| Background:Cancer mortality is significantly increased as aging population intensifies. Prostate cancer (PCa) is the most common male malignancy of the genitourinary system in the United States and Europe. In recent years, the PCa incidence is rapidly increasing in China, and at present, PCa has the highest incidence in Chinese men over 70 years old. Thus PCa has officially become one of the malignant tumors which impact significantly on Chinese men’s health. Unfortunately, when patients are diagnosed with PCa, the cancer has most likely metastasized and patients are in advanced stage. Over 50% of patients with advanced PCa may receive androgen deprivation therapy as the primary treatment, but this treatment has not approved to be beneficial. Many patients with advanced PCa receive conventional chemotherapy or targeted therapy, but sooner or later they will become resistant to these treatments, and therefore, other palliative treatments need to be urgently developed.In our prior published studies, we have established chemo-resistant prostate cells by gradually increasing paclitaxel concentration in the cell culture over time (9-12 months). Compared to parental PC3 and DU145cells, the chemo-resistant PC3-TxR and DU145-TxR cells appeared looser contact, narrower and more fusion form, suggesting that EMT may occur in chemo-resistant cell lines. These chemo-resistant cells were used as models in this experiment that aim to explore the association between PCa paclitaxel resistance and EMT. Finally, we tried to explore the mechanisms for PCa chemoresistance.This study includes three parts:Part 1. Analysis of EMT-associated characteristics in paclitaxel-resistant PCa cellsObjective:To determine whether EMT developes in the chemo-resistant PCa cells PC3-TxR and DU145-TxR, compared to their parental PC3 and DU145 cells.Methods:1) The morphological differences between the parental cells PC3, DU145 and their chemo-resistant cells PC3-TxR and DU145-TxR were observed under fluorescence microscopy.2) The EMT markers such as E-cadherin, N-cadherin, Snail, Vimentin were tested by RT-PCR, Real-time PCR and Western-blot.3) Metastasis and invasion between parental cells PC3, DU145, and their chemo-resistant cells PC3-TxR, DU145-TxR were tested by scratch assay and transwell assay.4) The luciferase transfected parental PC 3 and DU145 cells and chemo-resistant PC3-TxR, DU145-TxR cells were constructed. All cell lines were injected subcutaneously into the 6-week male SCID mice. Observed tumorigenesis by bioluminescence imaging system.Results:1) Compared to the parental PC3, DU145 cells, the chemo-resistant cells PC3-TxR, DU145-TxR were more evacuated and became narrow and assumed spindle shape-properties with the mesenchymal phenotype.2) The expression of E-cadherin was down-regulated, and the expressions of Vimentin, Snail, N-cadherin were up-regulated.3) Cell migration rate of DU145 cells in 24h was15.9±3.2%, compared to DU145-TxR cells,68.6±2.85%. Migration in chemo-resistant cells DU145-TxR was stronger than DU145 cells (P<0.01). The results of transwell showed that both the abilities of migration and invasion in chemo-resistant cells PC3-TxR were stronger than PC3 cells (P<0.01). And the abilities of migration and invasion in chemo-resistant cells DU145-TxR were stronger than DU145 cells too (P<0.01).4) Four luciferase transfected cells(PC3-luc, PC3-TxR-luc, DU145-luc, DU145-TxR-luc) were constructed successfully, and the luciferase index were 3.70×107,9.30×107,2.90×107, 4.22×107, The tumor volume of PC3-TxR-luc cells were larger than PC3-luc cells (P<0.01).Conclusion:An EMT happened in the chemo-resistant PCa cells PC3-TxR, DU145-TxR, compared to their parental prostate cancel cells PC3, DU145. The abilities of migration, invasion, and tumorigenicity in vivo were stronger in chemo-resistant cancer cells. results of transwell showed that both the abilities of migration and invasion in chemo-resistant cells PC3-TxR were stronger than PC3 cells (P<0.01). And the abilities of migration and invasion in chemo-resistant cells DU145-TxR were stronger than DU145 cells too (P<0.01).4) Four luciferase transfected cells(PC3-luc, PC3-TxR-luc, DU145-luc, DU145-TxR-luc) were constructed successfully, and the luciferase index were 3.70×107,9.30×107,2.90×107, 4.22×107, The tumor volume of PC3-TxR-luc cells were larger than PC3-luc cells (P<0.01).Conclusion:An EMT happened in the chemo-resistant PCa cells PC3-TxR, DU145-TxR, compared to their parental prostate cancel cells PC3, DU145. The abilities of migration, invasion, and tumorigenicity in vivo were stronger in chemo-resistant cancer cells.Part 2. E-cadherin contributes to migration, invasion and colony formation of PCa cells mediated by EMTObjective:To establish E-cadherin overexpressed or silenced PCa cells. Test the difference of cell migration, invasion, morphological changes, and paclitaxel sensitivity between the established cell lines and their parental cell lines. Verify the roles of EMT in the chemo-resistance.Methods:1) The E-cadherin overexpressed cells lines PC3-TxR-E-cadherin, DU145-TxR-E-cadherin and the control cell lines PC3-TxR-control, DU145-TxR-control were established by lentivirus, which were added in the cell lines for 48 hours, then screened by puromycin until all of the negative control cells were dead. Tested the expression of E-cadherin by real-time PCR and western-blot. Tested the