的发生发展有十分密切的关系,因此,当失去根治手术机会后,临床上多通过手术去势(切除睾丸)或药物去势(抗雄激素药物治疗)的方法阻断体内雄激素的产生及发挥作用,从而抑制癌细胞的生长,在一定程度上延缓晚期前列腺癌患者的病情进展。而长期应用抗雄激素药物去势治疗,则可使激素依赖性前列腺癌逐渐转变为激素非依赖性前列腺癌,预后极差。有研究表明,当癌细胞发生激素非依赖性的转变之后,患者多在18个月内死亡。
紫杉醇类药物是近年来用于激素非依赖性前列腺癌的一种有效的化疗药物,其作用机理是通过结合细胞中的微管蛋白来阻断细胞的有丝分裂,从而抑制肿瘤细胞生长。多西紫杉醇为半合成的紫杉醇类药物,许多研究证实其对前列腺癌细胞具有诱导凋亡作用。临床应用中发现,应用多西紫杉醇治疗前列腺癌,肿瘤体积平均减少至原来的1/3,少数患者可达到完全缓解,近40%的患者经系统治疗后血PSA可下降一半以上。其主要毒副作用为骨髓抑制及神经系统损害。在对激素非依赖性前列腺癌耐药性的研究上,国外学者发现了数个基因的过度表达(如bcl-2等)可能与耐药相关。目前国内研究多集中于多西紫杉醇的药效学上,主要是通过应用多西紫杉醇作用于前列腺癌细胞株来观察其生长抑制和诱导凋亡作用。多数研究把应用多西紫杉醇后前列腺癌细胞发生特征性凋亡现象做为目标,聚焦于细胞凋亡的形态学变化,而对凋亡及耐药过程中的某些重要蛋白质,如凋亡早期的凋亡启动蛋白及晚期的凋亡效应蛋白未加深入研究,而这些蛋白质正是前列腺癌细胞发生凋亡或耐药效应的最终执行者。因此,寻找此类在细胞凋亡及耐药前后含量发生显著变化的关键蛋白质对于筛选高效低毒抗肿瘤药物及研究肿瘤细胞耐药性形成机制均具有十分重要的意义。
随着人类基因组计划(HGP)的顺利实施及人类基因组序列草图的完成,生命科学的研究相应的进入了一个崭新的后基因组时代。功能基因组学逐渐代替结构基因组学成为了基因组学研究的重点,而功能基因组学研究的核心内容之一--蛋白质组学,可全景式的解析作为最终执行生命活动的蛋白质所产生的规律及其功能[5,61。目前国外研究中蛋白质组学技术主要用于寻找前列腺癌瘤标, Meehan等发现正常表达于前列腺中的NEDD8, calponin和follistatin相关蛋白在前列腺癌中表达下调或缺失。Senior等通过应用蛋白质芯片这一技术发现了6种具有潜在意义的前列腺癌的标记物。Alaiya等用二维电泳和生物质谱技术研究了前列腺癌的蛋白质谱,发现数种蛋白表达增高,这些研究也为进一步寻找差异蛋白打下了基础。有鉴于此,本实验应用差异蛋白质组学方法对雄激素非依赖性前列腺癌的凋亡机制及耐药性等问题进行了较为深入的探讨。
第一部分多西紫杉醇诱导PC3细胞凋亡机制的蛋白质组学研究
目的：比较多西紫杉醇(docetaxel)诱导激素非依赖性前列腺癌PC3细胞凋亡前后的差异表达蛋白,为进一步研究紫杉醇耐药的分子机制提供理论依据。
方法：以不同浓度的多西紫杉醇作用于激素非依赖性前列腺癌PC3细胞,并引起细胞凋亡,采用磺酰罗丹明B(SRB)方法检测不同浓度多西紫杉醇对前列腺癌PC3细胞的增殖抑制作用；提取凋亡前后细胞总蛋白,应用胶内差异凝胶电泳技术(differential in-gel electrophoresis,DIGE)对PC3细胞凋亡前后的蛋白质进行双向凝胶电泳图谱分析,分离并筛选鉴定相关差异蛋白。
结果：通过SRB法发现多西紫杉醇对前列腺癌PC3细胞的半数抑制浓度IC50为20nmol/L。应用该浓度多西紫杉醇处理前列腺癌PC3细胞,分别提取细胞凋亡前后的总蛋白,应用双向凝胶电泳图谱进行分析,找到表达差异较为明显的18个蛋白质位点,并进一步分析鉴定,发现包括葡糖糖调节蛋白78、抗氧化蛋白2等在内的8个蛋白表达上调,包括p-微管蛋白在内的10个蛋白表达下调。
结论：通过蛋白质组学技术,我们发现多西紫杉醇处理前后激素非依赖性前列腺癌PC3细胞系差异蛋白共18个,其中部分蛋白可能参与激素非依赖性前列腺癌对紫杉醇类药物的耐药作用。
第二部分耐多西紫杉醇前列腺癌细胞系PC3-R的建立
目的：诱导激素非依赖性前列腺癌PC3细胞对多西紫杉醇产生耐药,为进一步通过蛋白质组学研究耐药相关蛋白打下基础。
方法：以多西紫杉醇为诱导药物,人前列腺癌PC3细胞系为诱导对象,通过小剂量浓度递增法与大剂量冲击相结合的方法,建立耐药细胞株PC3-R。
结果：建立了稳定的多西紫杉醇耐药前列腺癌细胞株PC3-R,其半数抑制浓度IC50为142nmol/L,是敏感株的7倍,且耐药株与敏感株细胞生长速度和倍增时间相近。
结论：成功地通过小剂量浓度递增法与大剂量冲击相结合的方法建立了前列腺癌PC3细胞耐药株PC3-R,其半数抑制浓度IC50是敏感株的7倍。相比敏感株而言,耐药株在形态上无明显改变,细胞增殖未受影响。
第三部分多西紫杉醇诱导PC3细胞耐药机制的蛋白质组学研究
目的：比较激素非依赖性前列腺癌PC3细胞株对多西紫杉醇(docetaxel)耐药前后的蛋白质的差异表达,从蛋白质水平初步了解前列腺癌PC3细胞株产生耐药的机制。
