PC3 prostate cancer cell lines were obtained from ATCC. These cells are derived from a bone metastasis from a 62-year old prostate cancer patient. Human prostate fibroblasts, kindly provided by Dr J. Isaacs, were obtained from a prostate biopsy on a 62-year old prostate cancer patient with a Gleason score of 4. Both cell lines were grown and maintained in RPMI-1640 (Invitrogen) media supplemented with 10% fetal bovine serum. Cells were cultured in a humidified incubator at 37°C in a 5% CO₂ atmosphere.

Loss of CDK5 function was accomplished in PC3 cells by transfection of a dominant-negative construct containing a D144N mutation, kindly provided by Dr L.H. Tsai (Harvard Medical School) (18). The protocol used has been described previously (7). In brief, the construct was subcloned in a bidirectional Tet vector, pBI-EGFP (BD Biosciences), which had a zeocin resistance gene added for selection (kindly provided by Dr K. Schuebel, Johns Hopkins University School of Medicine). pBI-EGFP empty vector or pBI-EGFP CDK5dn vector was transfected into PC3 cells which contained a Tet-Off promoter construct, pTTa (BD Biosciences).

Western blotting was performed as described previously (19). Ten micrograms of protein was loaded on the gel. Primary antibodies were dissolved in blocking buffer [5% milk in TBST (100 mM Tris-HCl pH 7.4, 0.1% Tween-20, 150 mM NaCl in H₂O)]. A 1:1,000 dilution was used for anti- CDK5 (Sigma-Aldrich); anti-vinculin (Millipore, Upstate) was diluted 1:4,000. Secondary antibodies were diluted at a 1:4,000 dilution. Normalization of the band intensity was carried out with the housekeeper protein vinculin. Developed blots were scanned using a Microtek scanner.

Wound healing assays were performed with confluent PC3 control (containing the empty pBI-EGFP vector) or PC3 CDK5dn cells. A rubber-tipped scraper was used to scrape off an area of cells. Light microscopic images were captured immediately and 24 h after scraping.

The JHDL library has been described previously (13,14,17). Storage and screening of JHDL compounds were carried out as described previously (17). Briefly, PC3 control and CDK5dn cells were seeded in 96-well plates (1×10³ cells/well) and allowed to adhere overnight. Then 5 μl of drugs, stored as stock solutions of 200 μM in DMSO/H₂O, was added to complete RPMI media, so that cells were treated at a final concentration of 10 μM. After 48 h of treatment, 20 μl of MTS reagent from the CellTiter 96™ Aqueous Non-Radioactive Cell Proliferation Assay [a reagent containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS); Promega] was added to each well for a duration of 2–4 h at 37°C. Plates were analyzed using a SoftMax Pro plate reader (Molecular Devices). Proliferation of treated cells was compared with proliferation of DMSO-treated PC3 control or CDK5dn cells (proliferation index). Proliferation indices of PC3 CDK5dn cells were compared to the proliferation indices of PC3 control cells. A PubMed study was performed to assess the clinical use of potential hits.

MTS assays were performed to measure the antiproliferative effect of tilorone treatment. Tilorone dihydrochloride (Sigma-Aldrich) was stored as a 10 mM stock solution in DMSO at −20°C. One thousand PC3 cells were plated in 96-well plates containing 100 μl complete RPMI media. At circa 50% confluence, tilorone dihydrochloride was administered. For experiments the compound was diluted in complete RPMI media to obtain the desired final concentration. After treatment for 72 h (tilorone monotherapy), MTS reagent was added, and absorption at 490 nm was determined using a SoftMax Pro plate reader. Proliferation indices were calculated; untreated PC3 control or CDK5dn cells (in 103 μl complete RPMI media) were used as a control. Student’s t-tests were performed to assess p-values.

Clonogenic assays were performed to assess long-term survival after tilorone treatment. Prostate cancer cells were plated in 60 mm dishes and allowed to adhere. At 50–60% confluency, cells were treated with tilorone for 72 h. Subsequently, 1×10³ cells from each dish were plated in triplicate in 60-mm dishes and incubated in complete RPMI media for 12 days. Colonies were fixed and stained with a solution containing 90% methanol and 10% crystal violet solution (2.3% crystal violet, 0.1% ammonium oxalate and 20% ethyl alcohol; Sigma). Colonies were scanned with a computer scanner (Microtek) and counted manually. Student’s t-tests were performed to evaluate whether differences between cell lines were statistically significant.

