Figure 4.

Selective anti-tumor activity of lead hits on highly aggressive, metastatic prostate cancer cells. (A) ED50 of agents for highly metastatic (PC-3MLN4 and AT6.1) and less metastatic (PC-3M) prostate cancer cells after 72 h treatment, as determined using Cyquant proliferation assay. Data shown are mean ± 95% confidence interval, N=3 per group. (B) Percent apoptosis was determined by annexin V-stained cells after 72 hr treatment with 1 μM drug. Data shown are mean ± 95% confidence interval, N=4 per group. PC-3MLN4 were significantly (p < 0.01) more apoptotic than PC-3M when treated with fenbendazole, fluspirilene, suloctidil, and niclosamide. (C) Drug-induced apoptosis in PC-3MLN4 was significantly (p < 0.05) reduced by treatment with 20 µM caspase-3 inhibitor (Z-VAD-FMK) for fenbendazole, fluspirilene, and niclosamide. (D) PC-3M and PC-3MLN4 subcutaneous tumors were treated with 100 mg/kg fenbendazole, three times a week for five weeks. Percent tumor growth was calculated from the tumor volume measured on first day of treatment. Data shown are mean ± 95% confidence interval, N=10 per group. Tumor growth was significantly (p < 0.01) more reduced in PC-3MLN4 treated with FBZ (44.9%) than in PC-3M treated with FBZ. (E–G) AT6.1 cells were inoculated via tail vein 5 days prior to treatment with 100 mg/kg fenbendazole, given 3 times a week until day 22 when signs of morbidity were observed in the control group. (E) Secreted Gaussia luciferase activity in the peripheral blood, indicative of tumor burden in the mice was measured on the day of harvest, p < 0.001. (F) Representative staining of Ki-67 and activated caspase-3 on tumors in the lungs. (G) Ki-67 labeling and apoptotic index was determined using Imagescope software (Aperio). N=10 mice/group, p < 0.001.