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Combinatorial antitumor effect of HDAC and the PI3K-Akt-mTOR pathway inhibition in a Pten defecient model of prostate cancer.

Ellis L, Ku SY, Ramakrishnan S, Lasorsa E, Azabdaftari G, Godoy A, Pili R - Oncotarget (2013)

Bottom Line: In this study we utilized the AR negative PCa cell line and observed that re-expression of AR (PC3-AR) results in greater levels of apoptosis when treated with the pan-DACi, panobinostat (PAN).Combination of PAN with BEZ235 resulted in significant attenuation of the DNA damage repair protein ATM and significantly increased anti-tumor activity compared to each single treatment.Overall, superior anti-tumor activity with combination of PAN with BEZ235 was independent of AR status.

View Article: PubMed Central - PubMed

Affiliation: Genitourinary Program, Roswell Park Cancer Institute, Buffalo NY.

ABSTRACT
Increased expression of histone deacetylases (HDACs) and activation of the PI3K-Akt-mTORC1 pathway are common aberrations in prostate cancer (PCa). For this reason, inhibition of such targets is an exciting avenue for the development of novel therapeutic strategies to treat patients with advanced PCa. Previous reports demonstrated that HDAC inhibitors (HDACi) increases DNA damage and induce greater apoptosis in PCa cell lines that express androgen receptor (AR). In this study we utilized the AR negative PCa cell line and observed that re-expression of AR (PC3-AR) results in greater levels of apoptosis when treated with the pan-DACi, panobinostat (PAN). PAN mediated apoptosis in PC3 and PC3-AR cells was associated with increased levels of double strand DNA breaks, indicated by p-ɣH2AX. Further, PAN treatment in PC3-AR cells resulted in moderate attenuation of the ATM-Akt-ERK DNA damage response pathway. For this reason, we combined PAN with the dual PI3K-mTOR inhibitor, BEZ235. Combination of PAN with BEZ235 resulted in significant attenuation of the DNA damage repair protein ATM and significantly increased anti-tumor activity compared to each single treatment. Overall, superior anti-tumor activity with combination of PAN with BEZ235 was independent of AR status. These findings suggest that this therapeutic strategy should be further developed in clinical trials.

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PC3 and PC3-AR cells were treated with increasing concentrations of panobinostat for 24 and 48 hoursCells were trypsinized and washed in 1x PBS before being fixed in 70% ethanol and stained with PI. Cell cycle distribution was assessed by flow cytometry. (A) Representative histograms of cell cycle profile of PC3 and PC3-AR cells treated with 100nM panobinostat for 48 hours. (B-C) Quantitated measurements of G2M cell cycle distribution and (D-E) subG1 cell cycle distribution for PC3 and PC3-AR cells following panobinostat treatment. Columns represent mean of 3 independent experiments ±SE.* p < 0.05 (two-tailed t-test).
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Figure 2: PC3 and PC3-AR cells were treated with increasing concentrations of panobinostat for 24 and 48 hoursCells were trypsinized and washed in 1x PBS before being fixed in 70% ethanol and stained with PI. Cell cycle distribution was assessed by flow cytometry. (A) Representative histograms of cell cycle profile of PC3 and PC3-AR cells treated with 100nM panobinostat for 48 hours. (B-C) Quantitated measurements of G2M cell cycle distribution and (D-E) subG1 cell cycle distribution for PC3 and PC3-AR cells following panobinostat treatment. Columns represent mean of 3 independent experiments ±SE.* p < 0.05 (two-tailed t-test).

Mentions: Androgen receptor expression sensitizes PC3 cells to HDACi mediated apoptosis. PC3 and PC3-AR cells were exposed for 24 and 48 hours to increasing concentrations of the HDACi panobinostat (PAN) and plasma membrane integrity assessed by uptake of propidium iodide (PI). Initial confirmation of the functional status of AR expressed in PC3-AR cells is shown in supplement Fig. 1. As shown in Fig. 1A, only 100nM PAN slightly increased killing of PC3 cells. In contrast, low-nanomolar concentrations of PAN were sufficient to kill PC3-AR cells in a time dependent manner (Fig. 1B). We next assessed the biochemical changes that occurred after treatment of PC3 and PC3-AR cells with PAN. Only PC3-AR cells treated with 100nM PAN displayed hallmark features of apoptosis compared to the same treatment of PC3 cells. These included increased cell surface exposure of phosphatidyserine (Fig. 1C and 1D) and caspase activation (Fig. 1E). Further, treatment of PC3 and PC3-AR cells with 100nM PAN resulted in accumulation of subG1 population in PC3-AR cells indicating DNA fragmentation, whereas the same treatment resulted in cell cycle arrest of PC3 cells in the G2M phase of the cell cycle (Fig. 2).


