HHS Public Access Author manuscript Author Manuscript
Biotechnol Bioeng. Author manuscript; available in PMC 2016 December 06. Published in final edited form as: Biotechnol Bioeng. 2016 June ; 113(6): 1345–1356. doi:10.1002/bit.25898.
The Histone Deacetylase Inhibitor Entinostat Enhances PolymerMediated Transgene Expression in Cancer Cell Lines Jacob J. Elmer#1, Matthew D. Christensen#1, Sutapa Barua1, Jennifer Lehrman2, Karmella A. Haynes2, and Kaushal Rege1 1Chemical
Engineering, Arizona State University, Tempe, Arizona 85287
Author Manuscript
2Harrington
#
Biomedical Engineering, Arizona State University, Tempe, Arizona
These authors contributed equally to this work.
Abstract
Author Manuscript Author Manuscript
Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone-DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25-fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer-mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells.
Correspondence to: K. Rege, telephone: 480-727-8616; fax: 480-727-9321; [email protected]. Jacob J. Elmer’s present address is Department of Chemical Engineering, Villanova University, White Hall Room 119, 800 East Lancaster Ave, Villanova, PA 19085. Disclosure statement: Dr. Rege was an invited speaker at PepTalk: The Protein Science Week in 2015. All expenses for Dr. Rege were borne by Cambridge Healthtech Institute. Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Elmer et al.
Page 2
Author Manuscript
Keywords non-viral gene delivery; epigenetic silencing; transient protein expression; histones; acetylation
Introduction
Author Manuscript
If all of the DNAwithin a single human cell (~6 × 109 base pairs) was stretched out end to end, it would be nearly 2 m long (Annunziato, 2008). Since the diameter of most nuclei is only ~6 μm, this immense amount of DNA must be condensed by tightly wrapping it around histone proteins inside the nucleus. Humans have four core histones (H2A, H2B, H3, and H4) with positively charged tail domains that are rich in lysine and arginine residues that facilitate DNA binding. Histone octamers (H2A2H2B2H32H42) initiate DNA condensation by binding 147 base pairs of DNA to form nucleosomes. A special linker histone (H1) then connects the nucleosomes to produce chromatin fibers (30 nm) that are condensed even further into chromosomes (100–400 nm) during mitosis (Wolffe, 1999).
Author Manuscript
While DNA condensation allows cells to maintain vast amounts of genetic information, histone binding can also physically block gene expression. Cells address this problem by expressing a wide variety of enzymes that phosphorylate (Rossetto et al., 2012), methylate (Kouzarides, 2002), ubiquitinylate (Bonnet et al., 2012), or acetylate histones to regulate DNA binding and transcription (Bannister and Kouzarides, 2011). For example, histone acetyl transferases (HATs) use acetyl CoA to acetylate and neutralize the charge of ε-amino groups on lysine residues, thereby releasing DNA for transcription. In contrast, histone acetylation may be reversed by histone deacetylases (HDACs), which remove acetyl groups to restore the positive charge and DNA binding activity of histones (Xhemalce et al., 2011; Yang and Seto, 2007). HATs and HDACs are generally non-specific (Bannister and Kouzarides, 2011), but HDACs have been shown to form complexes with other proteins that target deacetylase activity to specific DNA sequences (Hayakawa and Nakayama, 2011; Nan et al., 1998). HDACs can also influence gene expression by deacetylating non-histone proteins like NF-κB and other transcription factors (Glozak et al., 2005; Hasselgren, 2007). Consequently, aberrations in HAT/HDAC activity have been implicated in several neurodegenerative diseases (Kumar and Rinwa, 2012) and cancer (Ropero and Esteller, 2007).
Author Manuscript
Histone acetylation can also regulate expression of foreign DNA. Bishop et al. showed that viral DNA is efficiently delivered to the nucleus, but it is then quickly bound and silenced by histones within the densely packed centromeric heterochromatin. However, the HDAC inhibitor trichostatin A (TSA) was able to restore viral gene expression (Bishop et al., 2006; Poleshko et al., 2008). HDAC inhibitors have also been shown to bring viruses out of latency (Archin et al., 2009; Danaher et al., 2005), while some viruses express proteins to specifically inhibit HDACs (Gu and Roizman, 2007; Tang and Maul, 2003). Aside from viral DNA, histones also bind bacterial plasmids with high affinity (Yaneva et al., 1995) and form nucleosomes in vitro (Nakagawa et al., 2001). Purified histone proteins or synthetic peptides with the cationic histone tail sequence have also been used for non-viral gene delivery (i.e., histonefection)(Kaouass et al., 2006; Reilly et al., 2012).
