extfieldsofstudy: Medicine Chemistry

text:
Layer-by-layer DNA films incorporating highly transfecting bioreducible poly(amido amine) and polyethylenimine for sequential gene delivery

Background The layer-by-layer (LbL) assembly method offers a molecular level control of the amount and spatial distribution of bioactive molecules. However, successful clinical translation of LbL film technology will most certainly require a better understanding and control of not only the film assembly process, but also film disassembly kinetics in physiologic conditions. Purpose This work focuses on the understanding and control of degradation properties of LbL films for localized gene delivery. Methods Bioreducible poly(amido amine)s (PAAs) containing cystaminebisacrylamide (CBA), methylenebisacrylamide, and 5-amino-1-pentanol (APOL) were synthesized by Michael addition polymerization for the construction of bioreducible LbL films capable of sequential gene delivery. Results The synthesized PAAs were screened for desirable buffering capacity, cell transfection, and cytotoxicity characteristics together with 25 kDa branched polyethylenimine (PEI) and cross-linked 800 Da PEI. By screening the various polycations we were able to identify a copolymer of CBA and APOL for the subsequent construction of the LbL films. By incorporating a highly transfecting polycation and a nondiffusing polycation we were able to improve the overall transfection of HEK293 and MC3T3 cells from the bioreducible LbL films. We also demonstrated the dual-stage release and transfection of two different DNAs from the LbL films. Conclusion The results indicate that LbL films consisting of bioreducible PAAs and non-diffusing polyelectrolytes have excellent degradation properties for the development of LbL coating technology for localized gene delivery applications.

Introduction
This work focuses on the design of layer-by-layer (LbL) films for localized gene delivery by understanding and controlling film degradation properties. LbL films consisting of polyelectrolyte multilayers (PEMs) 1-3 have become a popular coating method for the controlled release of DNA and other bioactive agents 4-6 from a variety of implantable devices such as titanium, 7,8 stainless steel stents, 6,9,10 and microneedles. 11,12 PEMs are assembled by a scalable process of repeated deposition of a polycation layer followed by a polyanion layer. The LbL assembly offers a molecular level control of the amount and spatial distribution of bioactive molecules unrivaled among biomedical coating methods. However, in order Polycations commonly used for systemic gene delivery, such as polyethylenimine (PEI), poly(l-lysine), and polysaccharides, form highly stable LbL films with DNA due to the polyvalent nature of their electrostatic interactions. The degradation of such films requires unphysiologic conditions. Strategies to design biodegradable LbL films include the use of hydrolytically and enzymatically degradable polycations. [14][15][16] Another particularly successful strategy involves the use of disulfide containing polycations (bioreducible polycations) whose degradation can be triggered by endogenous chemicals such as glutathione and thioredoxin reductases. 6,[17][18][19][20] Bioreducible LbL films can be degraded by redox-active thioredoxin enzymes in the cellular plasma membrane in contact with the LbL films during cell attachment and proliferation. [21][22][23] Relying on cellular exofacial redox activity of membrane proteins for LbL film disassembly offers major advantages over hydrolytic and enzymatic degradation mechanisms including stability in extracellular environment, responsiveness to redox gradients, and decreased cytotoxicity.
Successful applications of LbL films for gene delivery also require more efficient gene delivery vectors. PEI is one of the most successful polycation gene delivery vectors to date due to its buffering capability for endosomal escape of the gene payload and superior transfection efficiency. 24,25 However, high molecular weight PEI (branched polyethylenimine [bPEI]) in various forms is toxic to many cell lines. 26,27 Recent efforts have concentrated on reducing the cytotoxicity of PEI by using various low molecular weight PEI derivatives with biodegradable linkages. 28 Bioreducible poly(amido amine)s (PAAs), the focus of our work, have shown improved safety over PEI as gene delivery vectors. 6,13,29,30 Here, we examine a new type of PAA for LbL-based gene delivery. We applied the proton sponge hypothesis 24,31 to screen for PAAs with high buffering capacity. For comparison, we included bPEI (25 kDa), low molecular weight PEI (800 Da), and a degradable PEI (dPEI) in this study (Scheme 1). We synthesized dPEI by cross-linking 800 Da PEI with a biodegradable cross-linker. The screening enabled the selection of the best PAA for the subsequent LbL film construction. In order to avoid uncontrolled bulk peeling of the films, we constructed bioreducible PAA/DNA films with periodically inserted diffusion barrier layers (Scheme 2). LbL film 1 is made of alternating layers of PAA and DNA with periodically inserted bPEI while LbL film 2 is made of PAA/DNA layers with periodically inserted dPEI. The film assembly process was monitored by atomic force microscopy (AFM) while the film disassembly in a reducing solution was monitored by AFM, fluorescence, and dynamic light scattering (DLS). Transfection experiments were conducted on human embryonic kidney (HEK) 293 cell line, fibroblast NIH3T3 cell line, and osteoblast MC3T3 cell line. We show that acidbase titration can be used to screen polyelectrolytes for their buffering capacity and transfection efficiency. We further show that the incorporation of a polyelectrolyte layer to limit interlayer diffusion is essential in achieving sequential DNA release from biodegradable LbL films.

Synthesis of bioreducible poly (amino pentanol)
Bioreducible poly(amino pentanol) (PAAs) were synthesized by Michael addition copolymerization of equal molar ratios of monomer APOL and bisacrylamide monomers (CBA and MBA) by modifying literature procedures. 32,33 In a typical reaction, CBA (309 mg, 3 mmol, 1 equiv) was dissolved in CH 3 OH/H 2 O (4/1 v/v, 3 mL); then, APOL (781 mg, 3 mmol, 1 equiv) was added followed by degassing with N 2 . The vial was wrapped with foil and then placed in a 50°C oil bath. This mixture was stirred for 10 days at 50°C to yield a light yellow viscous solution. Additional APOL (10 mol%) was added and the reaction continued for another 2 days in order to consume all CBA. After evaporating CH 3 OH, the solution was added dropwise into 20 mL 4°C acetone. The oil phase was washed with 4°C acetone (3×5 mL). After acetone evaporation, 5 mL water was added, and the pH of the solution was adjusted to 4 using 1 M HCl. The final product was a solid foam (520 mg, 48% yield) after freeze drying.  Figure S1C).

Synthesis of dPEI
dPEI was synthesized by cross-linking 800 Da PEI using 1,6-hexanediol diacrylate. 34 One gram of 800 Da PEI was dissolved in 3 mL methylene chloride. Equimolar 1,6hexanediol diacrylate was added dropwise. The reaction was carried out for 6 hours at 45°C with constant stirring. The polymer was precipitated with hexane and collected by freeze drying. The product was stored in liquid N 2 until use. 1 Figure S1D).

acid-base titration
The buffering capacity of the polycations was determined by acid-base titration. PAA, bPEI, or dPEI containing 0.1 mmol protonable amine groups was dissolved in 10 mL of 150 mM NaCl aqueous solution. The pH of the solution was adjusted to 2 using 1 M HCl. The solution was titrated with 100 mM NaOH. Buffering capacity is defined as the percentage of protonable amine groups that become protonated from pH 7.4 to 5.1 and can be calculated as follows: 24,31 Buffering capacity (%) where ΔV NaOH is the titration volume of NaOH added to change the pH value from 5.1 to 7.4; N is the number of moles of protonable amine groups in the polymer.

Deposition of the LbL films
The LbL films were deposited by the dip coating method using a programmable Carl Zeiss HMS50 slide stainer (Carl Zeiss Meditec AG, Jena, Germany) with a homemade substrate holder following our previous work. 13 The substrate holder containing five-pieces substrate was first dipped into the PAA or PEI solution (0.5 mg/mL in 30 mM pH 5.5 acetate buffer and with 0.1 M NaCl) for 10 minutes and then in the plasmid DNA solution (0.25 mg/mL in 30 mM pH 5.5 acetate buffer and with 0.1 M NaCl) for 10 minutes. Between each polycation and DNA dipping, the film was rinsed by deionized water for 5 minutes. The polycation and DNA deposition process was repeated to construct the desired number of layers. The dipping solution was refreshed every eight dipping cycles in order to minimize the concentration variation. All the films were terminated with the polycation layer.

AFM imaging
AFM imaging was conducted using Veeco Dimension 3100 AFM (Veeco Instruments, Inc., Plainview, NY, USA). When the tapping mode was used in air to measure the film thickness, the AFM probe used was a silicon probe tap 300-G (BudgetSensors, Sofia, Bulgaria) with a factory-specified resonant frequency of 300 kHz and a spring constant of 40 N/m. In order to measure the LbL film thickness, the film was scratched with a razor blade to expose a part of the glass substrate. Film thickness was determined by the step height between the substrate and the film surface using the sectional height analysis command of the Nanoscope software version 5.12 (Veeco Instruments, Inc.). The film thickness was measured in triplicate. AFM contact mode in liquid was used to monitor film degradation in situ in 100 μL of 10 mM DTT in PBS buffer (pH 7.4) at room temperature using nonconductive silicon nitride probe NP-20 (Veeco Instruments, Inc.) with a nominal radius of 20 nm and a nominal cantilever spring constant of 0.32 N/m. AFM imaging ensued immediately after DTT solution injection. The surface was imaged continuously at an average rate of 1 Hz for 6 hours.
AFM contact mode in liquid and tapping mode in air were used to study polyplexes as well as the particles released from the bioreducible LbL films after the DTT reaction. In a typical experiment, a droplet of 50 μL polyplex (or solution containing the degradation product from the LbL films) was placed on a 1×1 cm 2 freshly cleaved mica substrate. After 5-minute incubation in room temperature, the substrate was rinsed by deionized water to remove the excess solution. Nonconductive silicon nitride probes (NP-20; Veeco Instruments, Inc.) were used for the liquid contact mode. PBS buffer (100 μL) was added on the sample surface to cover the sample and probe. Particle size and volume were analyzed

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Layer-by-layer DNA films for sequential gene delivery by Nanoscope software (version 5.12b; Veeco Instruments, Inc.). Bearing analysis command was used to calculate the particle volume. The analysis and calculation methods were the same as in our previous report. 33

Fluorescence spectroscopy
The amount of DNA released from the LbL films was determined by a fluorimeter. The LbL films were immersed in 10 mM DTT in PBS buffer (pH 7.4). After the film was completely degraded, the solution in contact with the film was concentrated to 250 μL before labeling the DNA in the solution with 10,000× SYBR safe DNA stain (Thermo Fisher Scientific). In a typical experiment, SYBR safe DNA stain was first diluted in Tris-acetate-EDTA buffer, and 100 μL of the diluted solution was added to 250 μL concentrated DNA solution. Then, 150 μL labeled sample was pipetted into 96-well plates. The same procedure was used to prepare standard DNA solutions of 0, 5,10,15,20,25,30,35, and 40 μL/mL concentrations in order to construct the standard curve. The standard curve varies linearly with concentration and was used to determine the DNA amount released from the LbL films.