方法：通过逐渐增加多西紫杉醇药物浓度的方法诱导前列腺癌PC3细胞产生耐药细胞株PC3-R,分别提取PC3及PC3-R的细胞总蛋白,利用胶内差异凝胶电泳(DIGE)技术筛选细胞敏感株PC3与耐药株PC3-R的差异蛋白,并用质谱技术(MALDI-TOF/TOF-MS)对相关差异位点进行蛋白分离及成分鉴定。
结果：利用DIGE技术及MALDI-TOF-MS质谱分析技术成功分离鉴定出耐药株PC3-R与敏感株存在差异的49种蛋白质,其中29种蛋白质发生表达上调,20种蛋白质发生表达下调。其中Galectin-1、ATP合酶等参与肿瘤血管的生成,coflin、Cathepsin D、Calreticulin等蛋白参与肿瘤的转移；Microtubule-associated protein-6、GRP78等参与肿瘤的耐药性调节。
结论：成功地通过蛋白质组学技术对PC3细胞株多西紫杉醇耐药前后表达存在明显差异的蛋白质进行鉴定分析,为进一步阐明前列腺癌细胞对多西紫杉醇耐药产生的机制提供了线索,也为发现前列腺癌转移及晚期激素非依赖性前列腺癌的靶向药物治疗提供实验依据。
Background
Prostate cancer has the highest incidence in all malignant tomors that in Europe and the United States adult males. It is also one of the important reasons that leads to death in the developed countrys. With the extension of average life expectancy and dietary structure change,the incidence and mortality rates of prostate cancer increased gradually in our country. We usually classified the periods of the prostate cancer with four stages in clinical. The treatments of early prostate cancer are usually with radical resection operation.while more than a stage of3a, patients lost the chance of operation because the cancer cells may invade the envelope of the prostate. As the incidences of prostate cancer in early states are not easy to notice, and the main symptoms,such as dysuria,are similar with benign prostatic hyperplasia,always be neglected. A lot of patients lost the opportunity of radical operation because the invasion and metastasis of the cancer cells have occured already. The development of prostate cancer cells and androgen secretion is closely related. So when loss the opportunity of radical operation, patients were uaually treated by operation (removal of the testes) or medical castration (antiandrogen medication) for blocking the in vivo androgen production and action. It may inhibit the growth of cancer cells, to a certain extent, delaying disease progression in patients with advanced prostate cancer. While the long-term application of antiandrogen drug castration, can make the hormone independent prostate cancer transforms gradually to hormone-independent prostate cancer, The prognosis is poor. Studies have shown that, when the cancer cells change to hormone-independent cessls, patients die within18months.