Biotechnol Bioeng. Author manuscript; available in PMC 2016 December 06.
Elmer et al.
Page 3
Author Manuscript
Since histones appear to significantly inhibit viral gene expression and bind plasmid DNA (pDNA) in vitro, we hypothesized that HDAC inhibition could potentially enhance polymermediated transgene delivery and/or expression. We have previously shown that inhibition of the cytoplasmic HDAC6 with tubacin increases polymer-mediated transgene expression by influencing intracellular plasmid trafficking on stabilized micro-tubules (Barua and Rege, 2010). In this study, we investigated the effects of Entinostat, a selective inhibitor of class 1 HDACs 1 and 3 (Hu et al., 2003). Previous studies with Entinostat have demonstrated that it effectively inhibits HDACs in vivo, resulting in hyperacetylation of histones (Camphausen et al., 2004) and expression of genes that were previously silenced (Duque-Afonso et al., 2011; Kasman et al., 2007, 2012). Our results show that Entinostat significantly enhances polymermediated transgene expression in both prostate and bladder cancer cells with moderate effects on cell viability. Therefore, Entinostat treatment may be an effective way to enhance transgene expression levels in transient systems.
Author Manuscript
Materials and Methods Polymer Synthesis
Author Manuscript
The 1,4C-1,4Bis and PA8 polymers were synthesized using methods similar to our previously published protocols (Barua et al., 2009; Potta et al., 2014; Vu et al., 2012). Briefly, the epoxide groups of diglycidyl ether (DGE) monomers 1,4 cyclohexanedimethanol DGE or ethylene glycol DGE, respectively, were reacted with polyamine monomers 1,4 bis(3-aminopropyl) piperazine and paromomycin, respectively, resulting in the formation of cationic polymers with molecular weights (MWs) >5,000 g/mol. Branched polyethyleneimine (PEI, MW = 25,000 g/mol) was purchased from Sigma (St. Louis, MO), and fresh stocks (50 ng/μL in HEPES buffer, pH 7.4) were prepared before every experiment to obviate any effects due to storage. Structures of monomers for synthesis of in-house polymers as well as the polymer structure for 25 kDa branched polyethylenemine are shown in Supporting Information Figures S1–3. Transfections in the Presence of Entinostat
Author Manuscript
Entinostat was kindly provided by Syndax Pharmaceuticals of Waltham, MA through an agreement with the Cancer Therapeutics Evaluation Program (CTEP) at NIH. Stocks were prepared in DMSO at concentrations ranging from 60 μM–20 mM and frozen at −80°C until needed. Human prostate (PC3 and PC3-PSMA) and murine bladder (MB49) cancer cells were seeded onto 24-well plates at a density of 50,000 cells per well with 500 μL RPMI (PC3 and PC3-PSMA) or DMEM (MB49) containing 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin. The PC3-PSMA cell line, derived by transducing PC3 cells for stable expression of the Prostate Specific Membrane Antigen (PSMA) receptor, was a generous gift from Dr. Michel Sadelain (Memorial Sloan Kettering Cancer Center, New York, NY) (Gong et al., 1999). All cell lines were incubated overnight (~18–20 h) at 37°C, and the serum-containing media (SCM) was replaced with serum-free media (SFM) immediately prior to transfection (except in the case of transfections performed in the presence of SCM). Polyplexes were prepared by incubating cationic polymers (1,4C-1,4Bis, PEI, or PA8) with pGL3-Control (luciferase reporter gene, Promega, Madison, WI) or pEGFP-C1 (enhanced green fluorescent protein or EGFP
Biotechnol Bioeng. Author manuscript; available in PMC 2016 December 06.
Elmer et al.