Dls
The particles in the LbL film degradation solution were analyzed using a Zetasizer (Nanosizer ZS; Malvern Instruments, Malvern, UK). In order to monitor the size of the particles released from the LbL films during reductive degradation in 10 mM DTT in PBS buffer (pH 7.4), the glass slide coated with the LbL films was cut into small pieces, 5 mm in diameter, to fit the microcuvette (ZEN0040; Malvern Instruments). We used three-dimensional printing to make a nylon microcuvette insert to hold the LbL sample in the top portion of the microcuvette. The microcuvette was filled with the DTT solution until just submerging the LbL films. The DLS measurements were performed immediately to determine the effective hydrodynamic diameter (D H ) of the particles released from the LbL film. The backscattering angle Θ was fixed at 172° with a laser wavelength λ=633 nm. The size measurement range was set between 1 nm and 6 μm. D H is a function of the diffusion coefficient (D), temperature (T), and viscosity (η) according to the Stokes-Einstein equation: D H = kT/3πηD where k is the Boltzmann constant, T is 25°C, and D is obtained from the autocorrelation function via the cumulate fitting. Data were collected every 15 minutes in the first 24 hours and every 30 minutes after that. The DTT solution was refreshed every 24 hours. The film degradation was continuously monitored for 14 days.
In vitro cell transfection HEK293 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). NIH3T3 (organism: Mus musculus) was purchased from ATCC through Dr Wei-Zen Wei's laboratory, Barbara Ann Karmanos Cancer Institute, Wayne State University. Osteoblast precursor MC3T3 cell line (from mouse calvaria) was purchased from ATCC through Dr Weiping Ren's laboratory, Department of Biomedical Engineering, Wayne State University. HEK293 and NIH3T3 cells were cultured and maintained in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and incubated at 37°C in a humidified incubator with 5% CO 2 . MC3T3 cells were cultured and maintained in MEM α supplemented with 10% FBS, 1% penicillin-streptomycin, and incubated at 37°C in a humidified incubator with 5% CO 2 . The medium was replaced every 2 days.
Transfection experiments using polyplexes were performed with HEK293, MC3T3, and NIH3T3 by using plasmid pEGFP-N1 as the report gene. Transfection experiments of each cell line were carried out in separate 24-well plates at an initial cell density of ~80,000 cells per well. Cells incubated for 24 hours (37°C in a humidified incubator with 5% CO 2 ) had to be ~80% confluent before the polyplexes were added to the cell medium. Different basic amino acid residue-to-DNA phosphate molar ratios, the N/P ratios, from 6/1 to 24/1 were used to prepare the polyplexes. The cell medium was replaced with 300 μL fresh medium (without FBS) per well just before adding the polyplexes. Then, 50 μL solution of the polyplexes containing 1 μg DNA was added to each well. The cells were incubated with the polyplex containing transfection medium for 3 hours in a humidified incubator (37°C, 5% CO 2 ). Next, the polyplex containing medium was removed from each well and replaced with 1 mL/well fresh cell culture medium containing FBS. After 24-hour incubation in the humidified incubator at 37°C with 5% CO 2 , cell images were captured by fluorescence microscopy. The polyplex transfection efficiency was determined by a flow cytometer, and cell viability was characterized by the MTT assay. All transfection experiments were carried out in triplicates.
In order to limit nonspecific cell attachments, the culture plates were coated with poly-HEMA. To apply the poly-HEMA coating, 100 mg poly-HEMA powder was dissolved in 5 mL ethanol and water mixture (v:v=95:1) by heating while stirring at 40°C. Then, 100 μL of filtered poly-HEMA solution was added into each well of the 24-well plate. The plate was kept in sterile conditions and dried overnight.
Transfection experiments on the LbL films were performed with HEK293 and MC3T3 using plasmid pEGFP-N1

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Xie et al and pEF.myc.ER-E2-Crimson as the reporter genes. LbL films deposited on 15 mm diameter glass coverslips were placed at the bottom of 24-well poly-HEMA-coated plates. The LbL films were sterilized under UV light for 1.5 hours before use. Fibronectin (40 μL) was added on the top of each film and allowed to dry in a sterilized environment for 2 hours. Then, the films were seeded with 1.5×10 4 cells in DMEM culture medium containing 10% FBS and incubated at 37°C in a humidified incubator with 5% CO 2 . The culture medium was refreshed every 2 days. Cell attachment and proliferation were checked and imaged daily with an inverted optical microscope.

cell viability assay
The number of viable cells was measured by using Vybrant MTT Cell Proliferation Assay Kit. 35,36 The MTT assay value for untreated cells (ie, cells not exposed to the transfection medium or LbL film) was taken as 100% cell viability. Briefly, after 24-hour incubation of polyplex-treated cells, the cell culture medium was removed and replaced with 100 μL fresh culture medium. Then, 10 μL of 12 mM MTT stock solution was added to each well. After 4-hour incubation at 37°C, 100 μL sodium dodecyl sulfate (SDS)-HCl was added to each well and mixed thoroughly with a pipette. Cell incubation continued at 37°C in the humidified chamber for 10 hours. The absorbance was measured at 570 nm. The cells cultured on the LbL surface were treated with the MTT assay kit on day 6. The method followed the same procedure as described above. All experiments were performed in triplicates.

Flow cytometry
Flow cytometry measurements were performed on triplicates immediately after each collection of cultured cells. All the cells were trypsinized and resuspended in Fluorescenceactivated cell sorting (FACS) buffer (1% BSA in PBS). All negative control experiments were conducted with cultures not exposed to polyplexes or grown on bare glass substrates without the LbL coating. All samples were measured with an Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific) using 488 nm for GFP excitation. GFP fluorescence was detected using a photomultiplier tube with a 530/30 bandpass filter. An electronic gate encompassed live cells based on the forward and side scatter properties of the population and a minimum of 10,000 gated events per sample was collected and stored in list mode files. Data analysis was performed with Attune software version 2.1 (Thermo Fisher Scientific). Nontransfected cells were used for background calibration.

Polymer synthesis and characterization
We employed Michael addition copolymerization of a primary amine monomer, APOL, and two bisacrylamide monomers, CBA and MBA, to prepare PAAs with different contents of disulfides by varying the CBA to MBA molar ratio (Scheme 1). The synthesized PAAs are linear polymers with various CBA/(CBA + MBA) percentages (33%, 67%, and 100%) according to the feed ratio. The polymer chemical composition was characterized by 1 H NMR using a spectrometer (400 MHz) ( Figure S1). The weight-average molecular weight (M w ) of three different poly(amino pentanol) (pAPOLs) (pAPOL-1, pAPOL-2, and pAPOL-3) is in the range of 1,200-3,200 with a polydispersity index (M w / [number-average molecular weight] M n ) range of 1.0-1.5 ( Table 1). The low molecular weight of the pAPOLs is attributed to primary amines, such as APOL, being less nucleophilic than secondary amines, which limits the reaction rate and molecular weight in Michael addition polymerization. 37 We synthesized dPEI by allowing 800 Da PEI to react with 1,6-hexanediol diacrylate. 38,39 The acrylate group reacts with either a primary amine or a secondary amine resulting in a highly branched structure. The ester bonds in dPEI are susceptible to hydrolysis at physiologic conditions. M w and M w /M n of PAAs and dPEI were determined by size exclusion chromatography in 0.3 sodium acetate (pH 4.4)/methanol (70/30, v/v) using Waters Alliance 2690 Separations Module (Waters Corporation, Milford, MA, USA) equipped with Waters Ultrahydrogel (500, 120 Å) column. Poly(ethylene glycol) (molecular weight of the peak maxima [Mp] =21,160 to 106 Da) was used as the standard.
We measured the buffering capacity of the synthesized PAAs in order to predict their gene delivery efficiency. The buffering capacity is defined as the percentage of amine groups becoming protonated when the pH changes from 7.4 to 5.1 during acid-base titration. 24,31 The buffering capacity of pAPOL-1

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Layer-by-layer DNA films for sequential gene delivery was calculated to be 82% based on the volume of 0.1 M NaOH solution used to increase the pH value of the pAPOL-1 solution from 5.1 to 7.4 ( Figure 1; Table 1). The pH range is chosen to correspond to the highest pH in the extracellular environment to the lowest endosomal pH. 40 Of all the polymers studied, pAPOL-1 with 100% CBA displays the highest buffering capacity followed by bPEI with pAPOL-3, and pAPOL-2 and dPEI displaying the lowest buffering capacity.

