Docetaxel (Taxotere^®) and MK571 were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Pomolic acid (PA), provided by BioBioPha Co. Ltd. (Yunnan, China), was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM, stored at −20°C and diluted in culture medium for use. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). DMSO, 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), propidium iodide (PI) and KO 143 were obtained from Sigma Chemical Co./Merck KGaA (Darmstadt, Germany). Rhodamine 123 (Rho123) and 5-carboxyfluorescein diacetate (5-CFDA) were obtained from Calbiochem/EMD/Merck KGaA. Doxorubicin (cat. no. ab120629), vincristin (cat. no. ab120226), and anti-N-cadherin antibody (polyclonal rabbit, cat. no. ab12221) were from Abcam (Cambridge, MA, USA). Verapamil was obtained from Alexis Biochemicals (San Diego, CA, USA). Mitoxantrone (cat. no. sc-207888) and PE-labeled anti-MRP1 (QCRL-2) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-E-cadherin/CDH1 antibody (HECD-1) (monoclonal mouse, cat. no. 13-1700) was obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-vimentin antibody, clone SP20 (rabbit monoclonal, cat. no. VPRM17) and Vectashield mounting medium (cat. no. H-1000) were from Vector Laboratories (Burlingame, CA, USA). Anti-N-cadherin, clone SP90 (rabbit monoclonal) was obtained from Sigma-Aldrich/Merck KGaA. Goat anti-rabbit IgG, F(ab′)₂ fragment conjugated to cyanine Cy™3 (111-166-047), and goat anti-mouse IgG, F(ab′)₂ conjugated to cyanine Cy™3 (111-586-072) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

The human prostate cell line PC3 (ATCC, CRL-1435^TM) was grown in DMEM, supplemented with 10% heat-inactivated FBS, 100 U penicillin and 100 mg/ml streptomycin in disposable plastic bottles at 37°C in 5% CO₂. Cells were sub-cultured using trypsin-EDTA every 3–4 days. The docetaxel-resistant PC-3 cell line was developed by stepwise increased concentrations of docetaxel, essentially as previously described (22). PC3 cells were continuously maintained in docetaxel, with treatments beginning at 4 nM. After treatment, the surviving cells were re-seeded into new flasks and allowed to recover. The procedure was repeated until the cells displayed resistance to treatment as evaluated by cellular viability (MTT assay). New cycles were performed, in which the docetaxel concentration was subsequently increased up to a final concentration of 20 nM. Surviving cells were termed PC3R.

Cell viability was assessed using the MTT assay as previously described (15). Briefly, 180 ml of a PC3 or PC3R cell suspension (10⁴ cells/well) was distributed in 96-well plates and incubated for 24 h at 37°C/5% CO₂ to allow the culture to stabilize. The cells were then treated with 20 ml of medium, various concentrations of docetaxel (0.5, 1, 2, 4, 8, 12, 16, 20, 100, 500 and 1,000 nM); PA (1, 2.5, 5, 7.5, 10, 12.5, 15 µg/ml equivalent to 5.29, 10.57, 15.86, 21.15, 26.44, 31.73 µM); DMSO concentrations for each dose were used as controls. After 72 h of incubation, the culture was treated with 20 µl MTT (2.5 mg/ml) and maintained for 4 h at 37°C in the dark before the supernatant being discarded. The formazan produced by reduction of the MTT by viable cells was dissolved in DMSO and the optical density was measured in a multi-mode microplate reader (Synergy™ HT; BioTek Instruments, Inc., Winooski, VT, USA) at 570 nm (reference filter 630 nm). Experiments were repeated at least 3 times. The results are expressed as the mean ± SD of percent inhibition of cell viability.

Apoptosis was assessed by cell cycle analysis using flow cytometry as previously described (15). After 24 h of resting, the plated prostate cells (2×10⁴/well) were treated with media or various concentrations of pomolic acid (PA) (2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 µg/ml) and incubated for another 48 h. After this time, cells were harvested and re-suspended in a hypotonic fluorescent solution (50 µg/ml PI and 0.1% Triton X-100 in 0.1% Na citrate buffer) for 1 h, at 4°C in the dark. The cell cycle was analyzed by flow cytometry (FL-2) (FACSCalibur; Becton Dickinson, San Jose, CA, USA) to determine the sub-G0/G1 DNA content. Sub-diploid populations were considered to be apoptotic. Data acquisition and analysis were carried out by BD CellQuest software, version 3.1f (BD Biosciences, San Jose, CA, USA). Each experiment was repeated at least 3 times. The results are presented as representative histograms and as mean ± SD of the percentage of the DNA that was fragmented.