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Author Manuscript
reporter gene, Clontech, Mountain View, CA) plasmid DNA. The concentration of plasmid DNA was kept constant at 200 ng/well, while the polymer:pDNA mass ratio varied for each polymer (PEI = 1:1, 1,4C-1,4bis = 10:1, PA8 = 50:1), depending on their previously determined optimal concentrations (Potta et al., 2014). Polyplexes and different doses (0, 0.33, 1, 3.3, 10, 33, and 100 μM) of the HDAC inhibitor Entinostat were simultaneously added to the cells while a constant DMSO (for solubilizing the drug) concentration of 0.5% (v/v) was employed in all cases. Following 6 h of incubation at 37°C with the polyplex and drug, serum-free media was exchanged with serum-containing media containing the corresponding Entinostat concentrations. The cells were then incubated at 37°C for an additional 48 h to allow for transgene expression. Transfections with 0 μM Entinostat with or without 0.5% DMSO were also performed as controls, and 0.5% DMSO was found to not have any significant effects on transgene expression efficacy (data not shown).
Author Manuscript
Luminescence Assay and Fluorescence Microscopy
Author Manuscript
Luciferase expression was quantified using the Luciferase Assay Kit from Promega. At 48 h after transfection, cell culture media was removed from each well, and the cells were washed once with phosphate- buffered saline (PBS) before adding 150 μL of the cell culture lysis reagent (Promega) to each well. The wells were then incubated at 37°C for 20 min to ensure complete cell lysis. Cell lysates (15 μL) were then mixed with luciferin solution (30 μL) and luminescence (LUM) was immediately measured using a Synergy 2 plate reader (Biotek, Winooski, VT). Luminescence values were divided by cell viability to obtain LUV values to account for differences in cell viability. Relative LUV (RLUV) values were then obtained by dividing by the LUV of each sample by the LUV of the polyplex control, which only consisted of the corresponding polymer (PEI, 1,4C-1,4Bis, or PA8) and plasmid DNA (i.e., 0 mM Entinostat). Therefore, the RLUV values presented here account for changes in cell density (e.g., a condition with luminescence similar to the control but with 50% viability will be multiplied by a factor of two) and illustrate the degree of enhancement for each condition relative to the control. Following transfections with the pEGFP-C1 plasmid, cells were examined with a Zeiss fluorescence microscope to visualize EGFP expression. All images were acquired within areas of 90–100% confluence near the center of each well. MTT Cell Viability Assay
Author Manuscript
Cell viability was quantified using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a yellow reagent which is converted to formazan (a purple dye) by living cells. This assay is a commonly used indicator of metabolic activity, which indirectly reports for cell viability. The MTT reagent was added to the cells (37°C for 2 h) and then a detergent from the kit (ATCC, Manassas, VA) was used to lyse the cells (additional 2 h). The formazan concentration was then quantified using by measuring the absorbance of the sample at 570 nm (A570), and cell viability was calculated by dividing the A570 value of each sample by the A570 value of the live cell control (no drug or polyplex added). Cell Cycle Analysis Cell cycle analysis was carried out by staining genomic DNA with propidium iodide (PI) with a few modifications of methods previously described in the literature (Krishan, 1975; Pozarowski and Darzynkiewicz, 2004). Briefly, PC3-PSMA human prostate cancer cells Biotechnol Bioeng. Author manuscript; available in PMC 2016 December 06.
Elmer et al.
Page 5
Author Manuscript
were seeded in 6-well plates at a density of 150,000 cells/well and cultured in the presence of 0.5% DMSO. The cells were treated with 0, 3.3, or 33 μM Entinostat for 48 h. Cells were harvested for flow cytometry analysis via trypsinization, rinsed once with PBS, and fixed with 70% EtOH. Cells were then permeabilized in a 0.001% Triton × solution, washed again in a PBS/FBS solution, and resuspended in a staining solution containing 5% FBS, 50 μg/mL PI, and 100 μg/mL RNase A for final flow cytometry analysis using an Attune® Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA). The distribution of cells in each phase of the cell cycle was then determined by measuring the intensity of PI fluorescence within each cell (20-fold enhancement observed with the prostate cancer cell lines at 33 μM Entinostat, the drug was only able to enhance luciferase expression by approximately seven-fold in MB49 cells at a much lower optimum concentration of 3.3 μM. This reduction in enhancement may be related to the sharp decrease in MB49 cell viability at Entinostat concentrations above 3.3 μM. This decrease in enhancement may also reflect differences between the interactions of human and murine HDACs with Entinostat, although additional cell lines of both species would need to be included in this study to verify this hypothesis. Regardless of the nature of these differences, it is still clear that Entinostat significantly enhanced transgene expression in each of the cell lines tested in a dose range from 3.3 μM to 33 μM. It is interesting to note that the optimum concentration of Entinostat observed in our experiments (3.3–33 μM) is much higher than the previously published submicromolar (
Biotechnol Bioeng. Author manuscript; available in PMC 2016 December 06. Published in final edited form as: Biotechnol Bioeng. 2016 June ; 113(6): 1345–1356. doi:10.1002/bit.25898.
