Small-molecule screening of PC3 prostate cancer cells identifies tilorone dihydrochloride to selectively inhibit cell growth based on cyclin-dependent kinase 5 expression

Abstract

Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice.

Figure 1

Suppression of CDK5 activity by transfection of a dominant-negative construct. (A) PC3 cells were transfected with a pBI-EGFP vector with or without a CDK5 dominant-negative construct; clones in which transfection was successful were selected. A western blotting for CDK5 was performed with these PC3 CDK5dn and control cell lysates. (B) Wound healing assay with PC3 CDK5dn and control cells to confirm CDK5 inactivity in PC3 CDK5dn cells. CDK5, cyclin-dependent kinase 5.

Figure 2

Screening of the JHDL identifies four small molecules that target PC3 cells selectively based on CDK5 activity. (A) Primary screen with all 3,360 compounds of the JHDL, by performing MTS assays after 48 h of treatment with the compounds at 10 μM. Compounds were selected when the proliferation index ratio CDK5dn/control was below 0.5 or above 1.5. Compounds were excluded as a hit when not resulting in a decreased cell proliferation of 10% in one of the cell lines compared to DMSO-treated controls. Compounds inhibiting cell proliferation by >70% were also selected. (B) Secondary screen by performing MTS assays in triplicate with PC3 cells treated with 41 selected compounds from the primary screen. Compounds were selected when the proliferation index ratio CDK5dn/control was below 0.7 or above 1.4. (C) Four compounds selected from the secondary screen. Tilorone was highly effective against PC3 cells, particularly PC3 CDK5dn cells. The other three compounds primarily targeted PC3 cells with active CDK5. Error bars display the standard deviation of three independent measurements. CDK5, cyclin-dependent kinase 5; JHDL, Johns Hopkins Drug Library.

Figure 3

Validation of tilorone dihydrochloride as a compound that selectively targets PC3 cells based on CDK5 activity. (A) MTS assays performed after treating PC3 CDK5dn and control cells for 72 h with tilorone. (B) MTS assays performed after 72 h treatment of healthy human prostate fibroblasts with tilorone to determine toxicity of tilorone in healthy cells. (C) Clonogenic survival assays performed after a 72-h treatment of PC3 cells with tilorone, further validating the screening results. Error bars display the standard deviation from at least three independent measurements. CDK5, cyclin-dependent kinase 5.

Figure 4

3D hanging drop growth assay performed to assess 3D spheroid growth and invasive potential of PC3 cells upon tilorone treatment. (A) Representative images of PC3 control (left) and CDK5dn (right) cells at day 0 and day 6, either untreated or treated with 5 μM tilorone. (B) Fold increase in spheroid and total size after six days of tilorone treatment at various concentrations. Error bars display the standard deviation of four independent measurements. CDK5, cyclin-dependent kinase 5.