| 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.Results1, 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.ConclusionsDocetaxel 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. |
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