The Histone Deacetylase Inhibitor Entinostat Enhances PolymerMediated Transgene Expression in Cancer Cell Lines Jacob J. Elmer#1, Matthew D. Christensen#1, Sutapa Barua1, Jennifer Lehrman2, Karmella A. Haynes2, and Kaushal Rege1 1Chemical
Engineering, Arizona State University, Tempe, Arizona 85287
Author Manuscript
2Harrington
#
Biomedical Engineering, Arizona State University, Tempe, Arizona
These authors contributed equally to this work.
Abstract
Author Manuscript Author Manuscript
Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone-DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25-fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer-mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells.
Correspondence to: K. Rege, telephone: 480-727-8616; fax: 480-727-9321; [email protected]. Jacob J. Elmer’s present address is Department of Chemical Engineering, Villanova University, White Hall Room 119, 800 East Lancaster Ave, Villanova, PA 19085. Disclosure statement: Dr. Rege was an invited speaker at PepTalk: The Protein Science Week in 2015. All expenses for Dr. Rege were borne by Cambridge Healthtech Institute. Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Elmer et al.
Page 2
Author Manuscript
Keywords non-viral gene delivery; epigenetic silencing; transient protein expression; histones; acetylation
Introduction
Author Manuscript
If all of the DNAwithin a single human cell (~6 × 109 base pairs) was stretched out end to end, it would be nearly 2 m long (Annunziato, 2008). Since the diameter of most nuclei is only ~6 μm, this immense amount of DNA must be condensed by tightly wrapping it around histone proteins inside the nucleus. Humans have four core histones (H2A, H2B, H3, and H4) with positively charged tail domains that are rich in lysine and arginine residues that facilitate DNA binding. Histone octamers (H2A2H2B2H32H42) initiate DNA condensation by binding 147 base pairs of DNA to form nucleosomes. A special linker histone (H1) then connects the nucleosomes to produce chromatin fibers (30 nm) that are condensed even further into chromosomes (100–400 nm) during mitosis (Wolffe, 1999).
Author Manuscript
While DNA condensation allows cells to maintain vast amounts of genetic information, histone binding can also physically block gene expression. Cells address this problem by expressing a wide variety of enzymes that phosphorylate (Rossetto et al., 2012), methylate (Kouzarides, 2002), ubiquitinylate (Bonnet et al., 2012), or acetylate histones to regulate DNA binding and transcription (Bannister and Kouzarides, 2011). For example, histone acetyl transferases (HATs) use acetyl CoA to acetylate and neutralize the charge of ε-amino groups on lysine residues, thereby releasing DNA for transcription. In contrast, histone acetylation may be reversed by histone deacetylases (HDACs), which remove acetyl groups to restore the positive charge and DNA binding activity of histones (Xhemalce et al., 2011; Yang and Seto, 2007). HATs and HDACs are generally non-specific (Bannister and Kouzarides, 2011), but HDACs have been shown to form complexes with other proteins that target deacetylase activity to specific DNA sequences (Hayakawa and Nakayama, 2011; Nan et al., 1998). HDACs can also influence gene expression by deacetylating non-histone proteins like NF-κB and other transcription factors (Glozak et al., 2005; Hasselgren, 2007). Consequently, aberrations in HAT/HDAC activity have been implicated in several neurodegenerative diseases (Kumar and Rinwa, 2012) and cancer (Ropero and Esteller, 2007).
Author Manuscript
Histone acetylation can also regulate expression of foreign DNA. Bishop et al. showed that viral DNA is efficiently delivered to the nucleus, but it is then quickly bound and silenced by histones within the densely packed centromeric heterochromatin. However, the HDAC inhibitor trichostatin A (TSA) was able to restore viral gene expression (Bishop et al., 2006; Poleshko et al., 2008). HDAC inhibitors have also been shown to bring viruses out of latency (Archin et al., 2009; Danaher et al., 2005), while some viruses express proteins to specifically inhibit HDACs (Gu and Roizman, 2007; Tang and Maul, 2003). Aside from viral DNA, histones also bind bacterial plasmids with high affinity (Yaneva et al., 1995) and form nucleosomes in vitro (Nakagawa et al., 2001). Purified histone proteins or synthetic peptides with the cationic histone tail sequence have also been used for non-viral gene delivery (i.e., histonefection)(Kaouass et al., 2006; Reilly et al., 2012).