The functional activity of the MDR proteins was determined based on the intracellular accumulation of specific substrates as previously described (15). For each experiment, cells (1×10⁵/well) were seeded into 24-well plates and pre-incubated for 24 h at 37°C/5% CO₂ to allow stabilization of the culture. Plated cells were then incubated for 30 min with substrates specific for P-gp/ABCB1 (200 ng/ml Rho123), MRP1/ABCC1 (5 µM CFDA) or BCRP/ABCG2 (3 µM mitoxantrone) in the presence of medium or the conventional inhibitor of these proteins, verapamil (50 µM), MK571 (50 µM) and KO143 (40 µM). Then the cells were washed in phosphate-buffered saline (PBS), harvested and kept on ice until flow cytometric analysis (FACSCalibur; Beckton-Dickinson cytometer). In this condition, an increase in cellular fluorescence correlates with transport activity. Results are presented as representative histograms or as the mean ± SD of arbitrary units of mean fluorescence intensity (MFI).

To assess the effect of PA on the activity of P-gp/ABCB1 and MRP1/ABCC1, plated cells were incubated for 30 min with medium (autofluorescence), with substrates specific for each protein in the presence or absence of specific inhibitors or, with substrate plus the desired concentration of PA. After harvesting, the cells were analyzed as described above.

PC3 or PC3R cells (2×10⁴/well) were seeded on coverslips in 24-well plates, left to rest for 24 h and then treated with medium or 5 µg/ml of PA. After 48 h of incubation, the cells were washed twice with PBS, pH 7.4, and fixed with a 4% buffered paraformaldehyde solution containing 4% sucrose for 40 min at 4°C. After washing in PBS, the coverslips were permeabilized with PBS-0.5% Triton, pH 7.0 for 15 min, and then blocked with PBS-5% BSA-0.1% Triton-Tween 0.05% for 1 h. After this time, coverslips were incubated with the diluted primary antibodies (E-cadherin 1:50; N-cadherin: 1:100; vimentin 1:50) at 4°C overnight. Then the coverslips were washed in PBS-0.25% Tween and incubated with secondary antibodies [goat anti-rabbit IgG, F(ab′)₂ fragment-Cy™3 (1:200), and goat anti-mouse IgG, F(ab′)₂ -Cy™3 (1:200)] diluted in PBS pH 7.4 for 1 h. Next, the coverslips were washed 2 times with PBS-0.25% Tween and then stained with 0.5 mg/ml DAPI for 5 min. Following washes with PBS and distilled water, the coverslips were mounted with Vectashield medium and observed in an epifluorescence microscope (Eclipse E-800; Nikon Corp., Tokyo, Japan). The quantitative analysis was performed using an image analysis system (Image-Pro Plus 4.5; Media Cybernetics, Inc., Rockville, MD, USA) composed of a digital camera (Evolution; Media Cybernetics, Inc.) coupled to a fluorescence microscope. High quality images of cells were captured (2048×1536 pixels buffer), using a 40× objective lens. At least 20 fields/coverslip were captured. For vimentin quantification, since all cells were labelled, the percentage of immunoreactive cells was calculated from the DAPI-positive cells.

The results are presented as mean ± standard deviation (SD) of at least 3 independent experiments. Student's t-test was used to analyze comparisons. One-way analysis of variance (ANOVA) and Dunnett and Tukey multiple comparison test were used to analyze differences among groups. P<0.05 was considered to refer to statistically significant differences. Statistical analysis was performed using GraphPad Prism 5 software (version 5.01; GraphPad Software, Inc., La Jolla, CA, USA).

The MTT assay was used to evaluate the response of PC3 cells to docetaxel (DTX). Cells were treated with different concentrations of DTX and viability was assessed 72 h later. Fig. 1 (left panel) shows that 2 nM of DTX reduced PC3 viability by approximately 50%, demonstrating the sensitivity of the cell line to DTX. Establishment of a docetaxel-resistant PC3 cell line, PC3R, was accomplished by 4–5 cycles of treatment with stepwise increasing concentrations of the drug from 4 to 20 nM, as described in Materials and methods. Higher concentrations were avoided due to their toxicity. At this point, cells were treated with DTX in the same conditions used for the parental line and the cell viability was measured by MTT assay. Fig. 1 (right panel) shows that the treated cells (PC3R) were resistant to low concentrations of DTX, being responsive only to higher concentrations of the drug.

To ascertain whether the PC3R cell line presents with a multidrug resistance phenotype, parental and resistant cells were treated with doxorubicin or vincristin (Fig. 2) and the cell viability was assessed by MTT assay, 72 h later. The results showed an increase in resistance of the PC3R cells to both drugs corroborating the multidrug resistance phenotype of this cell line.