Polyplex characterization
We studied the size and size distribution of polyplexes using AFM and DLS because polyplex size is one of the major factors determining DNA delivery efficiency. 41 The incubation time of polyplex formation was fixed at 30 minutes after the initial mixing because of the polyplex size dependence on its formation kinetics. 33,42 All the polycations studied were found to be capable of condensing DNA into polyplexes with an overall positive charge ( Table 2). Both bPEI and dPEI are effective in forming compact polyplexes with hydrodynamic size approaching 60 nm consistent with the literature values. 31 The smallest hydrodynamic size for the pAPOL polyplexes is around 100 nm. There was a significant reduction in size for pAPOL-1 and pAPOL-2 when the N/P ratio was increased from 8 to 12. The polyplexes formed by pAPOL-3 are larger and their size does not decrease with increasing N/P ratio.
To further investigate polyplex stability, we collected DLS data as a function of incubation time as shown in Figure 2.
The data show that the sizes of pAPOL-1, pAPOL-2, and dPEI polyplexes were unchanged during the 1-hour measurement time while the size of the pAPOL-3 polyplexes increased steadily with time. The increasing size is due to the agglomeration of the primary polyplex particles. Particle agglomeration is likely due to attractive polymer bridging interactions between loosely bound polyplexes. 33 A more rigid pAPOL-3 molecule may make it more difficult to fully condense DNA. Lastly, we found no clear correlation between the zeta potential and polyplex size stability. The polyplexes of pAPOL-1 with N/P ratio of 12 were imaged by AFM in air. Polyplexes 15 minutes post-formation tend to have a rod shape while those 45 minutes postformation show a spheroidal shape (Figure 3). These data are consistent with our previous observations of PAAs of different chemical compositions showing a strong morphological dependence on incubation time. 33

Polyplex transfection activity and cytotoxicity
To evaluate the transfection capability of the PAAs, their polyplexes containing pEGFP-N1 plasmid DNA were incubated with HEK293, MC3T3, and NIH3T3 cells. N/P ratios of the PAAs ranging from 12 to 16 were used. Polyplexes with N/P ratios of 8 and 24 did not show significant   Figure 4A) and mean fluorescence intensity (MFI) ( Figure 4B) are used here to express the transfection efficiency. The total number of events measured is 10,000 per sample. Of all the pAPOLs, pAPOL-1 exhibits the highest transfection efficiency; pAPOL-2 shows some transfection activities; and pAPOL-3 shows no transfection (data not shown). bPEI was able to transfect HEK293 cells and NIH3T3 cells but not as effectively in MC3T3 cells. Here, pAPOL-1 shows significant higher transfection activities than bPEI in MC3T3 cells. The biodegradable dPEI shows low transfection in all three cell lines. Figure 4C reports the cell viability data by the MTT assay. The control group consists of cells attaching and proliferating on the culture plate in the absence of the polyplexes. Polyplexes of pAPOL-1 at N/P=12 show the lowest toxicity in all three cell lines. Polyplexes of pAPOL-1 at N/P=16 and pAPOL-2 show significant toxicity to MC3T3 cells, which may be related to the lower rate of mitosis of MC3T3 compared with NIH3T3 and HEK293. 43 dPEI is less toxic to cells than bPEI as expected. 31 LbL film assembly and structural analysis The above polycation screening based on polyplexes has identified pAPOL-1 to be a suitable polycation with low cytotoxicity and high transfection efficiency for the

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Layer-by-layer DNA films for sequential gene delivery subsequent LbL film study. We prepared 16.5 bilayers of LbL films containing pAPOL-1 and DNA. In addition, bPEI or dPEI was inserted periodically as a barrier against interlayer diffusion (LbL film 1 and film 2 in Scheme 2). The LbL film deposition process was monitored by AFM. The film thickness was measured in AFM height images of samples where part of the film was removed so the height difference between the film surface and bare substrate can be determined. 13 Figure 5A and B shows the film growth patterns for LbL film 1 and film 2. Figure 5C and D shows AFM images of the films containing the scratched area for film thickness measurements. Both films show a relatively smooth texture. Both types of films exhibit linear growth behavior where the film thickness increases linearly with the number of layers. This linear growth behavior suggests minimal interlayer diffusion in the LbL films. [44][45][46] The total thickness for LbL film 1 is 77.5±1.9 nm, which yields an average polycation/DNA bilayer thickness of 4.6 nm. The total film thickness for LbL film 2 is 70.1±3.0 nm with the thickness per bilayer of 4.2 nm. Our data suggest that both bPEI and dPEI are effective diffusion barriers to maintain the layered structure in the LbL films. The same films were also measured in PBS buffer to show a wet film thickness of 103.7±3.9 nm for film 1 and 111.8±2.9 nm for film 2.

Film disassembly in solution
The LbL films immersed in reductive 10 mM DTT solution were taken out periodically to be imaged by AFM in air. Figure 6A shows AFM images captured during LbL film 1 degradation. The film (dry) thickness and surface roughness variation with degradation time are plotted in Figure 6B and C, respectively. At t=0, the film thickness is ~80 nm and the root mean square roughness is ~15 nm. Both decreased in values during the degradation period up to 225 hours. The film thickness decreased more rapidly during the first 24 hours indicating an initial burst release behavior and then decreased more slowly toward the limiting film thickness of 9.7±0.4 nm at 225 hours. The initial film roughness is 15.1±0.6 nm, which increased slightly to 19 nm in the first 4 hours, followed by a gradual decrease to ~15 nm at 50 hours and ~10 nm until the

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Layer-by-layer DNA films for sequential gene delivery end of the observation period. The initial roughness increase is due to many particles being released from the LbL film during the initial film degradation period. When LbL films undergo bulk degradation, we tend to observe large micron size pieces missing from the film. The fact that we did not observe such missing pieces from LbL film 1 and 2 indicates that the films degrade more gradually, following the LbL disassembly mechanism. LbL film 2 disassembles similarly as LbL film 1. During an 8-day degradation study, LbL 2 film (dry) thickness decreased gradually to 10.3±0.4 nm.
Our study indicates that both bPEI and dPEI are effective in preventing bulk degradation of the pAPOL/DNA films. Film degradation was also studied using real-time AFM imaging in DTT solution. We monitored film 2 disassembly in 10 mM DTT solution for 6 hours in order to capture the initial disassembly process in real time (Figure 7). Figure 7A shows the film surface morphological change in the reducing environment. We did not observe micrometer patches in the film indicating the absence of bulk film erosion. We focused our imaging on an area where part of the film was removed prior to the real-time experiment, by a razor blade, in order to monitor the film thickness change during film degradation in DTT solution. The starting film thickness is 104±4 nm with a surface roughness of 33.9 nm measured in PBS solution ( Figure 5D). The film thickness reduction is more pronounced in the first 150 minutes, by about 50% (Figure 7B). After 150 minutes, the film degradation rate starts to slow down. After 8 days of degradation, the thickness of film 2 was 30±11 nm.
DNA delivery efficiency is also impacted by the particle morphology released from the LbL films upon reductive degradation. Therefore, we characterized the particles released from the LbL films by DLS. The D H of the particles collected in the incubation solution during LbL film degradation was continuously monitored as a function of degradation time. We averaged the D H values in one 24-hour period and reported the averages every 24 hours as a function of degradation time in Figure 8A. LbL film 1 which contains bPEI released particles with a D H size range of 200-400 nm throughout the observation period of 360 hours. It should be pointed out that the large error bar in the first 24 hours is due to the high concentration of particles released during this initial period resulting in significant particle agglomeration ( Figure S2). LbL film 2 containing dPEI released larger particles in the range of 350-600 nm. After 9 days, the number of particles released from LbL film 2 was insufficient for DLS detection. AFM thickness analysis also shows that film 2 was fully degraded after 9 days with the remaining 8.9% of the film accounted for by the irreversibly adsorbed anchoring layer. To study the particle morphology, the released particles were collected, deposited on mica, and imaged by the AFM tapping mode in PBS buffer ( Figure 8B-E). Further analysis by AFM of the released particle volume in air shows that most particles are 2-5×10 4 nm 3 in particle volume, which corresponds to 2-3 plasmid DNA molecules per particle (Figure 9). Particles released in the first 6 hours are rod or spheroid shaped with a volume of 2-3×10 4 nm 3 ( Figure 9A and B). Particles collected on day 7 ( Figure 9C) have a similar morphology as those collected at 6 hours. Approximately 13% of the particles exhibit much larger particle volume equivalent to containing 10-12 DNA plasmids per particle ( Figure 9D), which is due to particle agglomeration. The AFM and DLS data suggest that LbL film 1 releases smaller particles for a longer period of time than film 2. dPEI   is less toxic than bPEI because of its biodegradable property; on the other hand, our data suggest that dPEI's biodegradable property also limits its ability to maintain a compact polyplex shape during a prolonged delivery process. The degradation kinetics was further analyzed by fluorescence spectroscopy. The DNA amount released from the LbL films in the presence of DTT was determined by fluorescent labeling of DNA with SYBR safe DNA stain. The fluorescence data were calibrated using a known amount of DNA as described in the "Materials and methods" section. Figure 10 shows the continuous release of DNA from the LbL film during the 300-hour observation period. The film exhibited a burst release behavior in the first 24 hours consistent with the film thickness data measured by AFM, probably due to excess polyelectrolytes quickly coming out of the film at the beginning of film degradation. After 24 hours, the film degradation slowed down significantly consistent with surface erosion mechanism. We achieved sequential DNA release of the pAPOL-1/DNA films by the insertion of diffusion barrier layers of bPEI and dPEI.

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Layer-by-layer DNA films for sequential gene delivery In vitro transfection activity and cytotoxicity of the LbL films The transfection study was carried out by culturing HEK293 and MC3T3 cells directly on the LbL films. A layer of fibronectin was applied to the film prior to cell culture because fibronectin has been found to improve cell attachment and proliferation. 47 Figure 11 shows the bright field (left column) and fluorescence images (right column) of proliferating HEK293 cells (left panel) and MC3T3 cells (right panel) on LbL film 1 ( Figure 11A) and LbL film 2 ( Figure 11B). When the cells were cultured on film 1, they attached and grew well on the films throughout the observation period of 6 days. GFP fluorescence images indicate transfection of pEGFP-N1 plasmid DNA from day 2 till the end of the cell culturing period. We observed a significant increase in the number of green cells in day 4. HEK293 and MC3T3 cells did not attach and grow well on LbL film 2. Cells were observed to detach and aggregate starting on day 2 (MC3T3) and day 4 (HEK293). On day 6, most cells appeared unhealthy and aggregated. Although HEK293 cells can be transfected when cultured on LbL film 2 ( Figure 11B, left panel), the transfection efficiency was significantly reduced due to poor cell attachment and growth.  Flow cytometry was used to characterize cell transfection efficiency on day 6. Day 6 was chosen because of the maximum fluorescence signal observed. The percentages of transfected cells and MFI are used here to quantify the transfection efficiency. The total event measured is 10,000 for each sample. Cells grown on 24-well culture plates without any transfection agent were used as a negative control. Figure 12A shows the percentage of transfected cells and relative MFI for each experimental group. HEK293 cells grown on LbL film 1 had 9.9% cells with green fluorescence (Figure 12A), and the corresponding MFI value was 4.6-fold higher than that of the control group ( Figure 12B). The MFI value of MC3T3 cells on LbL film 1 was only 2.5-fold higher than that of the control. For LbL film 2, HEK293 had a much lower percentage of transfected cells, ~0.9% ( Figure 12A). Relative MFI was 4.0-fold higher than the control ( Figure 12B). The MC3T3 cells grown on LbL film 2 showed ,0.5% transfection percentage ( Figure 12A). The relative MFI was 1.2-fold higher than the control ( Figure 12B). Figure 12C plots the cell viability data cultured on the LbL films by the MTT assay. For HEK293 cells, the cell viability of film 1 is 85.8% and for LbL film 2 it is 21.6%. MC3T3 cells showed 73.1% cell viability on film 1 and 11.6% on film 2. Our data suggest that LbL film 1 is more biocompatible than film 2, and HEK293 cell line is less adversely affected than MC3T3 cell line by the presence of the LbL films.
Lastly, to demonstrate the ability of LbL film 1 for sequential gene delivery, two different DNA plasmids were incorporated into the film as shown in Scheme 3. The bottom nine bilayers were constructed using the pEF.myc.ER-E2-Crimson plasmid and its transfection gave red fluorescence. The top 7.5 bilayers contain the green fluorescent pEGFP-N1 plasmid. HEK293 cells were cultured on this LbL film for 10 days. Figure 13 shows the dominance of the green fluorescence on day 4 and the emergence of red fluorescence on day 6. It shows the persistence of green fluorescence from day 4 to day 8 and delayed expression of the Crimson plasmid from day 4 to day 10. The data demonstrate the promise of such LbL films for controlled and sequential gene delivery for biomedical applications.

