Chemosensitizing Effect and Efficacy of Wilforlide A in Combination With Docetaxel in Drug-resistant Prostate Cancer

Materials and Methods

Materials. The six active components, celastrol, triptolide, pristimerin, triptonide, and demethylzeylasteral, were purchased from PI & PI Biotech Inc (Guangzhou, PR China) (Purity >95%, HPLC), while wilforlide A was obtained from the National Institutes for Food and Drug Control (Beijing, PR China) (Purity >95%, HPLC). Docetaxel was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The human androgen-independent prostate cancer cell line PC-3 and normal human prostate epithelial cells (HPrEpC) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The Dtx-resistant cell lines (PC3-TxR and DU145-TxR), that were established using a stepwise increase in exposure to Dtx were kindly provided by the Department of Medicine, University of Michigan. All the other solvents used were of HPLC grade purity or higher (VWR, Brisbane, CA, USA). Cell culture medium RPMI-1640, DMEM, fetal bovine serum (FBS), pyruvic acid, none-essential amino acids, penicillin-streptomycin, and 0.25% trypsin-EDTA solution were purchased from Invitrogen (Carlsbad, CA, USA).

Cytotoxicity of individual compounds. The cytotoxicity (IC₅₀) of each individual component and Dtx was determined in PC3 and DU145 and their corresponding drug resistant cell lines (PC3-TxR and DU145-TxR). The two cell lines were maintained in RPMI1640 medium supplemented with 10% FBS, 100 μM streptomycin and 100 units penicillin at 37°C in a 5% CO₂ humidified atmosphere. Upon reaching 80% confluence, the cells were trypsinized and seeded onto 96-well plates (3,000 cells/well) and incubated for another 24 h. The cells were then treated with 6 compounds at various concentrations for an additional 72 h (Dtx: 0.01, 0.1, 0.33, 1, 3.3, 10, 33, and 100 nM; pristimerin, triptolide, celastrol, and triptonide (0.1 to 100 ng/ml); WA (0.05 to 20 μg/ml); demethylzeylasteral (0.005 to 10 μg/ml).

The cell viability was measured using a proliferation assay with sulforhodamine B (SRB). The IC₅₀ was calculated using an Emax sigmoid model with the aid of GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

Chemosensitizing effect. The IC₅₀ of Dtx was determined for each combination of the herbal component with Dtx. The concentrations of each herbal component selected for the combination were 12.5, 25 or 50% of individual IC₅₀ values. The cytotoxicity of Dtx in combination with each herbal component was determined using a similar method as mentioned above. The drug combination effect of WA, the most chemosensitizing compound, was also determined in the corresponding sensitive cell line (PC3 and DU145) using the same method as above.

For all studies, the chemosensitizing effect (CE) was expressed as CE=IC_50D/IC_50HD, where IC_50D was the Dtx concentration alone that inhibited 50% proliferation, whereas IC_50HD was the Dtx concentration that inhibits 50% proliferation when used in combination with the herbal component.

Cytotoxicity of WA and Dtx in normal prostate cells. The cytotoxicity (IC₅₀) of WA, the best chemosensitizing component, was further determined in normal prostate and cancer cells. Normal prostate cells were cultured in prostate epithelial cell basal medium (PCS-440-030 from ATCC) supplemented with prostate epithelial cell growth kit (PCS-440-010 from ATCC) in a 5% CO₂ humidified atmosphere. Upon reaching 80% confluence, the cells were trypsinized and seeded onto 96-well plates (3,000 cells/well) and incubated for another 24 h. The cells were then treated with these compounds at various concentrations for additional 72 h (Dtx: 0.01, 0.1, 0.33, 1, 3.3, 10, 33, and 100 nM; WA: 0.0008, 0.002, 0.008, 0.02, 0.08, 0.2, 0.8, 2, 8, 20 μg/ml). Cell viability was measured and IC₅₀ calculated using the same method as mentioned above.

Identification of the potential mechanisms. PC3-TxR is a cell line known to overexpress P-glycoprotein (P-gp), which is associated with the mechanism of resistance to Dtx. Thus, this effect of WA on P-gp functional activity was investigated. The inhibition on P-gp activity was determined using a flow cytometry-based drug accumulation assay using a P-gp overexpressed cell line, K562/Dox. Daunorubicin (DNR), a well-known P-gp substrate with fluorescence, was used as the marker and PSC833 was used as a positive control (at a concentration of 2.5 mM). The K562/Dox cells were maintained by suspending culture in RMPI 1640 with 10% FBS and harvested by centrifugation (1,000 rpm for 5 min, room temperature). The cells were then re-suspended and adjusted to the cell density of 5×10³ cells/ml. The DNR accumulation was measured by incubating with DNR (5 μM) in the presence of various concentrations of WA (0, 0.675, 1.25, 2.5, and 5 μg/ml) for 2 h. Afterwards, the incubation was halted by washing with ice-cold PBS. The intra-cellular fluorescent intensity was measured using a Becton Dickinson FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with an Ultraviolet Argon laser (excitation at 488 nm, emission at 585/542 nm). The intracellular amount of DNR was expressed as the logarithm of fluorescent intensities of each treatment and normalized as the percentage of the positive control (PSC833, 2.5 mM).