Paclitaxel is a kind of effective chemotherapy drug for androgen-independent prostate cancer in recent years. Its mechanism of action is through a combination of cell microtubule proteins to block cell mitosis, thereby inhibiting the growth of tumor cells. Docetaxel is one of the semisynthesis of taxol drugs, and many studies have confirmed that it can induce apoptosis of prostate cancer cells. It was found in clinical that the application of docetaxel in the treatment of prostate cancer, tumor size was reduced from the original1/3, a few patients can achieve complete remission, and the serum PSA fell by more than half after systemic treatments in nearly40%of patients. In researchs of androgen-independent prostate cancer drug-resistance, foreign scholars have found a number of genes whose overexpression (such as Bcl-2) may be associated with drug-resistance. At present, the domestic researches focus on the effect of docetaxel, to observe the growth inhibition and apoptosis induced.by the application of docetaxel in prostate cancer cell lines. And most of them took the phenomenon of apoptosis as the research target. They focused on the morphological changes of apoptosis, but ignored some important proteins, such as the activating proteins in early stage of apoptosis and effector proteins in late stage of apoptosis,which might be the final performers of apoptosis or resistance in prostate cancer cells. Therefore, searching for such key proteins before and after the apoptosis and drug-resistance have very important significance for screening of efficient antineoplastic medicine and research of tumor cell drug resistance mechanism.
As the human genome project (HGP) carried out smoothly and the draft sequence of the human genome completed,life science research has entered into a new post genome era. The focus of genomics research were changed from structural genomics to functional genomics,and proteomics was one of the cores of functional genomics. The studies of proteomics technology is mainly used for prostate cancer targets In foreign countries. Meehan found that NEDD8, calponin and follistatin related proteins,which expressed in the normally prostate were loss or reduced in prostate cancer. Senior found six kinds of potential prostate cancer markers with the protein chip technology. Alaiya found the expression of several proteins increased when studied the protein mass spectrometry of prostate cancer by use of two-dimensional electrophoresis and biological mass spectrometry. These studies also made a foundation for further searching of differences in proteins.Because of this,we undertook relatively thorough research on the apoptosis and drug resistance in androgen independent prostate cancer cells with the application of proteomics.
Part I Proteomics Study on Apoptosis of Docetaxel Induced Prostate Cancer PC3Cells
Objective:To compare the protein expression profiles of androgen independent prostate cancer PC3cells and the cells that apoptosised by docetaxel.And to provide more theoretical foundations for the molecule mechanism of the docetaxel resistance in prostate cancer cells.
Methods:The apoptosis of the PC3cells were induced by different concentrations of docetaxel. Then sulforhodamine B(SRB) methods were used to investigate the inhibitive effects on proliferation of PC3cells with docetaxel. Total cellular proteins were extracted from the two cell lines which were separated by differential in-gel electrophoresis(DIGE).The profiles were obtained and analyzed to screen differentially expressed protein spots.
