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SD-208, a novel protein kinase D inhibitor, blocks prostate cancer cell proliferation and tumor growth in vivo by inducing G2/M cell cycle arrest.

Tandon M, Salamoun JM, Carder EJ, Farber E, Xu S, Deng F, Tang H, Wipf P, Wang QJ - PLoS ONE (2015)

Bottom Line: Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3.Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL.Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, United States of America.

ABSTRACT
Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.

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SD-208 arrested cells in G2/M and regulated the levels and activities of cell cycle regulatory proteins at the G2/M phase of cell cycle in prostate cancer cells.A-B. SD-208 induced G2/M cell cycle arrest in prostate cancer cells. DU145 cells (A) and PC3 cells (B) were treated with either vehicle (DMSO) or 30 μM SD 208 for 48 h. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. The cell cycle plots are representative of three independent experiments (left). Data in the bar graph are the mean ± SEM of three independent experiments (right). Statistical significance was determined using the unpaired t-test and is indicated. **, p<0.01; ***, p<0.001. C-D. Effects of SD-208 on the expression and activities of G2/M cell cycle regulatory proteins. DU145 (C) and PC3 (D) cells were treated with DMSO or 5–30 µM SD-208 for 24 and 48 h. At the end of each treatment, cells were harvested and subjected to immunoblotting for the cell cycle regulatory proteins as indicated. GAPDH was blotted as loading control. The densitometry data (mean ± SEM from four experiments) were plotted as ‘fold change’ over the control after normalization with GAPDH. The experiments were repeated four times and representative blots from one experiment are shown.
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pone.0119346.g005: SD-208 arrested cells in G2/M and regulated the levels and activities of cell cycle regulatory proteins at the G2/M phase of cell cycle in prostate cancer cells.A-B. SD-208 induced G2/M cell cycle arrest in prostate cancer cells. DU145 cells (A) and PC3 cells (B) were treated with either vehicle (DMSO) or 30 μM SD 208 for 48 h. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. The cell cycle plots are representative of three independent experiments (left). Data in the bar graph are the mean ± SEM of three independent experiments (right). Statistical significance was determined using the unpaired t-test and is indicated. **, p<0.01; ***, p<0.001. C-D. Effects of SD-208 on the expression and activities of G2/M cell cycle regulatory proteins. DU145 (C) and PC3 (D) cells were treated with DMSO or 5–30 µM SD-208 for 24 and 48 h. At the end of each treatment, cells were harvested and subjected to immunoblotting for the cell cycle regulatory proteins as indicated. GAPDH was blotted as loading control. The densitometry data (mean ± SEM from four experiments) were plotted as ‘fold change’ over the control after normalization with GAPDH. The experiments were repeated four times and representative blots from one experiment are shown.

Mentions: To gain insights in SD-208-induced growth inhibition, we determined the effect of SD-208 on cell cycle progression by flow cytometry. As shown in Fig. 5A-B, cell cycle analysis was conducted after treating DU145 and PC3 cells with 30 μM of SD-208 for 72 h. SD-208 hampered cell cycle progression by arresting the cells in G2/M phase of the cell cycle. Specifically, SD-208 significantly increased the proportion of cells in G2/M phase of the cell cycle from 18.78% (vehicle treated cells) to 30.09% in DU145 cells and 6.3% (vehicle treated cells) to 35.6% (30 µM SD-208 treated cells) in PC3 cells, implying that the growth inhibition caused by SD-208 in prostate cancer cells is a consequence of an arrest of cells in G2/M phase of cell cycle.


SD-208, a novel protein kinase D inhibitor, blocks prostate cancer cell proliferation and tumor growth in vivo by inducing G2/M cell cycle arrest.

Tandon M, Salamoun JM, Carder EJ, Farber E, Xu S, Deng F, Tang H, Wipf P, Wang QJ - PLoS ONE (2015)

SD-208 arrested cells in G2/M and regulated the levels and activities of cell cycle regulatory proteins at the G2/M phase of cell cycle in prostate cancer cells.A-B. SD-208 induced G2/M cell cycle arrest in prostate cancer cells. DU145 cells (A) and PC3 cells (B) were treated with either vehicle (DMSO) or 30 μM SD 208 for 48 h. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. The cell cycle plots are representative of three independent experiments (left). Data in the bar graph are the mean ± SEM of three independent experiments (right). Statistical significance was determined using the unpaired t-test and is indicated. **, p<0.01; ***, p<0.001. C-D. Effects of SD-208 on the expression and activities of G2/M cell cycle regulatory proteins. DU145 (C) and PC3 (D) cells were treated with DMSO or 5–30 µM SD-208 for 24 and 48 h. At the end of each treatment, cells were harvested and subjected to immunoblotting for the cell cycle regulatory proteins as indicated. GAPDH was blotted as loading control. The densitometry data (mean ± SEM from four experiments) were plotted as ‘fold change’ over the control after normalization with GAPDH. The experiments were repeated four times and representative blots from one experiment are shown.
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pone.0119346.g005: SD-208 arrested cells in G2/M and regulated the levels and activities of cell cycle regulatory proteins at the G2/M phase of cell cycle in prostate cancer cells.A-B. SD-208 induced G2/M cell cycle arrest in prostate cancer cells. DU145 cells (A) and PC3 cells (B) were treated with either vehicle (DMSO) or 30 μM SD 208 for 48 h. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. The cell cycle plots are representative of three independent experiments (left). Data in the bar graph are the mean ± SEM of three independent experiments (right). Statistical significance was determined using the unpaired t-test and is indicated. **, p<0.01; ***, p<0.001. C-D. Effects of SD-208 on the expression and activities of G2/M cell cycle regulatory proteins. DU145 (C) and PC3 (D) cells were treated with DMSO or 5–30 µM SD-208 for 24 and 48 h. At the end of each treatment, cells were harvested and subjected to immunoblotting for the cell cycle regulatory proteins as indicated. GAPDH was blotted as loading control. The densitometry data (mean ± SEM from four experiments) were plotted as ‘fold change’ over the control after normalization with GAPDH. The experiments were repeated four times and representative blots from one experiment are shown.
Mentions: To gain insights in SD-208-induced growth inhibition, we determined the effect of SD-208 on cell cycle progression by flow cytometry. As shown in Fig. 5A-B, cell cycle analysis was conducted after treating DU145 and PC3 cells with 30 μM of SD-208 for 72 h. SD-208 hampered cell cycle progression by arresting the cells in G2/M phase of the cell cycle. Specifically, SD-208 significantly increased the proportion of cells in G2/M phase of the cell cycle from 18.78% (vehicle treated cells) to 30.09% in DU145 cells and 6.3% (vehicle treated cells) to 35.6% (30 µM SD-208 treated cells) in PC3 cells, implying that the growth inhibition caused by SD-208 in prostate cancer cells is a consequence of an arrest of cells in G2/M phase of cell cycle.

Bottom Line: Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3.Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL.Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, United States of America.

ABSTRACT
Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.

Show MeSH
Related in: MedlinePlus