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Mutations in the p53 gene occur in a variety of human cancers with remarkably high frequencies (www-p53.iarc.fr). The majority of p53 mutations are missense that are localized to six ‘hotspot’ residues. Mutations in p53 result in the loss of the wild-type (WT) activity; however, these mutants exert either a ‘dominant-negative’ effect on the p53 WT activity or a ‘gain-of-function’ effects.1, 2, 3 Humans with a Li–Fraumeni syndrome, an autosomal-dominant disorder owing to germline mutations in p53 gene, are at an increased risk of tumorigenesis.4 Thus targeting p53 mutant offers a promising approach for cancer chemotherapeutics. However, the role of p53 mutant as a target for dietary-related cancer chemopreventive compounds remained to be investigated.

Phenethyl isothiocyanate (PEITC), abundantly present in watercress and cruciferous vegetables, exerts cancer chemopreventive effects in animal models, and epidemiological studies also support the role of dietary ITCs in protection against cancer in humans.5 In fact, PEITC has been studied in clinical phase 1 and phase 2 trials (http://www.clinicaltrials.gov/ct2/results?term=PEITC). The mechanisms proposed for PEITC include inhibition of cytochrome P450s, induction of phase II detoxifying enzymes, cell cycle arrest and apoptosis.6, 7, 8, 9, 10, 11, Immunofluorescent staining

SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC (4 or 6 μM) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific). Cells then were washed twice with 1 × PBS and fixed with formaldehyde (3.7%) at RT for 15 min. Fixed cells were washed four times with 1 × PBS and treated with 0.5% Triton X-100 (Sigma) at RT for 5 min. Cells were washed four times with 1 × PBS containing 0.5% Tween-20 and blocked with 10% goat serum (Sigma) overnight at 4 °C. Cells were washed four times with 0.1% Tween-20 and incubated with mouse PAB240 (1:300, Calbiochem, San Diego, CA, USA) or mouse PAB1620 (1 : 300, Calbiochem) that recognizes specifically the mutant or p53 WT, respectively, overnight at 4 °C. After four washes with 0.1% Tween-20, cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1 : 400, Invitrogen) at RT for 2 h. Cells were washed four times with 0.1% Tween-20 and coated with Prolong Gold Anti-Fade reagent (Invitrogen) containing DAPI. Coverslips were placed on the chamber slides, and cells were cured at RT in the dark for 24 h. Immunofluorescence analyses were performed with a Zeiss LSM 510 META NLO inverted Axiovert 200 M laser scan microscope (Thornwood, NY, USA) with a Plan-Apochromat 63 × 1.4 numerical aperture oil immersion objective lens. Images were captured using the Photomultiplier Tube detectors and analyzed using the Image J software (NIH, available at http://rsb.info.nih.gov/ij/). Fluorescent staining intensity was quantified using the Metamorph software (Sunnyvale, CA, USA).

To determine the effect of PEITC on γ-H2AX foci formation in SK-BR-3 cells transfected with p53 siRNA or NS siRNA or A549 cell lines, cells were treated with 4 μM PEITC or 1% DMSO as a control at 37 °C for 3 days. Cells were fixed with formaldehyde and processed for immunostaining to detect γ-H2AX foci as described above, except that mouse anti-γ-H2AX monoclonal antibody (1 : 300, Upstate, EMD Millipore, Billerica, MA, USA) was used as a primary antibody.

Co-immunoprecipitation

SK-BR-3, A549 and HOP92 cells were treated with the indicated concentration of PEITC or 1% DMSO as a control for 6 h. For preparation of cell lysates, cells were harvested and washed once with 1 × PBS, cell pellets were suspended in lysis buffer (20 mM Tris-Cl (pH 8.0), 137 mM sodium chloride, 10% glycerol, 1% NP-40, 2 mM EDTA) and Protease inhibitors (Roche, Indianapolis, IN, USA) and the cells were incubated on ice for 30 min. The cell suspension was centrifuged at 18 500 × g for 10 min at 4 °C, and the supernatant was collected. The supernatants were diluted in lysis buffer, and 200 μg of the lysate was gently tumbled at 4 °C for 1 h with protein G-agarose beads (Roche). The lysates obtained after preclearing were then gently tumbled at 4 °C for 2 h with mouse PAB240 antibody (2 μg, Calbiochem). Protein G-agarose beads were then added to the suspensions and incubation was performed for 2 h at 4 °C. The beads were washed four times with lysis buffer supplemented with protease inhibitors, and the immunoprecipitates were eluted by boiling in Laemmli buffer and resolved on 4–12% SDS-PAGE. Immunoprecipitated p53 was detected by western blotting using FL393 (Santa Cruz Biotechnology) as a primary antibody. For the secondary antibody, peroxidase-labeled anti-mouse IgG (1 : 2000, GE Healthcare, Pittsburgh, PA, USA) was used. The blot was developed using the ECL Prime Western Blot Detection Kit according to the manufacturer’s protocol (Amersham, GE Healthcare). As a control, the blot was stripped and then reprobed with anti-p53 (DO-1) antibody (1 : 1000, Santa Cruz Biotechnology) or anti-GAPDH antibody (1 : 2000, Novus Biologicals, Littleton, CO, USA). The density of the p53 bands in the PEITC treated samples relative to that of DMSO control was determined using the Gene Tools software (Cambridge, England, UK).

