Abstract 2976: Small molecule screening of prostate and pancreatic cancer cell lines identifies tilorone dihydrochloride as a compound that selectively inhibits growth in cells with inhibited cyclin-dependent kinase 5

Abstract

Abstract Cyclin-dependent kinase 5 (CDK5) has long been known to be an enzyme necessary for neuronal migration. In recent years, extraneural functions of CDK5 have been discovered, including CDK5 playing an essential role in invasion and migration in pancreatic and prostate cancer, and in tumor growth and Ras signaling through Ral in pancreatic cancer. This makes CDK5 an interesting target against cancer. We blocked CDK5 activity in prostate cancer cell line PC3 by dominant-negative constructs, and used a library consisting of 3360 compounds to screen for small molecules that differentially inhibit proliferation activity in wildtype PC3 (PC3wt) and CDK5 dominant-negative PC3 (PC3cdn) cells, by performing MTS assays. We selected for compounds in which the difference in proliferation activity between the two cell lines was more than 40% and/or compounds that inhibited both cell lines by more than 50%. With this selection, 32 compounds were identified. Topically acting compounds were excluded, the other compounds were sorted for difference in proliferation activity inhibition between the two cell lines, and a Pubmed search was performed on the compounds with the largest difference. This way, tilorone analog R 9536-DA was selected as the most potent compound, as it inhibited proliferation in PC3wt and PC3cdn cells by 72.5% (SD 0.5%) and 81.1% (SD 2.1%) respectively (a difference in proliferation activity of 32%). MTS and clonogenic assays confirmed the differential sensitivity of PC3wt and PC3cdn cells to tilorone dihydrochloride. The IC50 in PC3wt cells was around 16 μM, while in PC3cdn cells it was around 10 μM. This difference in sensitivity to tilorone dihydrochloride in the presence of CDK5 inhibition was confirmed in pancreatic and other prostate cancer cell lines as well. To examine the toxicity of tilorone dihydrochloride in normal cells, the compound was administered to normal human prostate fibroblasts, in which the IC50 was found to be higher than 20μM. Western blots were performed to investigate the mechanism behind this difference in sensitivity between cell lines. These experiments conclude that administration of tilorone dihydrochloride differentially induces PARP and the p38-MAPK signaling pathway, indicating the involvement of this pathway in tilorone induced cell death. Tilorone analogs previously have been shown in mice to inhibit metastasis and increase survival in several tumor types. This study suggests that the combination of tilorone and inhibition of CDK5 may be a promising therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2976. doi:10.1158/1538-7445.AM2011-2976