Results:The Median inhibitory concentration IC50is20nmol/L. We used docetaxel in this concentration to induce the PC3cells and extract total proteins both PC-3cells and the apoptosised cells. We obtained eighteen protein spots, among which eight proteins(such as78kD glucose-regulated protein and Peroxiredoxin-2) were up-regulated and ten proteins(such as Calreticulin) were down-regulated in apoptosised PC3cells.
Conclusions:We identified18proteins after apoptosis of docetaxel induced prostate cancer PC3cells by DIGE. Some of them may involved in the drug resistant mechanisms of docetaxel on the androgen independent prostate cancer treatment.
Part Ⅱ The Establishment of Docetaxel-resistant Cell Line PC3-R
Objective:To establish docetaxel-resisitant Androgen independent prostate cancer cell line(PC3-R), in order to further research for drug-resistance associated protein with proteomic.
Methods:Docetaxel-resisitant cell strain PC3-R, were induced by intermittent stepwised increasing with low concentration and impulse with high concentration of docetaxel.
Results:PC3-R cell strains, which were induced by intermittent stepwised increasing with low concentration and impulse with high concentration of docetaxel were established successfully. The IC50of cell strains was142nmol/L, which was7-times than PC-3cells.The cell growth velocity and doubling time were close of two cell strains.
Conclusions:We successfully established docetaxel-resistant cell strains PC3-R by intermittent stepwised increasing with low concentration and impulse with high concentration of docetaxel. The IC50of cell strains was7-times than docetaxel-sensitive cell strains. Compared with sensitive strains, Resistant strains showed no significant change in morphology and Cell proliferation was not affected.
Part Ⅲ Proteomic Analysis of Drug-resistant induced by Docetaxel in Androgen Independent Prostate Cancer PC3Cells
Objective:To compare the difference of protein expression between docetaxel-sensitive and docetaxel-resistant PCS cells, in order to find the mechanism of docetaxel-resistance of PC3cells.
Methods:Firstly, docetaxel-resistant PC3cells were produced by breeding with different dose of docetaxel. Then, total proteins were extracted from docetaxel-sensitive and docetaxel-resistant PC3cells in vitro. Secondly, the proteins were seperated with DIGE, and were analyzed by MALDI-TOF-Ms and bioinformation.
Results:We established docetaxel-resistant PC3-R cell strain, and the median inhibitory concentration (IC50) was7times than PC3. Forty nine differentially expressed proteins were found in docetaxel-resistant PC3cells by both DIGE and MALDI-TOF-TOF, compared with docetaxel-sensitive PC-3cells. Twenty nine proteins' expression was up-regulated and twenty proteins' expression was down-regulated. ATP synthase, Galectin-1were involved in the formation of tumor vessels; Calreticulin, Cathepsin D, Coflin were involved in tumor metastasis; GRP78, Microtubule-associated protein-6, and so on were involved in the drug resistance of tumor regulation.
Conclusions:Our study successfully established docetaxel-resistant prostate cancer PC3-R cell strain by combining gradually increasing concentration of docetaxel. It is suggested that an proteomic expression difference exists between docetaxel-sensitive and docetaxel-resistant PC3cells. It will be helpful for further understanding the molecular mechanisms of prostate cancer invasion and the drug resistance, and provide new experimental evidence for searching new drug therapy for advanced androgen independent prostate cancer.
