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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 inhibited prostate cancer cells proliferation, survival, and invasion and the anti-proliferative effect of SD-208 was mediated through the inhibition of PKD.A-B. SD-208 inhibited PC3 (A) and LNCaP (B) prostate cancer cell proliferation. PC3 and LNCaP cells were plated in triplicates in 24-well plates. Cells were allowed to attach overnight. A cell count at day 1 was made, and then either a vehicle (DMSO) or SD-208 at 30 μM was added. Cells were counted daily for a total of 6 days. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. C. SD-208 inhibited PC3 prostate cancer cell survival. PC3 cells were seeded into 96-well plates (3000 cells/well) and were then incubated in media containing 0.3–100 μM inhibitors for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The IC50 was determined as the mean of three independent experiments for each compound. D. SD-208 inhibited prostate cancer cell invasion. DU145 cells were incubated with 30 μM SD 208 in Matrigel inserts. After 20 h, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.4% hematoxylin solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 6 fields relative to the number of cells that migrated through the control insert. Percentage invasion was calculated as the percent of the cells invaded through Matrigel inserts vs. the total cells migrated through the control inserts. Data are the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ***, p<0.001. E-F. Overexpression of PKD1 and PKD3 in prostate cancer cells rescued the anti-proliferative effects of SD-208. PC3 (0.5 million) cells were seeded in a 60 mm dish and infected the next day with 50 and 100 MOI of PKD1 and PKD3 adenoviruses (Adv-PKD1 and Adv-PKD3). Empty adenovirus (Adv-) was used as control. After 24 h, 3000 cells/well were plated in 96-well plates and treated with and without 10 and 30 μM SD-208 for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis. This experiment was repeated three times and data are the mean ± S.E. of all three independent experiments. Statistical significance between DMSO and inhibitor treatment was determined using the unpaired t-test.*, p<0.05; **, p<0.01; ***, p<0.001. G. PKD mediated Hsp27 activity in prostate cancer cells was inhibited by SD-208. DU145 cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S910-PKD1 and p-S738/742-PKD1. GAPDH was blotted as loading control. The experiment was repeated two times and the representative blots are shown.
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pone.0119346.g004: SD-208 inhibited prostate cancer cells proliferation, survival, and invasion and the anti-proliferative effect of SD-208 was mediated through the inhibition of PKD.A-B. SD-208 inhibited PC3 (A) and LNCaP (B) prostate cancer cell proliferation. PC3 and LNCaP cells were plated in triplicates in 24-well plates. Cells were allowed to attach overnight. A cell count at day 1 was made, and then either a vehicle (DMSO) or SD-208 at 30 μM was added. Cells were counted daily for a total of 6 days. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. C. SD-208 inhibited PC3 prostate cancer cell survival. PC3 cells were seeded into 96-well plates (3000 cells/well) and were then incubated in media containing 0.3–100 μM inhibitors for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The IC50 was determined as the mean of three independent experiments for each compound. D. SD-208 inhibited prostate cancer cell invasion. DU145 cells were incubated with 30 μM SD 208 in Matrigel inserts. After 20 h, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.4% hematoxylin solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 6 fields relative to the number of cells that migrated through the control insert. Percentage invasion was calculated as the percent of the cells invaded through Matrigel inserts vs. the total cells migrated through the control inserts. Data are the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ***, p<0.001. E-F. Overexpression of PKD1 and PKD3 in prostate cancer cells rescued the anti-proliferative effects of SD-208. PC3 (0.5 million) cells were seeded in a 60 mm dish and infected the next day with 50 and 100 MOI of PKD1 and PKD3 adenoviruses (Adv-PKD1 and Adv-PKD3). Empty adenovirus (Adv-) was used as control. After 24 h, 3000 cells/well were plated in 96-well plates and treated with and without 10 and 30 μM SD-208 for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis. This experiment was repeated three times and data are the mean ± S.E. of all three independent experiments. Statistical significance between DMSO and inhibitor treatment was determined using the unpaired t-test.*, p<0.05; **, p<0.01; ***, p<0.001. G. PKD mediated Hsp27 activity in prostate cancer cells was inhibited by SD-208. DU145 cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S910-PKD1 and p-S738/742-PKD1. GAPDH was blotted as loading control. The experiment was repeated two times and the representative blots are shown.

Mentions: We next investigated the effects of targeted inhibition of PKD by SD-208 on prostate cancer cell proliferation, survival, and cell cycle progression. As shown in Fig. 4A-B, SD-208 at 30 μM caused significant reduction in PC3 prostate cancer cells proliferation starting at day 2 and persisted to the end of the experiment. SD-208 also concentration-dependently induced cell death with an IC50 of 17.0 ± 5.7 μM (n = 3) (Fig. 4C). The effect of SD-208 on tumor cell invasion was assessed using Matrigel invasion assay (cell invasion). As illustrated in Fig. 4D, treatment of cells with 30 μM SD-208 for 20 h resulted in over 60% inhibition of cell invasion compared with control, indicating that SD-208 significantly blocked tumor cell invasion. Taken together, our data demonstrates that SD-208 is a potent inhibitor of prostate cancer cell proliferation and invasion.