Combinatorial antitumor effect of HDAC and the PI3K-Akt-mTOR pathway inhibition in a Pten defecient model of prostate cancer.

Ellis L, Ku SY, Ramakrishnan S, Lasorsa E, Azabdaftari G, Godoy A, Pili R - Oncotarget (2013)

PC3 and PC3-AR cells were treated with increasing concentrations of panobinostat for 24 and 48 hoursCells were trypsinized and washed in 1x PBS before being fixed in 70% ethanol and stained with PI. Cell cycle distribution was assessed by flow cytometry. (A) Representative histograms of cell cycle profile of PC3 and PC3-AR cells treated with 100nM panobinostat for 48 hours. (B-C) Quantitated measurements of G2M cell cycle distribution and (D-E) subG1 cell cycle distribution for PC3 and PC3-AR cells following panobinostat treatment. Columns represent mean of 3 independent experiments ±SE.* p < 0.05 (two-tailed t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926822&req=5

Figure 2: PC3 and PC3-AR cells were treated with increasing concentrations of panobinostat for 24 and 48 hoursCells were trypsinized and washed in 1x PBS before being fixed in 70% ethanol and stained with PI. Cell cycle distribution was assessed by flow cytometry. (A) Representative histograms of cell cycle profile of PC3 and PC3-AR cells treated with 100nM panobinostat for 48 hours. (B-C) Quantitated measurements of G2M cell cycle distribution and (D-E) subG1 cell cycle distribution for PC3 and PC3-AR cells following panobinostat treatment. Columns represent mean of 3 independent experiments ±SE.* p < 0.05 (two-tailed t-test).
Mentions: Androgen receptor expression sensitizes PC3 cells to HDACi mediated apoptosis. PC3 and PC3-AR cells were exposed for 24 and 48 hours to increasing concentrations of the HDACi panobinostat (PAN) and plasma membrane integrity assessed by uptake of propidium iodide (PI). Initial confirmation of the functional status of AR expressed in PC3-AR cells is shown in supplement Fig. 1. As shown in Fig. 1A, only 100nM PAN slightly increased killing of PC3 cells. In contrast, low-nanomolar concentrations of PAN were sufficient to kill PC3-AR cells in a time dependent manner (Fig. 1B). We next assessed the biochemical changes that occurred after treatment of PC3 and PC3-AR cells with PAN. Only PC3-AR cells treated with 100nM PAN displayed hallmark features of apoptosis compared to the same treatment of PC3 cells. These included increased cell surface exposure of phosphatidyserine (Fig. 1C and 1D) and caspase activation (Fig. 1E). Further, treatment of PC3 and PC3-AR cells with 100nM PAN resulted in accumulation of subG1 population in PC3-AR cells indicating DNA fragmentation, whereas the same treatment resulted in cell cycle arrest of PC3 cells in the G2M phase of the cell cycle (Fig. 2).

Bottom Line: In this study we utilized the AR negative PCa cell line and observed that re-expression of AR (PC3-AR) results in greater levels of apoptosis when treated with the pan-DACi, panobinostat (PAN).Combination of PAN with BEZ235 resulted in significant attenuation of the DNA damage repair protein ATM and significantly increased anti-tumor activity compared to each single treatment.Overall, superior anti-tumor activity with combination of PAN with BEZ235 was independent of AR status.

View Article: PubMed Central - PubMed

Affiliation: Genitourinary Program, Roswell Park Cancer Institute, Buffalo NY.

ABSTRACT
Increased expression of histone deacetylases (HDACs) and activation of the PI3K-Akt-mTORC1 pathway are common aberrations in prostate cancer (PCa). For this reason, inhibition of such targets is an exciting avenue for the development of novel therapeutic strategies to treat patients with advanced PCa. Previous reports demonstrated that HDAC inhibitors (HDACi) increases DNA damage and induce greater apoptosis in PCa cell lines that express androgen receptor (AR). In this study we utilized the AR negative PCa cell line and observed that re-expression of AR (PC3-AR) results in greater levels of apoptosis when treated with the pan-DACi, panobinostat (PAN). PAN mediated apoptosis in PC3 and PC3-AR cells was associated with increased levels of double strand DNA breaks, indicated by p-ɣH2AX. Further, PAN treatment in PC3-AR cells resulted in moderate attenuation of the ATM-Akt-ERK DNA damage response pathway. For this reason, we combined PAN with the dual PI3K-mTOR inhibitor, BEZ235. Combination of PAN with BEZ235 resulted in significant attenuation of the DNA damage repair protein ATM and significantly increased anti-tumor activity compared to each single treatment. Overall, superior anti-tumor activity with combination of PAN with BEZ235 was independent of AR status. These findings suggest that this therapeutic strategy should be further developed in clinical trials.

Show MeSH
Related in: MedlinePlus