WL-276, an antagonist against Bcl-2 proteins, overcomes drug resistance and suppresses prostate tumor growth

Abstract

Patients with hormone-refractory prostate cancer (HRPC) have an estimated median survival of only 10 months because of acquired drug resistance, urging the need to develop therapies against the drug-resistant HRPC phenotype. Accumulating evidence suggests that overexpressing antiapoptotic Bcl-2 family proteins is at least partially responsible for the development of drug resistance among HRPC patients. Antagonizing the antiapoptotic Bcl-2 family proteins, therefore, is one potential approach to circumventing drug resistance in HRPC. WL-276 was developed as a small-molecule antagonist against antiapoptotic Bcl-2 family proteins, with binding potency comparable to (-)-gossypol. Overexpressing Bcl-2 or Bcl-X(L) failed to confer resistance to WL-276. WL-276 also effectively induced apoptosis in PC-3 cells. In addition, three PC-3 cell lines with acquired drug resistance against standard cancer chemotherapies were more sensitive to WL-276 than the parent PC-3 cell line. The increased cytotoxicity toward drug-resistant PC-3 cells shows the clinical potential of WL-276 against HRPC that is resistant to conventional therapies. The anticancer activity of WL-276 was manifested in its suppression of PC-3-induced prostate tumor growth in vivo. The selective toxicity of WL-276 against drug-resistant PC-3 cells and its in vivo suppression of PC-3 prostate tumor growth suggest that WL-276 is a promising lead candidate for the development of Bcl-2 antagonists against drug-resistant HRPC.

Figure 1

Syntheses of WL-276.

Figure 2

WL-276 inhibiting the binding of a Bak BH3 domain peptide to Bcl-2 or Bcl-X_L protein. Analysis was carried out as described in Materials and Methods. In brief, Bcl-2 or Bcl-X_L recombinant protein was first mixed with a fluorescein-labeled Bak BH3 domain peptide to form a protein-peptide complex, which resulted in fluorescence polarization (FP) increase. Upon the addition of WL-276, FP was measured, which reflects the amount of remaining protein-peptide complex.

Figure 3

in vitro cytotoxicity and apoptotic induction by WL-276 in PC-3 cell line. A. Dose-dependent effect of WL-276 on the cell viability of PC-3. The cells were exposed to WL-276 at the indicated concentrations for 48 hours and cell viability was assessed by a CellTiter Blue Viability Kit. Points, mean; bars, SD (n=3). B, C, and D Evidence of apoptotic death in drug-treated PC-3 cells. B. Levels of caspase-3/-7 induced by different doses of WL-276. Columns, mean, normalized to untreated cell sample; bars, SD (n=3, * P < 0.05, ** P < 0.01, *** P < 0.001). PC-3 cells were treated with WL-276 at the indicated concentration for 24 hours and caspase-3/-7 activity was evaluated by Apo-ONE® Caspase-3/-7 reagent. C, DNA fragmentation in PC-3 cells upon WL-276 treatment. PC-3 cells were treated with WL-276 at the indicated concentration for 6 hours and DNA fragmentation was assessed by using Apoptotic DNA Ladder Extraction Kit. D, Cytochrome c release in PC-3 cells upon WL-276 treatment. PC-3 cells were treated with WL-276 at the indicated concentration for 24 hours and cytochrome c in cytosol was assessed by Western Blot analyses.

Figure 4

Sensitivity of various anticancer agents and WL-276 to Jurkat cells with varied level of Bcl-2 and Bcl-X_L. Jurkat cells were exposed to the drugs at the indicated concentrations for 24 hours and cell viability was assessed by a CellTiter Blue Viability Kit. Columns, mean, normalized to untreated cell sample; bars, SD (n=3). IC₅₀s for taxol and fas ligand were not obtained because these two agents are cytostatic resulting in incomplete growth inhibition even at the highest concentration tested.

Figure 5

A. Sensitivities of drug-resistant PC-3 cells against the corresponding anticancer agents used for the development of drug resistance. PC-3 cells were exposed the drugs at the indicated concentrations for 24 hours. Cell viability was assessed by a CellTiter Blue Viability Kit. Point, mean, normalized to untreated cell sample; bars, SD (n=3). B. Sensitivities of drug-resistant PC-3 cells against WL-276. PC-3 cells were exposed the drugs at the indicated concentrations for 24 hours. Cell viability was assessed by a CellTiter Blue Viability Kit. Columns, mean, normalized to untreated cell sample; bars, SD (n=3, * P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 6

Effect of p.o. WL-276 at 50 and 100 mg/kg on the growth of PC-3 tumors in nude mice. Each mouse was inoculated s.c. in the right flank with 2 x 10⁶ PC3-LN4 cells suspended in 0.2 mL PBS. Seventy-two hours later, mice were randomized into three groups (n = 8) and were given daily WL-276 at 50 and 100 mg/kg body weight per day with intraperitoneal saline for 12 days. Control mice received saline. A. Tumor volumes. Points, mean; bars, SE (n = 8; * < 0.05 compared with the control group). B. Average body weight changes during treatment (Points, mean; bars, SE). C. Immunobloting of PARP in the tumor tissues. Reactive protein bands were visualized by enhanced chemilumiescence detection system, and membrane was stripped and probed with β-actin as loading control. Densitometric analysis of PARP - intensity adjusted with β-actin and normalized to that of saline control. D. Serum levels of WL-276 in mice. Column, mean; bars, SE (n = 8; * P = 0.023 between the two treated groups).