Lysate preparation and western blotting analysis

Different lysis buffers were used to prepare soluble, insoluble and WCL fractions. For lysate (soluble fraction) preparation, cells were harvested and washed twice with 1 × PBS. RIPA buffer (10 mM sodium phosphate (pH 7.2), 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 2 mM EDTA) was added to the cells, and the cells were incubated on ice for 30 min. Then the cell suspension was centrifuged at 18 500 × g for 10 min at 4 °C and the supernatant was collected, unless mentioned otherwise. The remaining pellet was defined as insoluble fractions. Insoluble fractions were dissolved in 2% SDS lysis buffer (65 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2% SDS, 50 mM DTT). For WCL fractions, cells were harvested and the pellets were dissolved in 2% SDS lysis buffer as described previously. The fractions were collected by centrifuging the lysate at 18 500 × g for 10 min at 4 °C. Then 30–50 μg of the lysates were loaded on 4–12% SDS/PAGE. Protein was transferred onto a PVDF membrane, and the blots were developed using the ECL Prime Western Blot Detection Kit according to the manufacturer’s protocol (Amersham). The antibodies for p21, Bax, ATM, pATM S1981, CHK2, pCHK2 Thr68 and p53 (DO-1) were purchased from Santa Cruz Biotechnology and GAPDH antibody was from Novus Biologicals. The antibody for ATG5 (1 : 1000, Cell Signaling, EMD Millipore, Danvers, MA, USA) was a gift from Dr. Shivendra V Singh.

Chromatin fractionation

SK-BR-3 cells were treated with the indicated concentrations of PEITC or DMSO as a control for 4 h. Cells were trypsinized and harvested by centrifugation at 500 × g for 5 min. Cell pellets were washed once with ice-cold PBS and transferred to 1.5-ml microcentrifuge tubes followed by centrifugation at 500 × g for 2 min. Pellets were stored at −80 °C prior to chromatin fractionation following the manufacturer’s instruction (Subcellular Protein Fractionation Kit, Thermo Scientific) to generate nuclear soluble and chromatin-bound protein fractions. Ten micrograms of protein from the soluble nuclear extract and the chromatin-bound nuclear extract for the samples from DMSO- or PEITC-treated cells were resolved on 4–12% SDS-PAGE and transferred to PVDF membranes. Blots were probed with p53 (DO-1) antibody (1 : 1000, Santa Cruz Biotechnology). Histone H3 and TopoIIB, which served as markers for the chromatin and soluble nuclear fractions, respectively, were detected with rabbit anti-Histone H3 polyclonal (Thermo Scientific) and mouse anti-TopoIIB monoclonal (Santa Cruz Biotechnology) antibodies.

RNA extraction and qRT-PCR

SK-BR-3, SK-BR-3 transfected with p53 siRNA or NS siRNA, H1299 and A549 cells were treated with 4 μM PEITC or DMSO as a control for 4 h. RNA was extracted from the cells using a Qiagen RNeasy Kit (Qiagen, Valencia, CA, USA), cDNA was synthesized by using High Capacity RNA to cDNA Kit (Applied Biosystems, Invitrogen, Thermofisher Scientific) and the gene expression level was measured by qRT-PCR using TaqMan gene expression assays (Applied Biosystems, Invitrogen). The gene expression level is normalized with GAPDH, and the average is presented with S.D. from triplicates of repeated experiments. RNA was extracted from the SK-BR-3 xenograft tissues by using Qiagen RNeasy Kit and was processed further for qRT-PCR as described for the SK-BR-3 cells. The gene expression levels were normalized with GAPDH. Fold changes in the gene expression levels were calculated for each tumor from the treated group relative to the tumors from the control group and the average is presented with S.D.