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 inhibited prostate cancer cells proliferation, survival, and invasion and the anti-proliferative effect of SD-208 was mediated through the inhibition of PKD.A-B. SD-208 inhibited PC3 (A) and LNCaP (B) prostate cancer cell proliferation. PC3 and LNCaP cells were plated in triplicates in 24-well plates. Cells were allowed to attach overnight. A cell count at day 1 was made, and then either a vehicle (DMSO) or SD-208 at 30 μM was added. Cells were counted daily for a total of 6 days. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. C. SD-208 inhibited PC3 prostate cancer cell survival. PC3 cells were seeded into 96-well plates (3000 cells/well) and were then incubated in media containing 0.3–100 μM inhibitors for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The IC50 was determined as the mean of three independent experiments for each compound. D. SD-208 inhibited prostate cancer cell invasion. DU145 cells were incubated with 30 μM SD 208 in Matrigel inserts. After 20 h, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.4% hematoxylin solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 6 fields relative to the number of cells that migrated through the control insert. Percentage invasion was calculated as the percent of the cells invaded through Matrigel inserts vs. the total cells migrated through the control inserts. Data are the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ***, p<0.001. E-F. Overexpression of PKD1 and PKD3 in prostate cancer cells rescued the anti-proliferative effects of SD-208. PC3 (0.5 million) cells were seeded in a 60 mm dish and infected the next day with 50 and 100 MOI of PKD1 and PKD3 adenoviruses (Adv-PKD1 and Adv-PKD3). Empty adenovirus (Adv-) was used as control. After 24 h, 3000 cells/well were plated in 96-well plates and treated with and without 10 and 30 μM SD-208 for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis. This experiment was repeated three times and data are the mean ± S.E. of all three independent experiments. Statistical significance between DMSO and inhibitor treatment was determined using the unpaired t-test.*, p<0.05; **, p<0.01; ***, p<0.001. G. PKD mediated Hsp27 activity in prostate cancer cells was inhibited by SD-208. DU145 cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S910-PKD1 and p-S738/742-PKD1. GAPDH was blotted as loading control. The experiment was repeated two times and the representative blots are shown.
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pone.0119346.g004: SD-208 inhibited prostate cancer cells proliferation, survival, and invasion and the anti-proliferative effect of SD-208 was mediated through the inhibition of PKD.A-B. SD-208 inhibited PC3 (A) and LNCaP (B) prostate cancer cell proliferation. PC3 and LNCaP cells were plated in triplicates in 24-well plates. Cells were allowed to attach overnight. A cell count at day 1 was made, and then either a vehicle (DMSO) or SD-208 at 30 μM was added. Cells were counted daily for a total of 6 days. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. C. SD-208 inhibited PC3 prostate cancer cell survival. PC3 cells were seeded into 96-well plates (3000 cells/well) and were then incubated in media containing 0.3–100 μM inhibitors for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The IC50 was determined as the mean of three independent experiments for each compound. D. SD-208 inhibited prostate cancer cell invasion. DU145 cells were incubated with 30 μM SD 208 in Matrigel inserts. After 20 h, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.4% hematoxylin solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 6 fields relative to the number of cells that migrated through the control insert. Percentage invasion was calculated as the percent of the cells invaded through Matrigel inserts vs. the total cells migrated through the control inserts. Data are the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ***, p<0.001. E-F. Overexpression of PKD1 and PKD3 in prostate cancer cells rescued the anti-proliferative effects of SD-208. PC3 (0.5 million) cells were seeded in a 60 mm dish and infected the next day with 50 and 100 MOI of PKD1 and PKD3 adenoviruses (Adv-PKD1 and Adv-PKD3). Empty adenovirus (Adv-) was used as control. After 24 h, 3000 cells/well were plated in 96-well plates and treated with and without 10 and 30 μM SD-208 for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis. This experiment was repeated three times and data are the mean ± S.E. of all three independent experiments. Statistical significance between DMSO and inhibitor treatment was determined using the unpaired t-test.*, p<0.05; **, p<0.01; ***, p<0.001. G. PKD mediated Hsp27 activity in prostate cancer cells was inhibited by SD-208. DU145 cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S910-PKD1 and p-S738/742-PKD1. GAPDH was blotted as loading control. The experiment was repeated two times and the representative blots are shown.
Mentions: We next investigated the effects of targeted inhibition of PKD by SD-208 on prostate cancer cell proliferation, survival, and cell cycle progression. As shown in Fig. 4A-B, SD-208 at 30 μM caused significant reduction in PC3 prostate cancer cells proliferation starting at day 2 and persisted to the end of the experiment. SD-208 also concentration-dependently induced cell death with an IC50 of 17.0 ± 5.7 μM (n = 3) (Fig. 4C). The effect of SD-208 on tumor cell invasion was assessed using Matrigel invasion assay (cell invasion). As illustrated in Fig. 4D, treatment of cells with 30 μM SD-208 for 20 h resulted in over 60% inhibition of cell invasion compared with control, indicating that SD-208 significantly blocked tumor cell invasion. Taken together, our data demonstrates that SD-208 is a potent inhibitor of prostate cancer cell proliferation and invasion.

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