Since we had previously identified several genes that contributed to the sensitizing effect of TW extract, including connective tissue growth factor (CTGF), cyclin E2 splice variant 1 mRNA (CCNE2) and Ankyrin repeat domain containing (ANKRD) (14), the effect of WA on these genes was investigated. The effect of WA on the expression level of these genes was determined using real-time PCR with a SYBR green protocol (Applied Biosystems 7300 Real-Time PCR System, Applied Biosystems, Carlsbad, CA, USA). The PC3-TxR cells were cultured and treated with 2.5 μg/ml WA for 6 and 24 h (n=3). The mRNA was then extracted, and the cDNA was obtained by reverse transcription. The gene expression was determined by using specific primers for each gene. The data were expressed as fold changes of these genes over the non-treated cells with GAPDH as the normalizing control.

In vivo efficacy study. Based on the results of the above in vitro studies, WA (the most active component) was selected for the in vivo study to investigate its antitumor effect in severe combined immunodeficiency (SCID) mice (Taconic Farms, Inc. Oxnard, CA, USA) implanted with Dtx-resistant tumor.

The dose and route of WA administration were based on our previous study (18). WA was found to be well tolerated in mice [oral dose: >30 mg/kg; intraperitoneal dose (i.p.): >6 mg/kg; intravenous dose (i.v.): >1.2 mg/kg] (18). However, the maximum concentration of WA following the 30 mg/kg oral dose was less than 30 ng/ml (which was far below the estimated effective concentration based on IC₅₀ determination). In view of the low oral bioavailability of WA (0.6%), i.p. and i.v. injections that produced sufficient in vivo plasma concentration in mice were used in the present efficacy study.

The study procedure was similar to our previous report (14). Male SCID mice (15 to 20 g, between 4-6 weeks old) were used. Before tumor implantation with PC3-TxR cells, they were housed in cages with HEPA-filtered air (12-h light/dark cycle) for one week after arrival at our animal vivarium. Then these cells were suspended in a 1:1 mixture of Matrigel (BC Biosciences, Franklin Lakes, NJ, USA) and RPMI 1640 (Mediatech, Manassas, VA, or Life Technologies, Grand Island, NY, USA) and subcutaneously implanted into planks of mice via injection. Mice that had consistently shown tumor growth for 14 days following the injection of cells were used in the efficacy studies. More specifically, the study was initiated when the tumors reached a size of about 120 mm³ (based on a formula for calculating semi-epplipoid: Volume=Width²×(Length/2).

The tumor-containing mice in the study were randomized into 7 groups with combination treatments consisting of high (H), medium (M), and low (L) dose WA as shown below (n=8). Since Dtx 20 mg/kg was effective in PC3 tumors (14), this dose was selected as the reference treatment. For WA, the i.p. dosing was chosen for convenient daily maintenance injection and the high dose (6 mg/kg) was chosen based on maximum solubility. This dose was expected to yield a WA plasma concentration of about 0.625 μg/ml (chemosensitizing concentration) based on our in vitro study.

• Group 1 (Control): no treatment;

• Group 2 (Dtx only): Dtx 20 mg/kg (i.v. once a week);

• Group 3 (Dtx+WA(H)): Dtx 20 mg/kg (i.v. once a week)+WA 1.2 mg/kg (i.v. once a week immediately after Dtx)+WA 6 mg/kg (i.p. once daily);

• Group 4 (Dtx+WA(M)): Dtx 20 mg/kg (i.v. once a week)+WA 0.6 mg/kg (i.v. once a week immediately after Dtx)+WA 3 mg/kg (i.p. once daily);

• Group 5 (Dtx+WA(L)): Dtx 20 mg/kg (i.v. once a week)+WA 0.3 mg/kg (i.v. once a week immediately after Dtx)+WA 1.5 mg/kg (i.p. once daily);

• Group 6 (WA(H)): WA 1.2 mg/kg (i.v. once a week)+WA 6 mg/kg (i.p. once daily);

• Group 7 (WA(L)): WA 0.3 mg/kg (i.v. once a week)+WA 1.5 mg/kg (i.p. once daily).

The changes in the tumor sizes of each mouse were measured over a 14-day time course, beginning on the first day of treatment.

Statistical analysis. All data from the study were expressed as mean±standard deviation (SD). The results among different groups were compared using a one-way analysis of variance (ANOVA), followed by the post-hoc Bonferroni test for multiple comparisons. The difference between the two independent groups was compared by the Student t-test. p<0.05 was considered statistically significant for all tests. All analyses were performed with the SPSS software (version 12.0; SPSS, Chicago, IL, USA).