Measurement of glutathione level

The levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured using the GSH/GSSG-Glo Glutathione Assay Kit (Promega). Briefly, SK-BR-3 cells were treated with PEITC or DMSO as a control for 4 h. Cells were then processed for the glutathione assay as per the manufacturer’s instructions (Promega).

Luciferase reporter assay

WWP-Luc (p21/WAF1 promoter) plasmid encoding p53 WT-binding element in the p21 promoter region was a gift from Bert Vogelstein (Addgene plasmid no. 16451, Cambridge, MA, USA).43 It was transfected into SK-BR-3, HOP92, AU565, H1299 and MEF ((10)3/175 and (10)/273) cells, followed by treatment with PEITC (4 or 6 μM, respectively) for 24 h. The cell lysate was made and the luciferase reporter assay was performed in accordance with the manufacturer's instructions (Luciferase assay, Promega).

Cell cycle analysis

SK-BR-3, SK-BR-3 transfected with p53 siRNA or NS siRNA and A549 cells were treated with PEITC, Nutlin-3 or both or DMSO as a control for 24 or 72 h. Cells then were prepared for flow cytometric analysis. Briefly, cells were washed with PBS free of Ca2+ and Mg2+, trypsinized for 5 min and harvested by centrifugation at 190 × g for 3 min at 4 °C. Cells were washed once with PBS and the pellets resuspended in 1 ml of 70% ethanol and stored at –20 °C overnight. Cells were harvested by centrifugation at 420 × g for 10 min. The cell pellets were washed once with 1 ml cold PBS and resuspended in 1 ml freshly prepared PI staining solution (PBS with 0.1% Triton X-100, 0.05 μg/ml propidium iodide, 0.1 mg/ml RNase (Sigma)). The cell suspension was incubated at RT for 30 min in the dark followed by incubation for 30 min at 4 °C. The samples were run on a Becton Dickinson FACS sorter (BD Biosciences, San Jose, CA, USA) and the data were analyzed using Mod Fit program (Verity Software House, Topsham, ME, USA).

Detection of ATM and CHK2 phosphorylation upon PEITC treatment

Cells were treated with either DMSO, ATZ, NAC, PEG-Catalase or PEITC alone or PEITC in combination with ATZ or NAC or PEG-Catalase for 4 h. Cells then were harvested by centrifugation at 1600 × g for 10 min at 4 °C, washed once with PBS and resuspended in RIPA buffer (10 mM sodium phosphate (pH 7.2), 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate and 2 mM EDTA) containing a protease and phosphatase inhibitor mixture and were incubated on ice for 30 min and centrifuged at 18 500 × g for 10 min at 4 °C. The supernatant was collected, and 200 μg of the lysate was loaded on 4–12% SDS/PAGE. Following electrophoresis, protein was transferred onto a PVDF membrane, and blot was probed with anti-pATM Ser1981 antibody (1 : 500) (Santa Cruz Biotechnology) or anti-pCHK2 Thr68 antibody (1 : 500) (Santa Cruz Biotechnology). For the secondary antibody, peroxidase-labeled anti-mouse IgG (1 : 1000, GE Healthcare) was used. The blot was developed using the ECL Prime Western Blot Detection Kit following the manufacturer’s protocol (Amersham). As a control, the blot was stripped and then reprobed with anti-ATM anti-body (1 : 500, Santa Cruz biotechnology) or CHK2 antibody (1 : 500, Santa Cruz biotechnology).

