A Proteomics Study Of Docetaxel-induced Apoptosis And Drug-resistant In Prostate Cancer PC3Cells

BackgroundProstate 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 PC3CellsObjective: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-RObjective: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 PC3CellsObjective: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.