Extrusion of drugs by transporter proteins of the ABC family is one of the main mechanisms of drug resistance. In order to investigate the involvement of the transporter proteins in DTX resistance, we analyzed the activity of P-gp/ABCB1, MRP1/ABCC1 and BCRP/ABCG2 on both parental and resistant cells. For this, cells were incubated for 30 min with substrates for BCRP/ABCG2, MRP1/ABCC1 and P-gp/ABCB1 (mitoxantrone, CFDA or Rho123) in the presence or absence of inhibitors specific for each transporter (KO143, MK571 or verapamil), and the cell fluorescence was evaluated by flow cytometry. As shown in Fig. 3, while no P-gp/ABCB1 activity was observed in the parental cell line (Fig. 3A), a significant increase in activity (P<0.001) was observed in the DTX-resistant PC3R cell line (Fig. 3B). To evaluate the involvement of P-gp/ABCB1 activity in DTX resistance, cells were treated with 8 nM DTX in the presence or absence of the P-gp inhibitor verapamil (VP) and cell viability was measured by MTT assay 72 h later. The results (Fig. 3C) suggest that inhibition of P-gp activity reverts the resistance of the PC3R cell line to DTX.

Different from P-gp, both PC3 and PC3R cell lines displayed MRP1/ABCC1 activity (Fig. 4A and B). To test the involvement of this protein in DTX response, we measured the viability of the cells after 72 h treatment with 8 nM DTX in the presence or absence of the MRP1/ABCC1 inhibitor MK571. Although the co-treatment induced a significant decrease in cell viability of both lineages this response was more evident in the PC3R cells (Fig. 4C).

However, even when these transporters were inhibited, approximately 40% of the cells remained alive suggesting the involvement of other resistance mechanisms in PC3 and PC3R cells. These mechanisms are not mediated by BCRP/ABCG2 as a negligible activity of this transporter was observed in the parental and DTX-resistant cell lines (data not shown).

In an attempt to identify options for CRPC treatment, we evaluated the effects of PA on the PC3R cell line. For this, parental and resistant cell lines were incubated with different concentrations of the triterpene and the cell viability was assessed by MTT assay 72 h later. PA appeared to be more active then DTX against PC as it acted on the two lineages in a similar way decreasing their viability up to 90% (Fig. 5).

In order to investigate whether the decrease in cell viability was due to apoptosis, we analyzed the DNA content of the sub-G0/G1 population in the cell cycle after 48 h of PA treatment. As shown in Fig. 6, PA increased the DNA fragmentation of both lineages in a dose-dependent manner indicating that death was mediated by apoptosis.

Since inhibition of P-gp/ABCB1 or MRP1/ABCC1 increases the susceptibility of the resistant cell line to DTX, we analyzed whether the enhanced cytotoxic effect of PA would be due to an effect on these proteins. PC3 and PC3R cells were incubated with substrates of each protein in the presence or absence of inhibitors or 15 µg/ml PA and accumulation of the substrate was evaluated by cytometry. The results obtained demonstrated that PA-induced death in PC3R is independent of the increase in P-gp/ABCB1 since it does not interfere with the protein activity of the cell line (Fig. 7). Moreover, PA was able to modulate MRP1/ABCB1 activity in both parental and PC3R cell lines (Fig. 8), although less efficiently then the commercial inhibitor MK571.

Progression from the localized to invasive and metastatic tumors is partially dependent on a process called epithelial-mesenchymal-transition (EMT) in which the tumor epithelial cells loose expression of adhesion molecules such as E-cadherin and express proteins characteristic of mesenchymal cells such as vimentin (23). This process is also involved in the deregulation of androgenic signalization that occurs during the development of CRPC and confers resistance to apoptosis and stem cell like properties to tumoral cells (12).

Morphological analyses of the cells showed that DTX treatment changed the shape of the parental cells to a more elongated, fibroblastic appearance suggestive of EMT. Indeed, PC3R cells showed an increased amount of N-cadherin and vimentin (P<0.001), and a slight decreased amount of E-cadherin (P<0.01) compared with the PC3 parental cells suggesting an EMT phenotype in PC3R cells. Since EMT is a process involved in the progression of PC to CRPC and therefore, a possible target for the treatment of this tumor, to explore the mechanism of action of PA on PC, we evaluate the effects of this compound on various markers of EMT. The treatment of PC3R cells with 5 µg/ml PA for 48 h, decreased N-cadherin, vimentin, and E-cadherin levels in PC3R cells compared with non-treated PC3R cells (Fig. 9) indicating that the triterpene partially reverts the EMT phenotype observed in the resistant cell line.