Discussion
Cell transfection by DNA from LbL films is a complex process and often a difficult task to achieve. LbL films need to disassemble in such a manner that generates transient condensed DNA nanoparticles to allow cell uptake, endosomal escape, and intracellular trafficking into the nucleus. Then, the nanoparticles need to unpack to enable DNA transfection in the cell nucleus. A prerequisite for transfection by LbL films is to use biodegradable polycations capable of condensing DNA, enabling endosomal escape of their gene payload, and achieving high transfection while maintaining

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Layer-by-layer DNA films for sequential gene delivery  low cytotoxicity. Here, we have applied acid-base titration as an effective screening tool for polymer gene delivery vectors.
A bioreducible PAA with APOL side groups, pAPOL-1, was selected for the subsequent build-up of LbL films, based on its performance in the polyplex form. The acid-base titration utilizes the proton sponge hypothesis, which relates the buffering capacity of the polycation gene delivery vectors to their transfection efficiency. 24,48 The proton sponge effect allows endosomal escape of the polyplexes by creating an osmotic pressure to burst the endosomes. The amount of titratable amines in the polymer is the main contributor to buffering capacity by reducing acidification, increasing Cl − accumulation, and increasing endosomal swelling and lysis. 50 Primary amines in the polymers are predominately protonated at neutral pH. 49 PEI and pAPOLs both possess higher order amines, which give rise to higher buffering capacity in the relevant pH range. 49,50 The homogeneous protonation distribution over the whole endolysosomal pH range is attributed to the continuous chains of ethylamine groups in bPEI and dPEI. 51 It is unclear why pAPOL-1 has the strongest buffering capacity. Compared to the other pAPOLs, the average distance between amine groups is longer in pAPOL-1 and it has the lowest molecular weight (more end groups containing secondary amines). Both factors could contribute to its high buffering capacity. 52 The poor buffering capacity of dPEI compared to bPEI is likely due to its tendency to hydrolyze in the titration pH range. Incidentally, polyplexes of pAPOL-1 show the highest transfection efficiency with the lowest cytotoxicity. The higher transfection efficiency of pAPOL-1 is consistent with its high buffering capacity and biophysical characteristics. For comparison, even though dPEI is less toxic to cells than bPEI, 31 it is also less effective than bPEI due to dPEI's hydrolytic instability thus limiting its use in the LbL films. Another focus of this work is the incorporation of diffusion barrier layers in order to achieve sequential DNA release from LbL films by limiting bulk film degradation. While DNA itself is a nondiffusing polyanion, 53 bioreducible PAAs are capable of interlayer diffusion as indicated by its exponential growth pattern. 13 There exists a diffusion zone in the LbL films with interlayer diffusion, which acts as a reservoir during layer growth until reaching a kinetic limit. 44,48 The growing reservoir with increasing number of layers results in the exponential growth of the LbL films. 48 The insertions of both bPEI and dPEI layers into the pAPOL-1/DNA LbL films change the film growth pattern to a linear growth pattern. Our results show that the linearly grown films also undergo more gradual film disassembly lasting for days when compared to bulk film degradation in minutes. Our results show that periodic insertions of the bPEI or dPEI layer are effective in achieving a linear film growth pattern and preventing bulk erosion. PEI has a smaller diffusion coefficient and a higher charge density than PAAs. It binds strongly to the surface during LbL assembly and acts as a separating layer to prevent interlayer diffusion of PAA molecules. PEI acts as a barrier by screening the residue charge on the film. 53 More importantly, it limits chain mobility during film disassembly thus allowing the gradual release of DNA nanoparticles at the film/ cell interfacial zone. 13 Our results demonstrate the strategy of combining highly transfecting bioreducible pAPOL-1 International Journal of Nanomedicine 2018:13 submit your manuscript | www.dovepress.com

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Xie et al and PEI in the LbL film assembly to achieve controlled gene delivery from the films.

Conclusion
Bioreducible PAAs containing CBA, MBA, and APOL were synthesized by Michael addition polymerization for the construction of bioreducible LbL films capable of sequential gene delivery. The synthesized PAAs together with 25 kDa branched PEI and cross-linked 800 Da PEI were evaluated prior to their incorporation into LbL films by acid-base titration, cell transfection, and MTT assay in their polyplex form. The prescreening identified pAPOL-1, made by the copolymerization of CBA and APOL, to exhibit high buffering capacity, high transfection efficiency, and low cytotoxicity. Therefore, pAPOL-1 was selected for the subsequent construction of the LbL films. In addition to pAPOL-1, nondiffusing PEI was incorporated into the LbL films in order to achieve sustained DNA release from the LbL films in the polyplex nanoparticle form. Cell transfections conducted on HEK293 and MC3T3 cells showed that LbL films containing pAPOL-1 and 25 kDa branched PEI are effective in DNA transfection while LbL films containing pAPOL-1 and cross-linked 800 Da PEI are less efficient. We also demonstrated the dual-stage release and transfection of two different DNAs from the LbL films. The results indicate that LbL films consisting of bioreducible PAAs and nondiffusing polyelectrolytes have excellent degradation properties for the development of LbL coating technology for localized gene delivery applications including tissue/ bone regeneration, wound healing, drug/vaccine delivery, and production of stem cells.

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The Effects of Combinatorial Genistein and Sulforaphane in Breast Tumor Inhibition: Role in Epigenetic Regulation

Dietary compounds that possess the properties of altering epigenetic processes are gaining popularity as targets for cancer prevention studies. These compounds when administered at optimal concentrations and especially in combination can have enhanced effects in cancer prevention or therapy. It is important to study the interaction of two or more compounds in order to assess their role in enhancing prevention. Genistein (GEN), found in soy, has been extensively studied for its role as an epigenetic modifier especially as a DNA methyltransferase (DNMT) inhibitor and sulforaphane (SFN), found in cruciferous vegetables, is known as a histone deacetylase (HDAC) inhibitor. However, very little is known about the effects of these two compounds in conjunction in breast cancer prevention or therapy. In our current study, we determined that, at certain doses, the compounds have synergistic effects in decreasing cellular viability of breast cancer cell lines. Our results indicate that the combination of GEN and SFN is much more effective than their single doses in increasing the rate of apoptosis and lowering the colony forming potential of these cells. We determined that these compounds inhibit cell cycle progression to G2 phase in MDA-MB-231 and G1 phase in MCF-7 breast cancer cell lines. Additionally, we determined that the combination is effective as an HDAC and histone methyltransferase (HMT) inhibitor. Furthermore, we demonstrated that this combination downregulates the levels of HDAC2 and HDAC3 both at the mRNA and protein levels. We also found that these compounds have the potential to downregulate KLF4 levels, which plays an important role in stem cell formation. The combination of GEN and SFN is also effective in downregulating hTERT levels, which is known to be activated when KLF4 binds to its promoter region. Our hypothesis is further strengthened by in vivo studies, where the combination is administered to transgenic mice in the form of genistein and SFN-enriched broccoli sprouts. We have demonstrated that the combination is more effective in preventing or treating mammary cancer via extending tumor latency and reducing tumor volumes/sizes than either of these dietary components administered alone. These results are consistent with our in vitro study suggesting potential preventive and therapeutic effects of this novel dietary combinatorial approach against breast cancer.

Introduction
According to recent statistics, though the breast cancer death rate has decreased over the years, overall breast cancer incidence rates have increased among most races [1]. The cause for breast cancer is still unknown though most of the evidence points toward genetic and epigenetic abnormalities. Epigenetic gene alterations are an important hallmark of breast cancer. A common cause of breast cancer is epigenetic modifications, especially increasing DNA methylation and histone deacetylation. Bioactive dietary compounds are of special interest because of their anticancer properties and numerous studies have suggested that these dietary compounds can alter abnormal epigenetic states in cancer [2,3]. Regular consumption of bioactive nutritional compounds can alter the epigenome and play an active role in cancer prevention and therapy [4]. Several dietary compounds administered singly or in conjunction with other dietary compounds or other chemotherapeutic agents can reverse abnormal gene activation or silencing in cancer cells [5].
Combinatorial epigenetic marks are gaining importance as biomarkers for breast cancer diagnoses [6]. Choosing an optimum concentration of dietary combination that can decrease DNA methylation and deacetylation levels may be effective to synergistically activate tumor suppressors [7]. Thus, combinatorial regimens are gaining increasing popularity in cancer research because of their effectiveness in preventing and treating cancer. The combination of two or more compounds has been found to be more effective than compounds acting singly in many cases [8,9] by the use of a quantitative measure named Combination Index. Combination Index measures the degree of interaction among drugs in the form of synergism or antagonism for a given end point of the effect measurement [10]. The effectiveness of single compounds is too often performed at very high doses that can confer potential side effects. Lower doses of the single compounds in combination may have a better chemopreventive potential and translational impact.
Genistein (GEN), found in natural soybean, is an isoflavone that is a known DNA methyltransferase (DNMT) inhibitor while sulforaphane (SFN), which is enriched in cruciferous vegetables such as broccoli sprouts (BSp) and kale, has been shown to have histone deacetylase (HDAC) inhibiting properties [11,12]. Figure 1 shows the structure of GEN and SFN. The inhibitory effects of GEN and SFN in tumor inhibition may be attributed to modulating epigenetic pathways by altering epigenetic marks in cancer cells. Our previous studies have shown that a combinatorial diet consisting of DNMT and HDAC inhibitors can alter the epigenetic machinery via direct and indirect inhibition of DNMTs and HDACs, thereby leading to modulating aberrations in DNA methylation and histone acetylation/deacetylation in tumor-related genes [13][14][15]. SFN is an inhibitor of HDAC activity, which results in an increase in global and local histone acetylation of a number of genes [16]. Unlike the single compounds, the combination of GEN and SFN has been understudied. GEN acts as an DNMT inhibitor reducing aberrant gene hypermethylation and SFN is an HDAC inhibitor that can remodel chromatin structure by affecting histone acetylation status in cancer cells and providing an open chromatin configuration.