Mouse SK-BR-3 xenograft model

Twenty female athymic nu/nu Balb/c mice (CAnN.Cg-Foxn1nu/Crl, 4–6-week old) were purchased from Charles River Laboratories (Wilmington, MA, USA). All in vivo studies and tumor harvest were performed in accordance with the Institutional Animal Care and Use Committee procedures and guidelines. Mice were weighed and housed under quarantine in polycarbonate cages (five mice/cage, equal average weight and variance among animals/cage) for 1 week. Mice were fed an AIN-93M diet and water ad libitum for 1 week of quarantine. After 1 week in quarantine, the mice were placed on either a control AIN-93M diet or an AIN-93M diet supplemented with PEITC (5 μmol PEITC/g diet) (10 mice/group). Both diets were prepared by Research Diets (New Brunswick, NJ, USA). The number of animals was decided based on previous studies, which used the SK-BR-3 xenograft mouse model to generate statistically significant data, and the concentration of the PEITC in diet was chosen based on our previous bioassay in mice.27, 44 The PEITC concentration in the diet was confirmed to be 4.97±0.16 μmol/g by ethyl acetate extraction followed by cyclocondesation reaction of 1,2-benzenedithiol with the ITCs as described.34 By single-factor ANOVA calculation, there was no statistically significant difference (alpha=0.05) between the PEITC samples. The food was replenished every alternate day. After 1 week of control and PEITC diets, the mice were injected with exponentially grown 2 × 106 SK-BR-3 cells (suspended in 50 μl of Matrigel) in their left and right mammary fat pads (‘cancer chemoprevention’ settings). No mortality or death was observed over the course of the experiment. Tumor formation was assessed externally weekly, and tumor dimensions were measured with Vernier calipers for a 10-week bioassay period. Tumor volumes were calculated with the formula L × W2 × 0.523. The tumors were small in size and were not easily measurable externally. Because of the small size, some of the tumors showed variations in the tumor volume measurements over the weeks. For this reason, only the tumors (n=7) that followed a growth pattern without fluctuations were considered for the final tumor volume calculations. However, no outliers were detected by using the GraphPad software (GraphPad Software, Inc., La Jolla, CA, USA). All the animals were killed, and the tumors were removed and were confirmed by H&E staining as described below. Animal weights (g) were measured weekly. The ITC concentration in the blood of the animals collected at the time of necropsy was evaluated by cyclocondensation reaction of 1,2-benzenedithiol with ITCs as described.34 PEITC concentration was found to be 1.13±0.15 μM (n=3) and 0.37±0.03 μM (n=2) in the blood of the PEITC-treated and control animals, respectively. Blinding to the groups was not possible because of the different diets; experiments were, however, blinded to tumor harvest and histopathological analysis. H&E-stained sections were blindly examined by pathologist for the incidence of tumors.

Histopathological analysis, immunohistochemistry, western blotting and qRT-PCR analysis of the SK-BR-3 tumor xenografts

H&E-stained slides were examined and tissues were confirmed as tumors by histopathological examination. Immunohistochemistry was performed at Histopathology and Tissue Shared Resources, Georgetown University following standard protocols. Briefly, tissues were sectioned at 5 μm, de-paraffinized with xylenes and rehydrated through a graded alcohol series. Heat-induced epitope retrieval was performed by immersing the tissue sections at 98 °C for 20 min in 10 mM citrate buffer (pH 6.0) with 0.05% Tween. Immunohistochemical staining was performed using a horseradish peroxidase-labeled polymer from Dako (Carpinteria, CA, USA) (K4001, K4003) according to the manufacturer’s instructions. Briefly, slides were treated with 3% hydrogen peroxide and 10% normal goat serum for 10 min each and exposed to primary antibodies p53 (DO-7) (1 : 800, Dako) for 1 h at RT and Ki-67 (1 : 15, Novus Biologicals) overnight at 4 °C. Slides were exposed to the appropriate HRP-labeled polymer for 30 min and DAB chromagen (Dako) for 5 min. Slides were counterstained with Hematoxylin (Fisher Scientific, Suwanee, GA, USA, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated and mounted with Acrymount (StatLab, Baltimore, MD, USA). Consecutive sections with the primary antibody omitted were used as negative controls. The sections were examined under an Olympus BX61 microscope at × 200 magnification. Representative images were captured from the entire tumor section using a DP70 camera and DP70 software (Olympus, Waltham, MA, USA), and images were analyzed using Image J software (NIH). Further, because of the small size of the tumors, four sections per tumors were analyzed for determining the cell number. The sections were stained with different antibodies as mentioned and for each antibody 20 pictures were taken from different areas on slide to count the total cell number. The data presented for each tumor are the average of the total number of cells from different antibody stainings.

As we were limited by the amount of tumor tissue, especially from the mice on the PEITC-supplemented diet, we divided the tumors randomly to perform western blotting and qRT-PCR analyses. For western blotting analysis, the SK-BR-3 tumor xenograft (n=12) lysate from each group was prepared by homogenizing the tissue in 20w/v of lysis buffer (Pierce, Rockford, IL, USA). Twenty-five micrograms of the lysates were loaded on 4–12% SDS/PAGE, transferred onto a PVDF membrane and the blot was probed with p53 (DO-1) antibody as described previously. To perform qRT-PCR assay with the SK-BR-3 tumor xenograft tissues, RNA was extracted from tumor tissues (n=4) using the Qiagen RNeasy Kit and gene expression level was measured as described previously.

Statistical analysis

Statistical differences in tumor volume and biological end points were evaluated with a two-tailed Student’s t-test. Differences were considered statistically significant at P-values of ≤0.05. All statistical tests were two-sided.