前列腺癌在美国和欧洲成年男性的恶性肿瘤中发病率最高,也是发达国家男性中因为癌症死亡的十分重要的原因之一。而随着人们生活条件逐渐提高,人均寿命不断延长以及饮食结构发生的巨大变化,前列腺癌的发病率和死亡率在我国均有逐渐升高的趋势。临床上通常将前列腺癌分为4期,其中早期前列腺癌(T2以下)的治疗多以根治性前列腺切除手术为主,而超过3期的前列腺癌,因癌细胞已经侵出前列腺包膜或外侵及转移,患者多失去手术机会。由于前列腺癌早期发病隐蔽,且在症状上表现为排尿困难,与良性前列腺增生症的主要症状类似,因此多被人们忽略,多数患者发现时肿瘤已有外侵及转移,失去了根治手术的时机。研究表明,人体内雄激素的分泌与前列腺癌的发生发展有十分密切的关系,因此,当失去根治手术机会后,临床上多通过手术去势(切除睾丸)或药物去势(抗雄激素药物治疗)的方法阻断体内雄激素的产生及发挥作用,从而抑制癌细胞的生长,在一定程度上延缓晚期前列腺癌患者的病情进展。而长期应用抗雄激素药物去势治疗,则可使激素依赖性前列腺癌逐渐转变为激素非依赖性前列腺癌,预后极差。有研究表明,当癌细胞发生激素非依赖性的转变之后,患者多在18个月内死亡。
紫杉醇类药物是近年来用于激素非依赖性前列腺癌的一种有效的化疗药物,其作用机理是通过结合细胞中的微管蛋白来阻断细胞的有丝分裂,从而抑制肿瘤细胞生长。多西紫杉醇为半合成的紫杉醇类药物,许多研究证实其对前列腺癌细胞具有诱导凋亡作用。临床应用中发现,应用多西紫杉醇治疗前列腺癌,肿瘤体积平均减少至原来的1/3,少数患者可达到完全缓解,近40%的患者经系统治疗后血PSA可下降一半以上。其主要毒副作用为骨髓抑制及神经系统损害。在对激素非依赖性前列腺癌耐药性的研究上,国外学者发现了数个基因的过度表达(如bcl-2等)可能与耐药相关。目前国内研究多集中于多西紫杉醇的药效学上,主要是通过应用多西紫杉醇作用于前列腺癌细胞株来观察其生长抑制和诱导凋亡作用。多数研究把应用多西紫杉醇后前列腺癌细胞发生特征性凋亡现象做为目标,聚焦于细胞凋亡的形态学变化,而对凋亡及耐药过程中的某些重要蛋白质,如凋亡早期的凋亡启动蛋白及晚期的凋亡效应蛋白未加深入研究,而这些蛋白质正是前列腺癌细胞发生凋亡或耐药效应的最终执行者。因此,寻找此类在细胞凋亡及耐药前后含量发生显著变化的关键蛋白质对于筛选高效低毒抗肿瘤药物及研究肿瘤细胞耐药性形成机制均具有十分重要的意义。
随着人类基因组计划(HGP)的顺利实施及人类基因组序列草图的完成,生命科学的研究相应的进入了一个崭新的后基因组时代。功能基因组学逐渐代替结构基因组学成为了基因组学研究的重点,而功能基因组学研究的核心内容之一--蛋白质组学,可全景式的解析作为最终执行生命活动的蛋白质所产生的规律及其功能[5,61。目前国外研究中蛋白质组学技术主要用于寻找前列腺癌瘤标, Meehan等发现正常表达于前列腺中的NEDD8, calponin和follistatin相关蛋白在前列腺癌中表达下调或缺失。Senior等通过应用蛋白质芯片这一技术发现了6种具有潜在意义的前列腺癌的标记物。Alaiya等用二维电泳和生物质谱技术研究了前列腺癌的蛋白质谱,发现数种蛋白表达增高,这些研究也为进一步寻找差异蛋白打下了基础。有鉴于此,本实验应用差异蛋白质组学方法对雄激素非依赖性前列腺癌的凋亡机制及耐药性等问题进行了较为深入的探讨。
第一部分多西紫杉醇诱导PC3细胞凋亡机制的蛋白质组学研究
目的：比较多西紫杉醇(docetaxel)诱导激素非依赖性前列腺癌PC3细胞凋亡前后的差异表达蛋白,为进一步研究紫杉醇耐药的分子机制提供理论依据。
方法：以不同浓度的多西紫杉醇作用于激素非依赖性前列腺癌PC3细胞,并引起细胞凋亡,采用磺酰罗丹明B(SRB)方法检测不同浓度多西紫杉醇对前列腺癌PC3细胞的增殖抑制作用；提取凋亡前后细胞总蛋白,应用胶内差异凝胶电泳技术(differential in-gel electrophoresis,DIGE)对PC3细胞凋亡前后的蛋白质进行双向凝胶电泳图谱分析,分离并筛选鉴定相关差异蛋白。
结果：通过SRB法发现多西紫杉醇对前列腺癌PC3细胞的半数抑制浓度IC50为20nmol/L。应用该浓度多西紫杉醇处理前列腺癌PC3细胞,分别提取细胞凋亡前后的总蛋白,应用双向凝胶电泳图谱进行分析,找到表达差异较为明显的18个蛋白质位点,并进一步分析鉴定,发现包括葡糖糖调节蛋白78、抗氧化蛋白2等在内的8个蛋白表达上调,包括p-微管蛋白在内的10个蛋白表达下调。
结论：通过蛋白质组学技术,我们发现多西紫杉醇处理前后激素非依赖性前列腺癌PC3细胞系差异蛋白共18个,其中部分蛋白可能参与激素非依赖性前列腺癌对紫杉醇类药物的耐药作用。
第二部分耐多西紫杉醇前列腺癌细胞系PC3-R的建立
目的：诱导激素非依赖性前列腺癌PC3细胞对多西紫杉醇产生耐药,为进一步通过蛋白质组学研究耐药相关蛋白打下基础。
方法：以多西紫杉醇为诱导药物,人前列腺癌PC3细胞系为诱导对象,通过小剂量浓度递增法与大剂量冲击相结合的方法,建立耐药细胞株PC3-R。
结果：建立了稳定的多西紫杉醇耐药前列腺癌细胞株PC3-R,其半数抑制浓度IC50为142nmol/L,是敏感株的7倍,且耐药株与敏感株细胞生长速度和倍增时间相近。
结论：成功地通过小剂量浓度递增法与大剂量冲击相结合的方法建立了前列腺癌PC3细胞耐药株PC3-R,其半数抑制浓度IC50是敏感株的7倍。相比敏感株而言,耐药株在形态上无明显改变,细胞增殖未受影响。
第三部分多西紫杉醇诱导PC3细胞耐药机制的蛋白质组学研究
目的：比较激素非依赖性前列腺癌PC3细胞株对多西紫杉醇(docetaxel)耐药前后的蛋白质的差异表达,从蛋白质水平初步了解前列腺癌PC3细胞株产生耐药的机制。
方法：通过逐渐增加多西紫杉醇药物浓度的方法诱导前列腺癌PC3细胞产生耐药细胞株PC3-R,分别提取PC3及PC3-R的细胞总蛋白,利用胶内差异凝胶电泳(DIGE)技术筛选细胞敏感株PC3与耐药株PC3-R的差异蛋白,并用质谱技术(MALDI-TOF/TOF-MS)对相关差异位点进行蛋白分离及成分鉴定。