Introduction
According to recent statistics, though the breast cancer death rate has decreased over the years, overall breast cancer incidence rates have increased among most races [1]. The cause for breast cancer is still unknown though most of the evidence points toward genetic and epigenetic abnormalities. Epigenetic gene alterations are an important hallmark of breast cancer. A common cause of breast cancer is epigenetic modifications, especially increasing DNA methylation and histone deacetylation. Bioactive dietary compounds are of special interest because of their anticancer properties and numerous studies have suggested that these dietary compounds can alter abnormal epigenetic states in cancer [2,3]. Regular consumption of bioactive nutritional compounds can alter the epigenome and play an active role in cancer prevention and therapy [4]. Several dietary compounds administered singly or in conjunction with other dietary compounds or other chemotherapeutic agents can reverse abnormal gene activation or silencing in cancer cells [5].
Combinatorial epigenetic marks are gaining importance as biomarkers for breast cancer diagnoses [6]. Choosing an optimum concentration of dietary combination that can decrease DNA methylation and deacetylation levels may be effective to synergistically activate tumor suppressors [7]. Thus, combinatorial regimens are gaining increasing popularity in cancer research because of their effectiveness in preventing and treating cancer. The combination of two or more compounds has been found to be more effective than compounds acting singly in many cases [8,9] by the use of a quantitative measure named Combination Index. Combination Index measures the degree of interaction among drugs in the form of synergism or antagonism for a given end point of the effect measurement [10]. The effectiveness of single compounds is too often performed at very high doses that can confer potential side effects. Lower doses of the single compounds in combination may have a better chemopreventive potential and translational impact.
Genistein (GEN), found in natural soybean, is an isoflavone that is a known DNA methyltransferase (DNMT) inhibitor while sulforaphane (SFN), which is enriched in cruciferous vegetables such as broccoli sprouts (BSp) and kale, has been shown to have histone deacetylase (HDAC) inhibiting properties [11,12]. Figure 1 shows the structure of GEN and SFN. The inhibitory effects of GEN and SFN in tumor inhibition may be attributed to modulating epigenetic pathways by altering epigenetic marks in cancer cells. Our previous studies have shown that a combinatorial diet consisting of DNMT and HDAC inhibitors can alter the epigenetic machinery via direct and indirect inhibition of DNMTs and HDACs, thereby leading to modulating aberrations in DNA methylation and histone acetylation/deacetylation in tumor-related genes [13][14][15]. SFN is an inhibitor of HDAC activity, which results in an increase in global and local histone acetylation of a number of genes [16]. Unlike the single compounds, the combination of GEN and SFN has been understudied. GEN acts as an DNMT inhibitor reducing aberrant gene hypermethylation and SFN is an HDAC inhibitor that can remodel chromatin structure by affecting histone acetylation status in cancer cells and providing an open chromatin configuration.  We hypothesized that the optimal concentration of the two compounds should be effective in synergistically decreasing cancer cell viability. The correct combination of two or more dietary compounds that act synergistically can be very effective in preventing and treating breast cancer. Previous studies have shown that SFN exhibits chemopreventive activity by inducing cell cycle arrest and induction of cyclin-dependent kinase inhibitor 1A (p21 waf1/cip1) via KLF4 transcription factor mediation [17].
The role of KLF4 in breast cancer is ambiguous [18]. Downregulation of KLF4 in several cancers, such as colon, gastric, esophageal, prostate and lung may contribute to cellular hyperproliferation and malignant transformation, which is consistent with its role in cell cycle arrest and apoptosis [19][20][21]. KLF4 is thought to act both as a tumor suppressor and as an oncogene in breast cancer. However, KLF4 is highly expressed in more than 70% of breast cancers and functions as an oncogene [21]. It is also highly expressed in cancer stem cells [22]. Previous studies have shown that KLF4 is essential for promoting migration and invasion, resulting in tumor formation in vivo [20,23]. It is believed to promote tumor initiation through nuclear reprogramming [24]. Moreover, KLF4 has been shown to act in concert with HDAC2 and HDAC3. Another study has shown that KLF4 recruits epigenetic modifiers HDAC2 and HDAC3 at the VEGF promoter [25]. KLF4 also activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in embryonic stem cells [26]. Since we found an optimum dosage using combinatorial GEN and SFN for decreasing cellular viability, we investigated the effect of this combination on KLF4 at the transcriptional as well as protein level. Recent studies have shown that the anticancer properties of benzyl isothiocyanate, a chemopreventive constituent derived from cruciferous vegetables, were enhanced by KLF4 knockdown in breast cancer cells [27]. We hypothesized that the combination of GEN and SFN causes breast cancer inhibition in vitro and in vivo via inhibition of KLF4 post-translationally as well as HDAC2 and HDAC3, which acts in conjunction with KLF4. Additionally, we sought to test whether the combination of GEN and SFN will also be effective in inhibiting hTERT, which is activated by recruitment of KLF4 to its promoter region. Moreover, we sought to evaluate whether a combinatorial GEN and SFN-enriched broccoli sprout diet could be effective in increasing tumor latency in spontaneous mouse models of mammary cancer and be more efficient in reducing tumor weight and volume as compared to mice groups administered a single compound or a control diet.

Effect of the Combination Treatment on the Cellular Viability of MCF-7 and MDA-MB-231 Breast Cancer Cells
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was performed to determine the dose-dependent effects of the combination of GEN and SFN on the viability of breast cancer cells. Figure 2A-C show the effects of various doses of the dietary combination on MCF-7 and MDA-MB-231 breast cancer cell lines. In order to determine the optimum combination to be used for the study, the effect of the combinations was compared to their respective single doses with respect to their efficacy in decreasing cellular viability. CompuSyn software Version 1.0 (ComboSyn, Inc., Paramus, NJ, USA) [28] was used to determine the combination index (CI). The doses of 5 µM SFN + 10 µM GEN and 5 µM SFN + 15 µM GEN were found to be synergistically decreasing cellular viability in both breast cancer cell lines. Additionally, from previous studies, we know that 5 µM SFN and 10 µM or 15 µM GEN is not toxic to cells. We tested the effect of the combination in decreasing the viability of MCF10A non-cancerous cell lines and found that the combination does not significantly decrease the cellular viability of MCF10A cell lines. The lower doses of SFN and GEN were not as effective in decreasing cellular viability in combination and were not showing synergy in both the cell lines. The combination of 5 µM SFN + 10 µM GEN and 5 µM SFN + 15 µM GEN was effective in decreasing cellular viability in both cell lines compared to the dimethyl sulfoxide (DMSO) control. The combinatorial regimen was also synergistically effective in decreasing cellular viability when compared to their single doses. The combination of 5 µM SFN and 15 µM GEN had a CI <1, which is indicative of synergy [29]. This combination was extremely synergistic in MCF-7 cell lines having a CI = 0.295. Therefore, for our further experiments to study the effects at the molecular level, we decided to move forward with 15 µM GEN instead of 10 µM GEN. Table 1 shows the CI indices for some of the combinations. Table 1  having a CI = 0.295. Therefore, for our further experiments to study the effects at the molecular level, we decided to move forward with 15 µM GEN instead of 10 µM GEN. Table 1 shows the CI indices for some of the combinations. Table 1

Combination of GEN and SFN Promote Cell Death as Evidenced through Cell Density Analysis and Apoptosis Assay
As evidenced through cell density analysis (by capturing photos of cultured cells) in Figure 3, both MCF-7 and MDA-MB-231 cells show a decrease in cell density and an increase in surface area between cells with the incorporation of the predetermined optimal concentrations of 5 µM SFN and 15 µM GEN in comparison with the MCF10A non-cancerous control cells. Both cell lines show these compounds to be effective in promoting apoptosis after three days of treatment. Furthermore, Figure  4 demonstrates an increase in apoptosis with the incorporation of combinatorial treatment when compared to treatment with single compounds further strengthening the hypothesis that the combination of GEN and SFN is more effective in increasing the rate of apoptosis in breast cancer cells than the same doses of the singly-administered compounds.

Combination of GEN and SFN Promote Cell Death as Evidenced through Cell Density Analysis and Apoptosis Assay
As evidenced through cell density analysis (by capturing photos of cultured cells) in Figure 3, both MCF-7 and MDA-MB-231 cells show a decrease in cell density and an increase in surface area between cells with the incorporation of the predetermined optimal concentrations of 5 µM SFN and 15 µM GEN in comparison with the MCF10A non-cancerous control cells. Both cell lines show these compounds to be effective in promoting apoptosis after three days of treatment. Furthermore, Figure 4 demonstrates an increase in apoptosis with the incorporation of combinatorial treatment when compared to treatment with single compounds further strengthening the hypothesis that the combination of GEN and SFN is more effective in increasing the rate of apoptosis in breast cancer cells than the same doses of the singly-administered compounds.

Combinatorial GEN and SFN Administration Decreases HDAC and HMT Expression
In order to observe the effect on epigenetic modifiers such as histone methyltransferases (HMTs) and HDACs, we treated the cells with the combination of GEN and SFN. In Figure 6, we found a significant decrease in enzymatic activity of HDACs in both cell lines. The combination treatment is more effective in the inhibition of overall HDAC activity. However, there was no significant downregulation of HDAC activity in the non-cancerous MCF10A cell line with the use of this combination. In Figure 7, we observed the effect of the combination of GEN and SFN in decreasing HMT activity in MCF-7 and MDA-MB-231 cell lines. The combination was effective in decreasing HMT activity more effectively than the DMSO control and single doses. There was, however, no significant decrease in HMT activity in the MCF10A control cell lines.

Combinatorial GEN and SFN Administration Decreases HDAC and HMT Expression
In order to observe the effect on epigenetic modifiers such as histone methyltransferases (HMTs) and HDACs, we treated the cells with the combination of GEN and SFN. In Figure 6, we found a significant decrease in enzymatic activity of HDACs in both cell lines. The combination treatment is more effective in the inhibition of overall HDAC activity. However, there was no significant downregulation of HDAC activity in the non-cancerous MCF10A cell line with the use of this combination. In Figure 7, we observed the effect of the combination of GEN and SFN in decreasing HMT activity in MCF-7 and MDA-MB-231 cell lines. The combination was effective in decreasing HMT activity more effectively than the DMSO control and single doses. There was, however, no significant decrease in HMT activity in the MCF10A control cell lines.

