Effects of imidazopyridine derivatives on the tumorigenicity of LNCaP C-81 cells.

<p><b>(A)</b> Clonogenic assay on plastic wares. LNCaP C-81 cells were plated in six-well plates at densities of 20, 200, and 2,000 cells/well. After 24 hours, attached cells were treated with respective compounds at 10 μM concentrations of imidazopyridine derivatives or solvent alone as control. Cells were fed on days 3, 6, and 9 with fresh culture media containing respective inhibitors. On day 10, cells were stained and the number of colonies counted. The photos of representative colony plates were taken from plates seeded with 2,000cells/well, and the number of colonies shown was counted also from plates seeded with 2,000cells/well. Minimal colony formation was observed at densities of 20 and 200 cells/well. Results presented are mean ± SE; n = 2x3. ***<i>p</i><0.0001. <b>(B)</b> Anchorage-independent soft agar assay. LNCaP C-81 cells were plated at a density of 5 x 10⁴ cells/35mm dish in 0.25% soft agar plates. The following day, cells in doublets or greater were marked and excluded from the study. Media were added every three days, and at the end of 5 weeks, colonies formed were stained and counted. Representative images of colonies are shown (above) and the colony number was counted (below). The experiments were performed in duplicate with 3 sets of independent experiments. Results presented are mean ± SE; n = 2x3. *** p<0.0001. <b>(C)</b>. Cell adhesion assay on plastic wares. Cells were suspended in treatment media for 30 minutes before being plated in 6-well plates at 3 x10³ cells/cm² using the same treatment media. Cells were allowed to adhere for one hour, fixed and stained by 0.2% crystal violet solution (50:50, water:MeOH). The total number of cells in five fields at 40x magnification for each well was counted. The experiments were performed in triplicate with 3 sets of independent experiments. Results presented are mean ± SE; n = 3x3. *<i>p</i><0.05; **<i>p</i><0.01. <b>(D)</b>. Cell migration transwell assay. Cell migration was assessed via Boyden chamber. An aliquot of 5 x 10⁴ C-81 cells was seeded in the insert of 24-well plates in media containing 10 μM respective compounds with solvent alone for control in both upper and lower chambers. After 24-hour incubation, the migrated cells were stained and those cells remaining in the upper chamber were removed via cotton swab. Cells which had migrated through to the lower chamber were counted. Representative images are shown at 40x magnification. The experiments were performed in triplicate with 3 sets of independent experiments, and the results presented are mean ± SE; n = 3x3, *<i>p</i><0.05; **<i>p</i><0.005.</p

Novel Imidazopyridine Derivatives Possess Anti-Tumor Effect on Human Castration-Resistant Prostate Cancer Cells

<div><p>Prostate cancer (PCa) is the second leading cause of cancer-related death afflicting United States males. Most treatments to-date for metastatic PCa include androgen-deprivation therapy and second-generation anti-androgens such as abiraterone acetate and enzalutamide. However, a majority of patients eventually develop resistance to these therapies and relapse into the lethal, castration-resistant form of PCa to which no adequate treatment option remains. Hence, there is an immediate need to develop effective therapeutic agents toward this patient population. Imidazopyridines have recently been shown to possess Akt kinase inhibitory activity; thus in this study, we investigated the inhibitory effect of novel imidazopyridine derivatives HIMP, M-MeI, OMP, and EtOP on different human castration-resistant PCa cells. Among these compounds, HIMP and M-MeI were found to possess selective dose- and time-dependent growth inhibition: they reduced castration-resistant PCa cell proliferation and spared benign prostate epithelial cells. Using LNCaP C-81 cells as the model system, these compounds also reduced colony formation as well as cell adhesion and migration, and M-MeI was the most potent in all studies. Further investigation revealed that while HIMP primarily inhibits PCa cell growth via suppression of PI3K/Akt signaling pathway, M-MeI can inhibit both PI3K/Akt and androgen receptor pathways and arrest cell growth in the G2 phase. Thus, our results indicate the novel compound M-MeI to be a promising candidate for castration-resistant PCa therapy, and future studies investigating the mechanism of imidazopyridine inhibition may aid to the development of effective anti-PCa agents.</p></div

Effects of imidazopyridine derivatives on PCa proliferative and apoptotic signaling under SR conditions.