结果：利用DIGE技术及MALDI-TOF-MS质谱分析技术成功分离鉴定出耐药株PC3-R与敏感株存在差异的49种蛋白质,其中29种蛋白质发生表达上调,20种蛋白质发生表达下调。其中Galectin-1、ATP合酶等参与肿瘤血管的生成,coflin、Cathepsin D、Calreticulin等蛋白参与肿瘤的转移；Microtubule-associated protein-6、GRP78等参与肿瘤的耐药性调节。
结论：成功地通过蛋白质组学技术对PC3细胞株多西紫杉醇耐药前后表达存在明显差异的蛋白质进行鉴定分析,为进一步阐明前列腺癌细胞对多西紫杉醇耐药产生的机制提供了线索,也为发现前列腺癌转移及晚期激素非依赖性前列腺癌的靶向药物治疗提供实验依据。
Background
Prostate cancer has the highest incidence in all malignant tomors that in Europe and the United States adult males. It is also one of the important reasons that leads to death in the developed countrys. With the extension of average life expectancy and dietary structure change,the incidence and mortality rates of prostate cancer increased gradually in our country. We usually classified the periods of the prostate cancer with four stages in clinical. The treatments of early prostate cancer are usually with radical resection operation.while more than a stage of3a, patients lost the chance of operation because the cancer cells may invade the envelope of the prostate. As the incidences of prostate cancer in early states are not easy to notice, and the main symptoms,such as dysuria,are similar with benign prostatic hyperplasia,always be neglected. A lot of patients lost the opportunity of radical operation because the invasion and metastasis of the cancer cells have occured already. The development of prostate cancer cells and androgen secretion is closely related. So when loss the opportunity of radical operation, patients were uaually treated by operation (removal of the testes) or medical castration (antiandrogen medication) for blocking the in vivo androgen production and action. It may inhibit the growth of cancer cells, to a certain extent, delaying disease progression in patients with advanced prostate cancer. While the long-term application of antiandrogen drug castration, can make the hormone independent prostate cancer transforms gradually to hormone-independent prostate cancer, The prognosis is poor. Studies have shown that, when the cancer cells change to hormone-independent cessls, patients die within18months.
Paclitaxel is a kind of effective chemotherapy drug for androgen-independent prostate cancer in recent years. Its mechanism of action is through a combination of cell microtubule proteins to block cell mitosis, thereby inhibiting the growth of tumor cells. Docetaxel is one of the semisynthesis of taxol drugs, and many studies have confirmed that it can induce apoptosis of prostate cancer cells. It was found in clinical that the application of docetaxel in the treatment of prostate cancer, tumor size was reduced from the original1/3, a