GEN and SFN in Combination Induces Changes in KLF4, HDAC2 and HDAC3 mRNA Levels
KLF4 has an ambiguous role in breast tumor inhibition. Since KLF4 acts as an oncogene in most breast cancer cells, we decided to check the effect of the combinatorial regimen in downregulating the oncogene at the transcriptional level. As hypothesized, in MCF-7 cells, the combination of 5 µM SFN and 15 µM GEN was effective in downregulating KLF4 more effectively than their singly-administered doses at the mRNA level ( Figure 8A). However, in the MDA-MB-231 cells, we did not observe a significant downregulation with the combination at the mRNA level. Since KLF4 works in concert with HDAC2 and HDAC3, we sought to observe its effect on the two epigenetic modifying enzymes at the transcriptional level. As expected, the combination was effective in downregulating HDAC2 and HDAC3 at the transcriptional level. Figure 8B,C show the effect of the combination on HDAC2 and HDAC3 mRNA level.

GEN and SFN in Combination Induces Changes in KLF4, HDAC2 and HDAC3 mRNA Levels
KLF4 has an ambiguous role in breast tumor inhibition. Since KLF4 acts as an oncogene in most breast cancer cells, we decided to check the effect of the combinatorial regimen in downregulating the oncogene at the transcriptional level. As hypothesized, in MCF-7 cells, the combination of 5 µM SFN and 15 µM GEN was effective in downregulating KLF4 more effectively than their singlyadministered doses at the mRNA level ( Figure 8A). However, in the MDA-MB-231 cells, we did not observe a significant downregulation with the combination at the mRNA level. Since KLF4 works in concert with HDAC2 and HDAC3, we sought to observe its effect on the two epigenetic modifying enzymes at the transcriptional level. As expected, the combination was effective in downregulating HDAC2 and HDAC3 at the transcriptional level. Figure 8B,C show the effect of the combination on HDAC2 and HDAC3 mRNA level.

Combination Treatment Induces Changes in KLF4, hTERT, HDAC2 AND HDAC3 Protein Levels
Since KLF4 is known to be recruited to the promoter region of hTERT for its activation [31], we sought to evaluate the effect of the combination treatment on hTERT protein levels. We found that, the combination was effective in downregulating KLF4 protein levels and hTERT protein levels. Figure 9A shows that the combination is more effective than the single doses in inhibiting KLF4. The densitometry analysis of the image using imageJ software shows the quantitative analyses of the downregulation. Figure 9B shows the protein level downregulation of hTERT using the combination and the corresponding quantitative densitometry analyses of the images. Figure 10A shows the protein level downregulation of HDAC2 and HDAC3 which is believed to act in concert with KLF4. Since KLF4 is downregulated by the combinatorial regimen, we tested for downregulation of HDAC2 and HDAC3. The western blot analyses show downregulation of HDAC2 ( Figure 10A) and downregulation of HDAC3 ( Figure 10B)

Combination Treatment Induces Changes in KLF4, hTERT, HDAC2 AND HDAC3 Protein Levels
Since KLF4 is known to be recruited to the promoter region of hTERT for its activation [31], we sought to evaluate the effect of the combination treatment on hTERT protein levels. We found that, the combination was effective in downregulating KLF4 protein levels and hTERT protein levels. Figure 9A shows that the combination is more effective than the single doses in inhibiting KLF4. The densitometry analysis of the image using imageJ software shows the quantitative analyses of the downregulation. Figure 9B shows the protein level downregulation of hTERT using the combination and the corresponding quantitative densitometry analyses of the images. Figure 10A shows the protein level downregulation of HDAC2 and HDAC3 which is believed to act in concert with KLF4. Since KLF4 is downregulated by the combinatorial regimen, we tested for downregulation of HDAC2 and HDAC3. The western blot analyses show downregulation of HDAC2 ( Figure 10A) and downregulation of HDAC3 ( Figure 10B

Combination Treatment Induces Changes in KLF4, hTERT, HDAC2 AND HDAC3 Protein Levels
Since KLF4 is known to be recruited to the promoter region of hTERT for its activation [31], we sought to evaluate the effect of the combination treatment on hTERT protein levels. We found that, the combination was effective in downregulating KLF4 protein levels and hTERT protein levels. Figure 9A shows that the combination is more effective than the single doses in inhibiting KLF4. The densitometry analysis of the image using imageJ software shows the quantitative analyses of the downregulation. Figure 9B shows the protein level downregulation of hTERT using the combination and the corresponding quantitative densitometry analyses of the images. Figure 10A shows the protein level downregulation of HDAC2 and HDAC3 which is believed to act in concert with KLF4. Since KLF4 is downregulated by the combinatorial regimen, we tested for downregulation of HDAC2 and HDAC3. The western blot analyses show downregulation of HDAC2 ( Figure 10A) and downregulation of HDAC3 ( Figure 10B)

Combinatorial Dietary Regimen of GEN and Broccoli Sprouts (BSp) Is Effective in Reducing Mammary Tumor Incidence and Delaying Tumor Latency in a Spontaneous Breast Cancer Mouse Model
In order to test the efficacy of the combinatorial dietary regimen of GEN and BSp in an in vivo model system, we treated the transgenic mouse model, C(3)1-SV40, that can spontaneously develop mammary cancer in early age, with either GEN and BSp alone or with a combination treatment of these diets. The special diets were supplemented with 250 mg/kg of GEN and/or 13% BSp in corn-oilbased AIN-93G diet from four weeks of age and continued throughout the study, which mimics the processes of prevention and therapeutic effects by these treatments. The concentrations of the diets used in this study were corresponding to ~2 g of isoflavone intake and/or consuming 133 g (~2 cups) broccoli sprout/per day by an adult human, respectively.
We found that the combination treatment of GEN and BSp was the most effective in reducing tumor incidence and tumor volume as compared to the control and either single treatment alone during the whole course of the experiment ( Figure 11A,B). Figure 11C shows the actual tumor comparison in different treatment groups at the endpoint and the combination treatment resulted in the smallest tumor sizes compared to the other treatment groups. More importantly, our results indicate that only the combinatorial treatment with GEN and BSp can lead to a significant delay of tumor latency (p < 0.05). These results suggest that the combination treatment with soybean GEN and SFN-enriched BSp are more effective in both preventing mammary cancer via extending tumor latency and reducing tumor volumes/sizes than either of these treatments alone. These results are consistent with the in vitro study suggesting potential preventive and therapeutic effects of this novel dietary approach on breast cancer.

Combinatorial Dietary Regimen of GEN and Broccoli Sprouts (BSp) Is Effective in Reducing Mammary Tumor Incidence and Delaying Tumor Latency in a Spontaneous Breast Cancer Mouse Model
In order to test the efficacy of the combinatorial dietary regimen of GEN and BSp in an in vivo model system, we treated the transgenic mouse model, C(3)1-SV40, that can spontaneously develop mammary cancer in early age, with either GEN and BSp alone or with a combination treatment of these diets. The special diets were supplemented with 250 mg/kg of GEN and/or 13% BSp in corn-oil-based AIN-93G diet from four weeks of age and continued throughout the study, which mimics the processes of prevention and therapeutic effects by these treatments. The concentrations of the diets used in this study were corresponding to~2 g of isoflavone intake and/or consuming 133 g (~2 cups) broccoli sprout/per day by an adult human, respectively.
We found that the combination treatment of GEN and BSp was the most effective in reducing tumor incidence and tumor volume as compared to the control and either single treatment alone during the whole course of the experiment ( Figure 11A,B). Figure 11C shows the actual tumor comparison in different treatment groups at the endpoint and the combination treatment resulted in the smallest tumor sizes compared to the other treatment groups. More importantly, our results indicate that only the combinatorial treatment with GEN and BSp can lead to a significant delay of tumor latency (p < 0.05). These results suggest that the combination treatment with soybean GEN and SFN-enriched BSp are more effective in both preventing mammary cancer via extending tumor latency and reducing tumor volumes/sizes than either of these treatments alone. These results are consistent with the in vitro study suggesting potential preventive and therapeutic effects of this novel dietary approach on breast cancer.

Discussion
The side effects of toxic drugs and chemotherapy have led to a growing interest in compounds that provide non-toxic and safer therapy. The use of dietary compounds as chemotherapeutic and chemopreventive agents is becoming very popular as an additional form of therapy. A healthy diet still remains among the most effective chemoprevention methods [32]. At the molecular level, a healthy diet consisting of bioactive nutritional compounds ensures proper epigenetic regulation of the system, which can facilitate prevention of cancer. Breast cancer is one of the leading causes of cancer death in women. The most prominent epigenetic features altered in breast cancer are DNA methylation patterns and chromatin states [33]. Therefore, the study of bioactive dietary compounds has gained prominence because they may have the ability to modulate epigenetic aberrations in cancer and restore the normal epigenetic state of oncogenes or tumor suppressor genes [34].
Breast cancer statistics have revealed that the incidence of breast cancer is much lower among Asians as compared to Caucasians and African Americans [35]. Previous studies have attributed this variation in breast cancer incidence to differences in diet and lifestyle [36]. The Asian diet in general is rich in soy and cruciferous vegetables and as a result may provide protection against breast cancer [37]. Our current study focuses on assessing the impact of consumption of GEN and cruciferous vegetables in breast tumor prevention.
GEN, which is an isoflavone found in soybeans, is an anticancer agent due to its property of being a DNMT inhibitor. GEN has also shown HDAC inhibiting properties. Additionally, GEN has been known to resensitize breast cancer cells to estrogen-targeted chemotherapy by activation of estrogen receptor in ER-negative breast cancer [38]. GEN has been known to block breast cancer cells in G2/M phase of the cell cycle by induction of p21 WAF1/CIP1 expression [39] and increase

