Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

Abstract

Background: There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel.

Results: The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel.

Conclusion: This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

Trial registration: ClinicalTrials.gov NCT00571675.

Figures

Figure 1

Effects of Docetaxel on apoptosis and proliferation in the PC-3, DU-145 and 22RV1 Docetaxel resistant sublines. A. The PC-3 Ag, PC-3 D8, PC-3 D12, DU-145 Ag, DU-145 R, 22RV1 Ag and 22RV1 R cell lines were treated with Docetaxel (20 and 80 nM) for 48 hrs. Apoptosis was assessed by propidium iodide DNA staining and flow cytometry. Values are expressed as mean ± standard deviation. An independent samples t-test was used to compare the mean percentage apoptosis for control, 20 nM and 80 nM for each cell line. **p < 0.001. B. Cell proliferation was measured using the MTT Assay. The PC-3 sublines (PC-3 Ag, PC-3 D8 and PC-3 D12) were treated with increasing doses of Docetaxel (10, 20, 50 & 100 nM) for 24, 48 and 72 hrs. Data shown is representative of three independent experiments.

Figure 2

P-gp expression levels in the Doctaxel resistant cell lines. A. Total cellular protein was extracted from the cells and 50 μg was assessed by western blotting. P-gp was not expressed in the PC-3 Ag, PC-3 D8, PC-3 D12 sublines or the DLKP (40 μg) negative control cell line, but was expressed in the DLKP-A (5 μg) drug resistant variant of the DLKP cell line. β-actin the house keeping protein was used as loading control for P-Gp. B. The PC-3 Ag, PC-3 D12 and NCI-ADR/RES cells lines were treated with Docetaxel (20 nM) alone or in the presence of the P-gylcoprotein inhibitor Elacridar (0.25 and 0.5 μM) and Docetaxel (20 nM) and assessed for apoptosis by flow cytometry. An independent samples t-test was used to compare the average percentage of apoptosis between the control groups and each of the treated groups for the PC-3 Ag, PC-3 D12 and NCI-ADR/RES cell lines *p- < 0.05. C. Total cellular protein was extracted from the cells and 50 μg was assessed by western blotting. P-Gp was weakly expressed in the DU-145 R and strongly expressed in the 22RV1 R sublines. No expression was seen in the Aged matched. β-actin the house keeping protein was used as loading control for p-glycoprotein. D. The DU-145 Ag, DU-145 R and 22RV1 Ag and 22RV1 R cells lines were treated with Docetaxel (20 nM) alone, or the P-Gp inhibitor Elacridar (0.25 μM) alone or in the presence of Elacridar (0.25 μM) and Docetaxel (20 nM) and assessed for apoptosis by flow cytometry. Blots shown are representative of three independent experiments.

Figure 3

Cellular sensecence in response to Docetaxel treatment. The PC-3 Ag, PC-3 D8 and PC-3 D12 cells were plated in 6 well plates at 300,000/well and grown for 24 hours. The cells were then treated with Docetaxel (100 nM) for 48 hours and fixed with 2% formaldehyde and 0.2% glutaraldehyde. Senescent cells were then assessed by staining cells for β-galactosidase expression. Positive cells were counted and expressed as a percentage of the total number of cells in the well. Pictures are a representation of 5 independent experiments.

Figure 4

Autophagy as a mechanisms of Docetaxel resistance. Following treatment of the Aged matched controls (Upper panel) and PC-3 D12 resistant sublines (Bottom panel) with 50 nM of Docetaxel for 1, 3, 6, 12 and 24 hrs, total cellular protein was extracted and assessed for LC3II by western blotting using the relevant anti-body and conditions as described in the methods section. For positive controls cells were either treated with Rapamycin (R, 0.2 μM) for 4 hrs, Starvation (S, the cells were starved in EBSS (Earls Balanced Salt Solution) medium for 2 hrs), or Starvation plus Bafilomycin (S+B, 20 nM) for 2 hrs. β-actin the house keeping protein was used as loading control. Blots shown are representative of three independent experiments.

Figure 5

Validation of Low Density Arrays. Total cellular protein was extracted from the PC-3 sublines (PC-3 Ag, PC-3 D8 & PC-3 D12) and 50 μg was loaded onto a western blot to assess the expression of Clusterin, HSP90, Id-1, Bcl-2, Bcl-xL, Bax, and Bid using their respective antibodies as described in the Method section. The house keeping protein β-actin was used as a loading control. Blots shown are representative of three independent experiments.

Figure 6

NF-κB transcriptional activity and Baseline NFkB subunit activity. A. PC-3 Ag and PC-3 D12 cells were plated at 100,000/per well and grown for 24 hours. The baseline NFκB activity was assessed by luciferase assay, using a Dual Luciferase Assay from Promega following the manufactures instructions. Averages from 3 independent experiments are shown. Mean values were compared using t-test assuming equal variances. B. The PC-3 Ag and PC-3 D12 cells were grown to confluency and nuclear extracts prepared as per the TransAM™ Assay Kit manual. Averages from 3 independent experiments are shown. Mean values were compared using t-test assuming equal variances.

Figure 7

NF-κB transcriptional activity following docetaxel treatment. A. The PC-3 and DU-145 aged matched and Docetaxel resistant sublines were treated with 50 uM concentrations of Docetaxel for 3, 6 and 24 hours at which time NFκB activity was assessed by luciferase assay, using a Dual Luciferase Assay from Promega following the manufactures instructions. Graphs are generated from 3 independent experiments. Mean values were compared using t-test assuming equal variances. * p < 0.01; ** p < 0.0001. B. Cells were treated as above and at the different time points total cellular protein was extracted and 50 ug loaded onto western blots and assessed for P-IkBα and IkBα. β-actin was used as an additional loading control. Blots are representative of three independent experiments.

Figure 8

NF-κB inhibitor studies following docetaxel treatment. The PC-3 sublines where pre-treated with the NFκB inhibitor (BAY 11-7082, 5 μM) for 24 hours followed by Docetaxel (20 nM) for a further 48 hours and assessed for apoptosis by flow cytometry. Graphs represent the results of 3 independent experiments. Mean values were compared using t-test assuming equal variances. * p < 0.05.