few patients can achieve complete remission, and the serum PSA fell by more than half after systemic treatments in nearly40%of patients. In researchs of androgen-independent prostate cancer drug-resistance, foreign scholars have found a number of genes whose overexpression (such as Bcl-2) may be associated with drug-resistance. At present, the domestic researches focus on the effect of docetaxel, to observe the growth inhibition and apoptosis induced.by the application of docetaxel in prostate cancer cell lines. And most of them took the phenomenon of apoptosis as the research target. They focused on the morphological changes of apoptosis, but ignored some important proteins, such as the activating proteins in early stage of apoptosis and effector proteins in late stage of apoptosis,which might be the final performers of apoptosis or resistance in prostate cancer cells. Therefore, searching for such key proteins before and after the apoptosis and drug-resistance have very important significance for screening of efficient antineoplastic medicine and research of tumor cell drug resistance mechanism.
As the human genome project (HGP) carried out smoothly and the draft sequence of the human genome completed,life science research has entered into a new post genome era. The focus of genomics research were changed from structural genomics to functional genomics,and proteomics was one of the cores of functional genomics. The studies of proteomics technology is mainly used for prostate cancer targets In foreign countries. Meehan found that NEDD8, calponin and follistatin related proteins,which expressed in the normally prostate were loss or reduced in prostate cancer. Senior found six kinds of potential prostate cancer markers with the protein chip technology. Alaiya found the expression of several proteins increased when studied the protein mass spectrometry of prostate cancer by use of two-dimensional electrophoresis and biological mass spectrometry. These studies also made a foundation for further searching of differences in proteins.Because of this,we undertook relatively thorough research on the apoptosis and drug resistance in androgen independent prostate cancer cells with the application of proteomics.
Part I Proteomics Study on Apoptosis of Docetaxel Induced Prostate Cancer PC3Cells
Objective:To compare the protein expression profiles of androgen independent prostate cancer PC3cells and the cells that apoptosised by docetaxel.And to provide more theoretical foundations for the molecule mechanism of the docetaxel resistance in prostate cancer cells.