Discussion
The side effects of toxic drugs and chemotherapy have led to a growing interest in compounds that provide non-toxic and safer therapy. The use of dietary compounds as chemotherapeutic and chemopreventive agents is becoming very popular as an additional form of therapy. A healthy diet still remains among the most effective chemoprevention methods [32]. At the molecular level, a healthy diet consisting of bioactive nutritional compounds ensures proper epigenetic regulation of the system, which can facilitate prevention of cancer. Breast cancer is one of the leading causes of cancer death in women. The most prominent epigenetic features altered in breast cancer are DNA methylation patterns and chromatin states [33]. Therefore, the study of bioactive dietary compounds has gained prominence because they may have the ability to modulate epigenetic aberrations in cancer and restore the normal epigenetic state of oncogenes or tumor suppressor genes [34].
Breast cancer statistics have revealed that the incidence of breast cancer is much lower among Asians as compared to Caucasians and African Americans [35]. Previous studies have attributed this variation in breast cancer incidence to differences in diet and lifestyle [36]. The Asian diet in general is rich in soy and cruciferous vegetables and as a result may provide protection against breast cancer [37]. Our current study focuses on assessing the impact of consumption of GEN and cruciferous vegetables in breast tumor prevention. GEN, which is an isoflavone found in soybeans, is an anticancer agent due to its property of being a DNMT inhibitor. GEN has also shown HDAC inhibiting properties. Additionally, GEN has been known to resensitize breast cancer cells to estrogen-targeted chemotherapy by activation of estrogen receptor in ER-negative breast cancer [38]. GEN has been known to block breast cancer cells in G2/M phase of the cell cycle by induction of p21 WAF1/CIP1 expression [39] and increase acetylation of histones 3 and 4 and H3-lysine4 at the p21 WAF1/CIP1 and p16 INK4a transcription start sites, mediated by induction of histone acetyltransferases (HATs) in prostate cancer cells [40]. In renal cancer, GEN increases histone acetylation by enhancing HAT activity and causes acetylation of histones 3 and 4, dimethyl-H3K4 and trimethyl-H3K4 near the transcription start site at the BTG3 gene promoter [41]. Our lab has recently shown that GEN alters the gut microbiome of breast cancer patients possibly via epigenetic modulation brought about by metabolites produced by bacteria residing in the gut [42]. SFN, which is abundant in cruciferous vegetables such as broccoli or kale, is an HDAC inhibitor and is known to be an effective inhibitor of several class I and II HDAC inhibitors [43]. It inhibits breast cancer stem cells in vitro and in vivo by suppressing the Wnt/ β-catenin pathway [44,45]. Our earlier studies have shown that SFN can cause epigenetic repression of hTERT expression in breast cancer cells [46]. It exhibits chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21 WAF1/CIP1 ). Higher levels of HDAC1 and HDAC2 expression are significantly associated with tumor differentiation [47]. Previous studies have shown that SFN binds to the HDAC pocket and inhibits HDAC activity [48]. In our current study, we used a combination of an HDAC inhibitor with the use of SFN along with a DNMT inhibitor with the use of GEN as this approach may have more tumor-inhibiting potential. Our rationale for using this combination is that the HDAC inhibitor will cause acetylation of histones and as a result a more open chromatin configuration and thus provide more accessibility to the DNA for the DNMT inhibitor to act upon.
Here, we report for the first time that at optimal concentrations, the combination of GEN and SFN can have synergistic effects in reducing the viability of breast cancer cells with no significant effect on the viability of non-cancerous control cells. We have used a much lower than usual concentration of GEN and SFN where they effectively decrease the viability of breast cancer cells. The study is particularly important as we are able to show that in MCF-7 ER (+) and MDA-MB-231 ER (−) breast cancer cells, the combination is much more effective than singly administered doses of these compounds both in vitro and in vivo studies. At a concentration of 5 µM SFN and 15 µM GEN, the cellular viability of breast cancer cells decreases synergistically. The combination is effective in promoting apoptosis much more effectively than separate doses. Cell cycle analyses reveal that the cells are arrested at G2/M phase for MDA-MB-231 cells and at G1 phase for MCF-7 cells. The molecular mechanisms reveal that at the transcriptional level, the combinatorial regimen can decrease the levels of HDACs and a similar observation can be seen at the post-translational level. Upon further assessing HDACs and HMTs, it can be observed that the activity of HDACs and HMTs is decreased with the combination therapy. However, the combination was not effective in synergistically downregulating DNMT activity. In addition to being a DNMT inhibitor, GEN is also known to possess histone modifying properties [40,41,49]. Previous studies have shown that GEN increased acetylated histones 3, 4, and H3/K4 at the p21 and p16 transcription start sites and also increased the expression of histone acetyltransferases that function in transcriptional activation [50]. Thus, the combination of GEN and SFN acting synergistically may be due to the histone acetyltransferase promoting activity of GEN that activates tumor suppressor genes such as p21 and p16, whereas SFN may be acting as an HDAC inhibitor and suppressor of the activity of oncogenes. Together the combination of GEN and SFN is effective in altering epigenetic modifications in breast cancer cell lines. In our current study we observed the downregulation of kruppel like factor-KLF4 using this combination. Additionally, we have observed that in vivo the combination was much more efficacious in reducing tumor volume and causing tumor latency in mice that that were fed the combinatorial diet as compared to the mice on a control diet or the diets consisting of the singly administered compounds.
In our study, we sought to assess the role of dietary phytochemicals in breast tumor inhibition. Therefore, our goal was to use compounds in their natural form. However, it was not possible to use soy to assess the efficacy of GEN since soy contains a mixture of compounds such as diadzein, glycetein and genistein. Therefore, it is difficult to elucidate the role of GEN in this complex mixture. Hence, we chose to use the purified compound. However, broccoli sprouts contain 50 times higher concentration of glucoraphanin, which is converted to isothiocyanate by the enzyme myrosinase [51,52]. Thus, the use of broccoli sprout would be effective in assessing the role of SFN. Therefore, we used broccoli sprout instead of the purified compound. The dose of broccoli sprouts used is 13% BSp which is equivalent to 0.45% of SFN or 350 ppm. Studies in colorectal cancer have shown that dietary intake of 300 ppm SFN prevent adenoma formation in mice [53]. Further studies have shown that in vivo 250 mg/kg GEN was effective in mammary cancer chemoprevention [54,55].
Kruppel-like factors are transcriptional regulators that influence proliferation and several cellular functions in cancer. One family member, KLF4, can function both as a tumor suppressor and as an oncogene. KLF4 can activate and/or repress transcription depending on which transcription factors it interacts with and also on its target gene. Studies have indicated that p21 acts like a switch and modulates the activity of KLF4 [56]. Some studies have reported that KLF4 activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in embryonic stem cells [57,58]. Recent studies have indicated that poly (ADP-ribose) polymerase (PARP1) interacts with KLF4, where knockdown of PARP1 reduces hTERT expression [26]. PARP1 recruits KLF4 to activate telomerase expression in cancer cells. GEN has been previously shown to decrease the expression of PARP1 [59]. In our current study we observed a downregulation of KLF4 at the mRNA level for the MCF-7 cell line with the combination treatment. The combination was effective in downregulating KLF4 levels post-translationally for both MCF-7 and MDA-MB-231 cell lines. Previously, it has been reported that KLF4 expression is required to prevent epithelial-to-mesenchymal transition in MDA-MB-231 mammary cell line. The forced expression of KLF4 suppressed cell migration and invasion by restoring E-cadherin expression [60]. Therefore, we assessed the level of E-cadherin in the treated cells and observed an upregulation of E-cadherin in the cells treated with the combinatorial regimen ( Figure S1). This may suggest that the upregulation could be independent of KLF4 downregulation. Previous studies have shown that GEN and SFN alone have increased the expression of E-cadherin through mechanisms not related to KLF4 regulation. GEN increases E-cadherin levels in vivo by directly targeting it and regulating the Wnt signaling pathway and prevented accumulation of β-catenin in the cytoplasm [61]. SFN has also been known to increase E-cadherin expression and inhibit prostate carcinogenesis and lung metastases in TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse models along with an increase in apoptotic bodies [62]. Thus, the reports point towards an alternative pathway by the dietary phytochemicals to increase E-cadherin expression in cancer cells which may not necessarily involve KLF4 mediation. However, an in-depth study is warranted to study the association between KLF4 and E-cadherin expression in breast cancer cells.
The combination was also effective in downregulating HDAC2 and HDAC3 that act in concert with KLF4. Since KLF4 activates hTERT, with the downregulation of KLF4, hTERT should also be downregulated. Therefore, we assessed the levels of hTERT in breast cancer cells. As hypothesized, hTERT levels were significantly reduced using the combinatorial regimen in both of the breast cancer cell lines we tested.
The combinatorial dietary regimen of GEN and SFN was also administered to an ER (−) transgenic mammary cancer mouse model by orally feeding the mice with either GEN or SFN-enriched broccoli sprouts (BSps) diets alone or in combination. Our results showed combinatorial treatment with GEN and BSp diet led to the most effective response in both preventive and therapeutic effects on inhibition of mammary tumor in vivo as compared to either non-combinatorial treatment or control diet. The dietary combinatorial regimen consists of relatively low concentrations of GEN and SFN that are physiologically accessible by daily consumption of soy and BSp [63,64]. Thus, our novel combination dietary regimen shows an important bioavailability and translational potential toward chemoprevention and/or treatment of breast cancer. It would be interesting to design studies in the future to assess the influence of the combinatorial dietary regimen and compare their effects in ER positive, ER negative and BRCA1 mutated triple negative breast cancers.