Biotechnol Bioeng. Author manuscript; available in PMC 2016 December 06.
Elmer et al.
Page 3
Author Manuscript
Since histones appear to significantly inhibit viral gene expression and bind plasmid DNA (pDNA) in vitro, we hypothesized that HDAC inhibition could potentially enhance polymermediated transgene delivery and/or expression. We have previously shown that inhibition of the cytoplasmic HDAC6 with tubacin increases polymer-mediated transgene expression by influencing intracellular plasmid trafficking on stabilized micro-tubules (Barua and Rege, 2010). In this study, we investigated the effects of Entinostat, a selective inhibitor of class 1 HDACs 1 and 3 (Hu et al., 2003). Previous studies with Entinostat have demonstrated that it effectively inhibits HDACs in vivo, resulting in hyperacetylation of histones (Camphausen et al., 2004) and expression of genes that were previously silenced (Duque-Afonso et al., 2011; Kasman et al., 2007, 2012). Our results show that Entinostat significantly enhances polymermediated transgene expression in both prostate and bladder cancer cells with moderate effects on cell viability. Therefore, Entinostat treatment may be an effective way to enhance transgene expression levels in transient systems.
Author Manuscript
Materials and Methods Polymer Synthesis
Author Manuscript
The 1,4C-1,4Bis and PA8 polymers were synthesized using methods similar to our previously published protocols (Barua et al., 2009; Potta et al., 2014; Vu et al., 2012). Briefly, the epoxide groups of diglycidyl ether (DGE) monomers 1,4 cyclohexanedimethanol DGE or ethylene glycol DGE, respectively, were reacted with polyamine monomers 1,4 bis(3-aminopropyl) piperazine and paromomycin, respectively, resulting in the formation of cationic polymers with molecular weights (MWs) >5,000 g/mol. Branched polyethyleneimine (PEI, MW = 25,000 g/mol) was purchased from Sigma (St. Louis, MO), and fresh stocks (50 ng/μL in HEPES buffer, pH 7.4) were prepared before every experiment to obviate any effects due to storage. Structures of monomers for synthesis of in-house polymers as well as the polymer structure for 25 kDa branched polyethylenemine are shown in Supporting Information Figures S1–3. Transfections in the Presence of Entinostat
Author Manuscript
Entinostat was kindly provided by Syndax Pharmaceuticals of Waltham, MA through an agreement with the Cancer Therapeutics Evaluation Program (CTEP) at NIH. Stocks were prepared in DMSO at concentrations ranging from 60 μM–20 mM and frozen at −80°C until needed. Human prostate (PC3 and PC3-PSMA) and murine bladder (MB49) cancer cells were seeded onto 24-well plates at a density of 50,000 cells per well with 500 μL RPMI (PC3 and PC3-PSMA) or DMEM (MB49) containing 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin. The PC3-PSMA cell line, derived by transducing PC3 cells for stable expression of the Prostate Specific Membrane Antigen (PSMA) receptor, was a generous gift from Dr. Michel Sadelain (Memorial Sloan Kettering Cancer Center, New York, NY) (Gong et al., 1999). All cell lines were incubated overnight (~18–20 h) at 37°C, and the serum-containing media (SCM) was replaced with serum-free media (SFM) immediately prior to transfection (except in the case of transfections performed in the presence of SCM). Polyplexes were prepared by incubating cationic polymers (1,4C-1,4Bis, PEI, or PA8) with pGL3-Control (luciferase reporter gene, Promega, Madison, WI) or pEGFP-C1 (enhanced green fluorescent protein or EGFP
Biotechnol Bioeng. Author manuscript; available in PMC 2016 December 06.
Elmer et al.