Methods:The apoptosis of the PC3cells were induced by different concentrations of docetaxel. Then sulforhodamine B(SRB) methods were used to investigate the inhibitive effects on proliferation of PC3cells with docetaxel. Total cellular proteins were extracted from the two cell lines which were separated by differential in-gel electrophoresis(DIGE).The profiles were obtained and analyzed to screen differentially expressed protein spots.
Results:The Median inhibitory concentration IC50is20nmol/L. We used docetaxel in this concentration to induce the PC3cells and extract total proteins both PC-3cells and the apoptosised cells. We obtained eighteen protein spots, among which eight proteins(such as78kD glucose-regulated protein and Peroxiredoxin-2) were up-regulated and ten proteins(such as Calreticulin) were down-regulated in apoptosised PC3cells.
Conclusions:We identified18proteins after apoptosis of docetaxel induced prostate cancer PC3cells by DIGE. Some of them may involved in the drug resistant mechanisms of docetaxel on the androgen independent prostate cancer treatment.
Part Ⅱ The Establishment of Docetaxel-resistant Cell Line PC3-R
Objective:To establish docetaxel-resisitant Androgen independent prostate cancer cell line(PC3-R), in order to further research for drug-resistance associated protein with proteomic.
Methods:Docetaxel-resisitant cell strain PC3-R, were induced by intermittent stepwised increasing with low concentration and impulse with high concentration of docetaxel.
Results:PC3-R cell strains, which were induced by intermittent stepwised increasing with low concentration and impulse with high concentration of docetaxel were established successfully. The IC50of cell strains was142nmol/L, which was7-times than PC-3cells.The cell growth velocity and doubling time were close of two cell strains.
Conclusions:We successfully established docetaxel-resistant cell strains PC3-R by intermittent stepwised increasing with low concentration and impulse with high concentration of docetaxel. The IC50of cell strains was7-times than docetaxel-sensitive cell strains. Compared with sensitive strains, Resistant strains showed no significant change in morphology and Cell proliferation was not affected.
Part Ⅲ Proteomic Analysis of Drug-resistant induced by Docetaxel in Androgen Independent Prostate Cancer PC3Cells
Objective:To compare the difference of protein expression between docetaxel-sensitive and docetaxel-resistant PCS cells, in order to find the mechanism of docetaxel-resistance of PC3cells.
Methods:Firstly, docetaxel-resistant PC3cells were produced by breeding with different dose of docetaxel. Then, total proteins were extracted from docetaxel-sensitive and docetaxel-resistant PC3cells in vitro. Secondly, the proteins were seperated with DIGE, and were analyzed by MALDI-TOF-Ms and bioinformation.
Results:We established docetaxel-resistant PC3-R cell strain, and the median inhibitory concentration (IC50) was7times than PC3. Forty nine differentially expressed proteins were found in docetaxel-resistant PC3cells by both DIGE and MALDI-TOF-TOF, compared with docetaxel-sensitive PC-3cells. Twenty nine proteins' expression was up-regulated and twenty proteins' expression was down-regulated. ATP synthase, Galectin-1were involved in the formation of tumor vessels; Calreticulin, Cathepsin D, Coflin were involved in tumor metastasis; GRP78, Microtubule-associated protein-6, and so on were involved in the drug resistance of tumor regulation.
Conclusions:Our study successfully established docetaxel-resistant prostate cancer PC3-R cell strain by combining gradually increasing concentration of docetaxel. It is suggested that an proteomic expression difference exists between docetaxel-sensitive and docetaxel-resistant PC3cells. It will be helpful for further understanding the molecular mechanisms of prostate cancer invasion and the drug resistance, and provide new experimental evidence for searching new drug therapy for advanced androgen independent prostate cancer.
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