Materials and Methods
Cell Lines: We used the ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cells for this study. MCF10A human mammary epithelial cells were used as a non-cancerous control. All cell lines were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA). MCF-7 cells were derived from a 69-year-old Caucasian female from an excision of a chest wall nodule and from a pleural effusion [65]. MDA-MB-231 cells are relatively aggressive in comparison to MCF-7 cells and have a mutated p53 status [66]. MCF10A are non-tumorigenic cells derived from benign proliferative breast tissue and are spontaneously immortalized. They do not express estrogen receptors [67].
Chemicals: Genistein (≥95% pure), C 15 H 10 O 5 with a molecular weight of 270.241 g/mol was purchased from Sigma-Aldrich (St. Louis, MO, USA) and R,S-sulforaphane (≥98% pure), C 6 H 11 NOS 2 with a molecular weight of 177.28 g/mol, was purchased from LKT Laboratories, Minneapolis, MN, USA. Each compound was diluted in DMSO and stored in stocks of 10 mmol/L at −20 • C.
Cell Culture and Treatment: The media to culture the MCF-7 and MDA-MB-231 cells was made using Dulbecco's Modified Eagle's Medium DMEM 1X (Corning cellgro-Thermo Fisher Scientific, Waltham, MA, USA), 10% by volume of fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA) and 1% by volume of 50X penicillin-streptomycin (Corning cellgro). The media for culturing MCF10A cells was made using DMEM F12 media (Corning cellgro-Thermo Fisher Scientific, Waltham, MA, USA), 5% Donor Horse Serum (Corning cellgro-Thermo Fisher Scientific, Waltham, MA, USA), 100 unit/mL penicillin streptomycin, 100 µL of 0.05 µg/mL hydrocortisone, 100 µL of 20 ng/mL EGF, 0.292 g of 2 mmol/L L-glutamine and 100 ng/mL cholera endotoxin. Incubation was done in an incubator that maintained a temperature of 37 • C and provided an environment having at 5% CO 2 and 95% air. Cells were sub-cultured when they were about 90% confluent, counted using a hemocytometer and seeded into 6-well plates. After 24 h, they were treated with individual as well as combination doses of GEN and SFN for 3 days. DMSO was used as a control. Media and compounds were replaced every 24 h during the 3-day treatment.
Cell Density: For the cell density analyses, approximately 200,000 cells were plated in 6-well plates. They were incubated for 24 h and then treated with GEN and/or SFN each day at the same time for 72 h. Media was replaced each day. On day four, cells were viewed under a microscope and images were taken at 100× magnification.
MTT Assay: MTT assay was performed to assess the percentage of viability of breast cancer cells and the control cells after treatment with GEN and SFN. Tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide (MTT) was obtained from Sigma Aldrich (St. Louis, MO, USA) and diluted to 1mg/mL using PBS (Phosphate buffered saline). Cells were seeded in 96 well plates and treated with appropriate concentrations of GEN and SFN for 3 days. On the fourth day, 50 µL of MTT solution was added to each well and incubated for approximately 4 h at 37 • C. MTT solution was removed and DMSO was added and incubated for about 15 min. The color of the solution changed to shades of purple. The absorbance was measured using a spectrophotometer (BioRad; Hercules, CA, USA) at 595 nm.
RNA Isolation: RNA was isolated using the RNeasy kit from Qiagen (Valencia, CA, USA). MDA-MB-231 and MCF-7 cells were plated in 6-well plates and allowed to adhere to the plate for 24 h. The cells were then treated with individual and combination doses of GEN and SFN for 3 days. On the fifth day, the cells were collected using trypsin. After the cells detached from the plate, fresh media was added and then centrifuged at 300× g for 5 min. The pellets were used for further analysis using manufacturer's protocol.
Quantitative Real-Time PCR: cDNA synthesis kit (Bio-Rad) was used to reverse transcribe RNA (made using the protocol above) to cDNA. For the PCR setup, 1 µL of cDNA was used, along with 5 µL of SYBR green, 2 µL of nuclease-free water, 1 µL of reverse and forward primers specific for KLF4, HDAC2 and HDAC3. The total volume was made upto10 µL. PCR was performed using CFX Connect Real Time system (Bio-Rad) where the thermal cycling initiation (4 min at 94 • C) preceded 35 cycles of PCR (94 • C, 15 s; 60 • C, 30 s, 72 • C, 30 s). GAPDH (Glyceraldehyde 3-phospate dehydrogenase) was used as an endogenous control. Fold change was calculated using ∆∆Cq method [68]. All of the primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). The primer sequences used were as follows: GAPDH Cell Cycle Analysis: Approximate 5 × 10 4 cells were plated in 6-well plates. Furthermore, 2 mL of media was added to the wells and allowed to attach to the surface of the plate for 24 h. After 24 h, the cells were treated with GEN and SFN for 3 days at approximately the same time each day. On the fifth day, the cells were collected and fixed with 70% ethanol. Cells were prepared for analyses with a flow cytometer by treating them with propidium iodide (0.04 mg/mL), 0.1% TritonX-100 and RNase (100 mg/mL). Fluorescent signals were read at the UAB Comprehensive Flow Cytometry Core.
Annexin V Apoptosis Assay by Fluorescence-activated Cell Sorting (FACS): MCF-7 and MDA-MB-231 cells were seeded in 6-well plates. After 24 h, the cells were attached to the bottom of the well. The cells were then treated with the GEN and SFN for 3 days. On the fifth day, the cells were collected and prepared for apoptosis assay by using the Annexin V-conjugated Alexafluor 488 Apoptosis assay kit. Cells were treated with Alexa 488 and propidium iodide. The treated cells were then sent to UAB Comprehensive Flow Cytometry Core where they were analyzed using FACS-Caliber instrument (BD Biosciences, San Jose, CA, USA). The cells in Q1 phase were used in analysis of necrosis, whereas the ones in Q2 (early stage apoptosis) and Q4 (late stage apoptosis) phase were used for apoptotic analysis.
Western Blot Analysis: Breast cancer cells were treated with individual doses of GEN and SFN and their combination for 3 days, followed by extraction of protein by RIPA (Radioimmunoprecipitation buffer) lysis buffer. The concentration of protein was then determined by performing Bradford Assay using Bio-Rad protein assay kit (Bio-Rad; Hercules, CA, USA). 4-15% Tris-HCL gel was used for electrophoresis. The wells were loaded with equal concentration of protein mixed with loading buffer. After separation of the proteins by electrophoresis (200 V), the protein was transferred onto nitrocellulose membrane using the Trans Turbo Blot (BioRad). After successful transfer, the membrane was blocked with blocking buffer using a MilliPor SnapID. The milk buffer was made using dry milk powder (0.5%) and Tween (1%) dissolved in Tris Buffered Saline solution. Primary antibodies (Cell Signaling Technology, Danvers, MA, USA) specific to the genes of interest was incubated for 20-30 min and washed for four times using 30 mL of TBST (1X Tris-buffered saline solution with 1% Tween). Secondary antibodies were then added for 15-20 min followed by washing three-four times with TBST. Chemiluminescent substrate was added to the blot according to manufacturer's suggestion and images were captured using an imager (BioRad Chemidoc XRX+ system).
HDAC Activity Assay: Breast cancer cells MCF-7 and MDA-MB-231 as well as control cells MCF10A were treated for 3 days with individual doses (Control, 5 µM SFN, 15 µM GEN) as well as their combination doses (5 µM SFN + 15 µM GEN). After treatment, the cells were collected and the nuclear extract was obtained using an EpiQuik Nuclear Extraction Kit from EpiGenTek (Farmingdale, NY, USA). An EpiQuik HDAC Activity/Inhibition Assay Kit obtained from EpiGentek (Catalog # P-4002) was used to colorimetrically measure HDAC activity using the protocol provided by the manufacturer.
HMT Activity Assay: Breast cancer cells MCF-7 and MDA-MB-231 as well as control cells MCF10A were treated for 3 days with single doses (Control, 5 µM SFN, 15 µM GEN) as well as their combination doses-5 µM SFN + 15 µM GEN. After treatment, the cells were collected and the nuclear extract was obtained using EpiQuik Nuclear Extraction Kit from EpiGenTek (Farmingdale, NY, USA). EpiQuik Histone Methyltransferase Activity/Inhibition Assay Kit (H3-K27) obtained from EpiGentek (Catalog # P-3005) was used to colorimetrically measure HMT activity using the protocol provided by the manufacturer.
Statistics and Data Analysis: Any conclusion in the study was derived from three independent experimental replicates. Data were represented as mean + SD. Statistics for the study were performed using Microsoft Excel. Significance was determined using a Student's t-test in Microsoft Excel. p < 0.05 (indicated by *) is considered statistically significant. All comparisons were done with the DMSO control. For evaluating the nature of interaction between GEN and SFN, CompuSyn software was used. The dose of each compound was entered individually since they are non-constant ratio combos. The CompuSyn software gives combination index (CI) values. CI < 1 indicates synergism between the compounds, CI = 1 indicates additive interaction between the compounds and CI > 1 indicates antagonism between the compounds. The interaction between the dietary phytochemicals is represented in the form of a graph called isobologram. CI index analyses are similar to isobologram analysis and provide a quantitative assessment of the interaction between drugs. The isobologram equation is a special case of the combination index equation. The isobologram assesses the interaction between the compounds at median effect concentration.
Ethics: The in vivo experiments using mouse models were performed by strictly adhering to the rules and guidelines of UAB's Institutional Animal Care and Use Committee (IACUC-131009970 21-Oct-2013).
Animal Experimental Design: The C3(1)-SV40 Tag transgenic mouse model (SV40) was used to study the efficacy of the combination treatment of GEN and SFN. The SV40 mouse model can spontaneously develop hormone-independent invasive mammary cancer (ERα-negative breast cancer) at an early age of around 20 weeks. The SV40 mice at 4 weeks of age were randomly divided into different experimental groups (9-12 mice/group) and diets were administered from 4 weeks of age and continued throughout the study.
The experimental groups were as follows: Group (1). Control group: Mice were fed with modified corn oil-based AIN-93G diet; Group (2). GEN group: Mice were fed with 250 mg/Kg GEN diet; Group (3). BSp group: mice were fed with 13% BSp diet (w/w); (4). GEN + BSp group: Mice were administered the combined GEN and BSp diet with concentrations as described above. All diets were prepared using a modified corn oil-based AIN-93G diet as a base by a commercially available company (TestDiet, St. Louis, MO, USA).
Tumor volumes and body weight were measured weekly. Tumor volumes were calculated using the formula: tumor volume (cm 3 ) = (length × width 2 ) × 0.523 [69]. The experiment was terminated when the mean of tumor diameter in the control mice exceeded 1.0 cm. At the end of the experiment, the primary mammary tumors were excised, weighed and appropriately stored in liquid nitrogen for further analysis.

Conclusions
In conclusion, the current study highlights the efficacy of this combinatorial diet consisting of GEN and SFN in enhancing the translational chemopreventive potential during breast tumorigenesis. In vitro, the combination has downregulated KLF4, which acts as an oncogene in breast cancer. Additionally, the combination was effective in downregulating HDAC activity, especially HDAC2 and HDAC3, which act in concert with KLF4. KLF4 is known to activate hTERT, which is highly activated in breast cancer. Our studies have shown that hTERT is downregulated with the combination. Thus, this combination is very effective in inhibiting breast tumorigenesis without having any toxicity on the normal cells. Future studies will involve assessing KLF4 protein expression in mice tumors as well as the expression of HDAC2, HDAC3 and hTERT in the tumor tissues. Additionally, we will perform experiments to study in depth the p21 gene, which acts as a switch in KLF4 regulation by studying the p21 promoter region. We will also study the hTERT promoter region to resolve the interaction among KLF4, hTERT, HDAC2 and HDAC3.