Page 4
Author Manuscript
reporter gene, Clontech, Mountain View, CA) plasmid DNA. The concentration of plasmid DNA was kept constant at 200 ng/well, while the polymer:pDNA mass ratio varied for each polymer (PEI = 1:1, 1,4C-1,4bis = 10:1, PA8 = 50:1), depending on their previously determined optimal concentrations (Potta et al., 2014). Polyplexes and different doses (0, 0.33, 1, 3.3, 10, 33, and 100 μM) of the HDAC inhibitor Entinostat were simultaneously added to the cells while a constant DMSO (for solubilizing the drug) concentration of 0.5% (v/v) was employed in all cases. Following 6 h of incubation at 37°C with the polyplex and drug, serum-free media was exchanged with serum-containing media containing the corresponding Entinostat concentrations. The cells were then incubated at 37°C for an additional 48 h to allow for transgene expression. Transfections with 0 μM Entinostat with or without 0.5% DMSO were also performed as controls, and 0.5% DMSO was found to not have any significant effects on transgene expression efficacy (data not shown).
Author Manuscript
Luminescence Assay and Fluorescence Microscopy
Author Manuscript
Luciferase expression was quantified using the Luciferase Assay Kit from Promega. At 48 h after transfection, cell culture media was removed from each well, and the cells were washed once with phosphate- buffered saline (PBS) before adding 150 μL of the cell culture lysis reagent (Promega) to each well. The wells were then incubated at 37°C for 20 min to ensure complete cell lysis. Cell lysates (15 μL) were then mixed with luciferin solution (30 μL) and luminescence (LUM) was immediately measured using a Synergy 2 plate reader (Biotek, Winooski, VT). Luminescence values were divided by cell viability to obtain LUV values to account for differences in cell viability. Relative LUV (RLUV) values were then obtained by dividing by the LUV of each sample by the LUV of the polyplex control, which only consisted of the corresponding polymer (PEI, 1,4C-1,4Bis, or PA8) and plasmid DNA (i.e., 0 mM Entinostat). Therefore, the RLUV values presented here account for changes in cell density (e.g., a condition with luminescence similar to the control but with 50% viability will be multiplied by a factor of two) and illustrate the degree of enhancement for each condition relative to the control. Following transfections with the pEGFP-C1 plasmid, cells were examined with a Zeiss fluorescence microscope to visualize EGFP expression. All images were acquired within areas of 90–100% confluence near the center of each well. MTT Cell Viability Assay
Author Manuscript
Cell viability was quantified using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a yellow reagent which is converted to formazan (a purple dye) by living cells. This assay is a commonly used indicator of metabolic activity, which indirectly reports for cell viability. The MTT reagent was added to the cells (37°C for 2 h) and then a detergent from the kit (ATCC, Manassas, VA) was used to lyse the cells (additional 2 h). The formazan concentration was then quantified using by measuring the absorbance of the sample at 570 nm (A570), and cell viability was calculated by dividing the A570 value of each sample by the A570 value of the live cell control (no drug or polyplex added). Cell Cycle Analysis Cell cycle analysis was carried out by staining genomic DNA with propidium iodide (PI) with a few modifications of methods previously described in the literature (Krishan, 1975; Pozarowski and Darzynkiewicz, 2004). Briefly, PC3-PSMA human prostate cancer cells Biotechnol Bioeng. Author manuscript; available in PMC 2016 December 06.
Elmer et al.
Page 5
Author Manuscript
were seeded in 6-well plates at a density of 150,000 cells/well and cultured in the presence of 0.5% DMSO. The cells were treated with 0, 3.3, or 33 μM Entinostat for 48 h. Cells were harvested for flow cytometry analysis via trypsinization, rinsed once with PBS, and fixed with 70% EtOH. Cells were then permeabilized in a 0.001% Triton × solution, washed again in a PBS/FBS solution, and resuspended in a staining solution containing 5% FBS, 50 μg/mL PI, and 100 μg/mL RNase A for final flow cytometry analysis using an Attune® Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA). The distribution of cells in each phase of the cell cycle was then determined by measuring the intensity of PI fluorescence within each cell (20-fold enhancement observed with the prostate cancer cell lines at 33 μM Entinostat, the drug was only able to enhance luciferase expression by approximately seven-fold in MB49 cells at a much lower optimum concentration of 3.3 μM. This reduction in enhancement may be related to the sharp decrease in MB49 cell viability at Entinostat concentrations above 3.3 μM. This decrease in enhancement may also reflect differences between the interactions of human and murine HDACs with Entinostat, although additional cell lines of both species would need to be included in this study to verify this hypothesis. Regardless of the nature of these differences, it is still clear that Entinostat significantly enhanced transgene expression in each of the cell lines tested in a dose range from 3.3 μM to 33 μM. It is interesting to note that the optimum concentration of Entinostat observed in our experiments (3.3–33 μM) is much higher than the previously published submicromolar (