3D growth assays were performed utilizing the same protocol as described previously (17). In short, spheroids were generated by culturing PC3 cells for 16 h as a hanging drop over a humidified plate in a CO₂ incubator in complete RPMI media containing 0.5% methylcellulose. Spheroids were embedded in collagen matrix (BD Biosciences), treated with tilorone, and imaged using a Nikon Eclipse Ti microscope (Nikon) on the day of treatment and six days after treatment start. Spheroid and total (spheroid plus sprouts) areas were measured with ImageJ. Fold increases were calculated by dividing the spheroid/total area at day 6 by the spheroid/total area on day 0 for each individual spheroid. For each cell line and time point, fold increases of four spheroids were averaged. Statistical analyses were performed using Student’s t-tests.

PC3 prostate cancer cells were chosen for the JHDL compound screen due to their highly metastatic potential and androgen independence, thereby resembling aggressive metastatic castrate-resistant prostate cancer. CDK5 activity was inhibited by transfection and selection of a dominant-negative mutation (CDK5 144N). These PC3 CDK5dn cells had a higher protein level of total CDK5 as compared to the PC3 control cells (PC3 cells transfected with an empty vector) (Fig. 1A). A wound healing assay (8) confirmed that CDK5 was functionally inactive in these cells; unlike the PC3 control cells, PC3 CDK5dn cells did not have the ability to invade the scraped surface area (Fig. 1B).

A high-throughput screening assay was performed to select compounds that target PC3 cells based on CDK5 activity. PC3 control and CDK5dn cells were treated with all compounds of the JHDL at 10 μM for 48 h. To identify hits that selectively target PC3 cells based on CDK5 expression, we selected all compounds in which the proliferation index ratio (CDK5dn/control) was below 0.5 or above 1.5 (Fig. 2A). Furthermore, hits had to inhibit cell proliferation of PC3 cells by at least 10%, as we were specifically interested in compounds that inhibited cell growth (horizontal and vertical line in graph). We also selected all compounds that inhibited cell proliferation in PC3 cells by 70% (bottom left corner of the graph), as we were interested in identifying potential highly effective antitumor agents. In total, 41 hits were selected for further evaluation.

A secondary screen was performed in which selected hits from the primary screen were added at 10 μM for 48 h to PC3 control and CDK5dn cells in triplicate, to weed out false positive results (Fig. 2B). Cutoff values were slightly less strict than in the primary screen; compounds were considered a hit when the ratio of proliferation indices (CDK5dn/control) was below 0.7 or above 1.4. This resulted in the identification of three compounds that selectively target CDK5-expressing PC3 cells: rutilantin, ethacridine lactate and cetalkonium chloride (Fig. 2C). These compounds have not been used as antitumor agents and their potential clinical use as intravenous antitumor agents seems limited (20–23). Another compound, tilorone analog R9536-DA, was highly effective in inhibiting both isogenic PC3 cell lines (>70% inhibition), but it inhibited proliferation of PC3 CDK5dn cells somewhat more effectively (ratio CDK5dn/control: 0.687). Tilorone and its analogs have antiviral activity, acting at least in part as interferon inducers (24–26) and have been shown preclinically and clinically to have antitumor activity as well (27,28).

We continued our experiments with freshly dissolved tilorone dihydrochloride. After 72 h of tilorone treatment at various concentrations, its IC₅₀ was established at 8–12 μM in PC3 CDK5dn cells and 15 μM in PC3 control cells in MTS assays (Fig. 3A). At 8 μM, proliferation activity was decreased by 24 and 47% in the PC3 control and CDK5dn cells, respectively (p=0.001). To assess toxicity of tilorone in normal prostate cells, MTS assays were performed with tilorone treatment of human prostate fibroblasts (Fig. 3B). Sensitivity of these cells to tilorone was similar to that of the PC3 control cells.

The inhibitory effect of tilorone in PC3 cells was further assessed by performing clonogenic assays (Fig. 3C). PC3 CDK5dn cells were also significantly more sensitive than PC3 control cells to tilorone in this assay. Treatment with 10 μM tilorone resulted in clonogenic survival of 40% in PC3 CDK5dn cells and 72% in PC3 control cells (p=0.002).

A spheroid growth assay was performed to assess 3D tumor growth and invasion of PC3 cells upon tilorone treatment (Fig. 4). Both PC3 control and PC3 CDK5dn cells had comparable increases in spheroid size over six days. However, total size (the size of spheroids plus sprouts) had a higher fold increase in PC3 control cells, confirming that untreated PC3 CDK5dn cells had a decreased invasive potential as compared to PC3 control cells. When tilorone was administered at 5 μM, PC3 control spheroids had a similar growth and invasive pattern as the untreated PC3 control cells (p=0.59) (Fig. 4B, left graph). However, when tilorone was administered at the same concentration to PC3 CDK5dn cells, a significant decrease in both spheroid size and total size was observed (p<0.01), suggesting that tilorone successfully inhibits spheroid growth and invasion of PC3 CDK5dn cells when administered at 5 μM (Fig. 4B, right graph). At 10 μM, both isogenic cell lines had a decreased invasive potential.