<p><b>(A)</b> LNCaP C-81 cells were plated in triplicate in T25 flasks at 4 x 10³ cells/cm² in regular medium, grown for 72 hours then steroid starved for 48 hours. Cells were then treated with 10 μM imidazopyridine derivatives or DMSO as control for an additional 72 hours under SR conditions. Cells were trypsinized and live cell numbers counted via Trypan Blue assay. The experiments were performed in triplicate with 3 sets of independent experiments. *** <i>p</i><0.0005. <b>(B)</b> MDA PCa2b-AI cells were grown, treated, and counted under conditions as described above in (A) for LNCaP C-81 cells. Results presented are mean ± SE; n = 3x3. *<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.0005. <b>(C)</b> LNCaP C-81 total cell lysate proteins were collected from (A) after cell number counting. Those cells were grown in SR conditions and analyzed for phosphorylated Akt and STAT5, as well as total AR, Akt, Shc, p53, cyclin B₁, cyclin D₁, PCNA, Bcl_XL, and Survivin protein levels. β-actin protein level was used as a loading control. Similar results were observed in two sets of independent experiments. <b>(D)</b> MDA PCa2b-AI total cell lysate proteins from (B) after cell number counting. Cells were grown in SR conditions and analyzed for phosphorylated STAT5, as well as total AR, Shc, p53, cyclin B₁, cyclin D₁, PCNA, Bcl_XL, and Survivin protein levels. β-actin protein level was used as a loading control. Similar results were observed in three sets of independent experiments. <b>(E)</b> MDA PCa2b-AI total cell lysate proteins from cells grown in regular conditions were analyzed for phosphorylated Akt and total Akt. β-actin protein level was used as a loading control. Similar results were observed in three sets of independent experiments.</p

Kinetic analysis of HIMP and M-MeI’s effects on LNCaP C-81 cells under steroid-deprived conditions.

<p><b>(A)</b> Cells were plated in six-well plates at 2 x 10³ cells/cm² in regular medium for three days, then steroid-starved for 48 hours followed by treatment with 10 μM HIMP or M-MeI in SR medium containing 1 nM DHT. Solvent alone was used for controls. On day 0, 1, 3, 5 and 7, one set of cells in duplicates from each group was harvested for live cell counting. Remaining cells were replenished with fresh respective medium. The experiments were performed in duplicates with 3 sets of independent experiments. Results presented are mean ± SE; n = 2x3. *<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.0005. <b>(B)</b> Total cell lysate proteins from HIMP- and M-MeI-treated C-81 cells from (A) were collected and analyzed for AR, cPSA, cyclin B₁, PCNA, Bax, p53, and Bcl-X_L proteins. β-actin protein level was used as a loading control. <b>(C)</b> Histograms of cell cycle distributions of LNCaP C-81 cells upon 7 days of HIMP and M-MeI treatments. Cells were plated in T25 flasks at 2 x 10³ cells/cm² in regular medium for three days, then steroid-starved for 48 hours followed by treatment with 10 μM HIMP or M-MeI in SR medium with 1 nM DHT and solvent DMSO alone as control. One set of cells from each group was harvested after 3, 5, and 7 days treatment for flow cytometric analysis. Similar results were obtained from two sets of independent experiments. The data shown were representative results of 7-day treatments.</p

Effects of imidazopyridine derivatives on the proliferation of prostate epithelial cells.

<p><b>(A)</b> Dosage effect of Imidazopyridine derivatives on LNCaP C-81 cells. Cells were plated in six-well plates at 2 x 10³ cells/cm² in regular medium and grown for 72 hours. One set of cells was then fed with fresh regular medium containing 0,1,5, or 10 μM imidazopyridine derivatives with solvent alone for control and grown for an additional 72 hours. Another set of cells was first steroid starved in SR medium for 48 hours then treated with respective compounds in fresh SR media containing 1 nM DHT for 72 hours. All cells were trypsinized and live cell numbers were counted. The experiment was conducted in duplicate wells with 3 sets of independent experiments. The results presented are mean ± SE; n = 2x3. *<i>p</i><0.05 **<i>p</i><0.005 ***<i>p</i><0.0005. <b>(B)</b> Effects of imidazopyridine derivatives on the growth of various PCa cells and immortalized prostate epithelial cells. All cells were plated in six-well plates at the noted density in their respective medium for three days, steroid-starved for two days, then fed with fresh SR medium with 1 nM DHT containing 10 μM imidazopyridine derivatives and grown for three additional days. Cells were trypsinized and live cell number was counted. LNCaP C-81–2 x 10³ cells/cm², MDA PCab2b AI—3 x 10³ cells/cm², PC-3–2 x 10³ cells/cm², RWPEI– 7.5 x10³ cells/cm². All experiments were performed in triplicate wells with 3 sets of independent experiments. Results presented are mean ± SE; n = 3x3. *<i>p</i><0.05; **<i>p</i><0.001; ***<i>p</i><0.0001.</p