expression of EMT-associated markers and signaling pathways by Western-blot.2) The abilities of metastasis, invasion and colony formation between parental chemoresistant cells PC3-TxR, DU145-TxR and the constructed cells were tested by scratch assay, and transwell assay. The paclitaxel sensitivities were tested by MTS assay.3) Silenced the expression of E-cadherin in PC3, DU145 cell lines by siRNA. Extracted the mRNAs and proteins after 48 hours. Tested the expression of E-cadherin by Real-time PCR and Western-blot. Tested the expression of EMT-associated marks by Real-time PCR.4) The abilities of metastasis and colony formation between parental cells PC3, DU145 and the silenced cells were tested by scratch assay and colony formation assay. The paclitaxel sensitivities between the silenced cells and parental cells were tested by MTS assay.Results:1) E-cadherin overexpressed cells lines (PC3-TxR-E-cadherin, DU145-TxR-E-cadherin) and the control cell lines (PC3-TxR-control, DU145-TxR-control) were successfully established, and the expression of E-cadherin in PC3-TxR-E-cadherin, DU145-TxR-E-cadherin were up-regulated, compared to PC3-TxR-control, DU145-TxR-control cells and PC3-TxR, DU145-TxR cells (P<0.01). There were no differences of the E-cadherin expression between PC3-TxR-control, DU145-TxR-control cells and PC3-TxR, DU145-TxR cells. The expression of Vimentin, Claudin-1 and Notch-1 were down-regulated in the E-cadherin overexpressed cells.2) Cell migration rate in 24h were:DU145-TxR,68.8±2.86%. DU145-TxR-control,66.6±2.88%. DU145-TxR-E-cadherin,38.3±1.63%. The ability of migration in chemo-resis-tant cells DU145-TxR was stronger than DU145-TxR-E-cadherin cells (P<0.01).The results of transwell showed that both the abilities of migration and invasion in chemo-resistant cells were stronger than E-cadherin overexpressed chemo-resistant cells cells (P<0.01). the results of MTS showed that the IC50 in 72hours were:PC3-TxR,146.81±1.46nmol/L. PC3-TxR-contro1139.13±4.60 nmol/L. PC3-TxR-E-cadherin,96.2±15.03nmol/L. DU145-TxR,3831.95±65.69 nmol/L. DU145-TxR-control,3725.45±87.36 nmol/L.DU145-TxR-E-cadherin, 3022.10±34.01nmol/L. The tolerance to paclitaxel were both reversed in two different E-cadherin overexpressed chemo-resistant cells (P<0.05).3) The expression of E-cadherin in PC3-si-E-cadherin, DU145-si-E-cadherin cells were down-regulated, compared to PC3-si-control, DU145-si-control cells and PC3, DU145 cells. And there were no differences of the E-cadherin expression between PC3-si-control, DU145-si-control cells and PC3, DU145 cells. The expression of Vimentin and Snail were up-regulated in the E-cadherin silenced cells.4) Cell migration rate of in 48h were:DU145,56.5±1.4%. DU145-si-control,58.2±2.36%. DU145-si-E-cadherin,85.1±2.9%.The ability of migration in DU145-si-E-cadherin cells was stronger than DU145 cells (P<0.01). The abilities of colony formations were stronger in both E-cadherin silienced cells thanparental cells (P<0.01). The results of MTS showed that the IC50 in 72hourswere:PC3,9.49±0.89nmol/L. PC3-nc,9.71±2.38nmol/L. PC3-si-E-cadherin,14.73±1.58nmol/L. The tolerance to paclitaxel was enhanced in E-cadherin silenced PC3 cells (P<0.05). DU145,8.31±1.24nmol/L. DU145-nc, 8.77±2.40nmol/L. DU145-si-E-cadherin,17.03±1.54nmol/L. The tolerance to paclitaxel was enhanced in E-cadherin silenced DU145 cells (P<0.01).Conclusion:E-cadherin was closely related with EMT in PCa chemo-resistant cells and also correlated with invasion, metastasis, and clone formation ability in PCa cells and chemo-resistant cells; EMT occurring plays an important role in PCa cells paclitaxel resistance.Part 3. The roles of Notch signaling pathway on chemo-resistance of PCa cellsObjective:To investigate the relation between Notch-1 and EMT, and the role of Notch-1 in the chemo-resistance of PCa.Methods:1) The expression of Notch-1 in the E-cadherin overexpressed chemo-resistant prostate cells and E-cadherin silenced parental prostate cells were tested by Real-time PCR.2) The inhibition efficiency of the members of Notch signaling pathway family in chemo-resistant PCa cells, which were cause by y-secretase inhibitor (GSI) in different concentrations and times, were tested by Western-blot.3) The drug combination effection of paclitaxel and GSI were tested by MTS assay.Results:1) Compared with normal chemo-resistant PCa cells, Notch-1 expression was down-regulated in E-cadherin overexpressed PCa chemo-resistant cells; and was up-regulated in E-cadherin silenced parental PCa cells.2) Notch-1 expression were inhibited in both PC3-TxR and DU145-TxR cells after GSI was added (20μmol/L,72H). And Notch-4 expression was diminished in both PC3-TxR and DU145-TxR cells after GSI was added (20μmol/L,24H).3) The results of MTS showed that IC50 of the chemotherapeutic combination at 72hours were:PC3-TxR+GSI,13.9±1.59nmol/L. DU145-TxR+GSI,838.0± 134.4nmol/L. Compared with paclitaxel treated cells PC3-TxR,191.2±11.9 nmol/L. DU145-TxR,3957.8±100.8nmol/L. The GSI could significantly recover the sensitivity of paclitaxel for chemo-resistant prostate cells ((P<0.01).Conclusion:The expression of Notch-1 was correlated with EMT phenotypes in chemo-resistant PCa cells. GSI inhibited the expression of Notch-1 and Notch-4 in chemo-resistant PCa cells. Notch signaling pathway may play an important role in PCa cells chemo-resistance. |
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