Preparation of extracts
RAN were ground into fine powder and 100 g of the powder were extracted with 125 ml of distilled water and mixed thoroughly to give a smooth paste for preparation of an aqueous extract of RAN. After 24 h at 4oC, the paste was stirred for 3 h at room temperature and the aqueous extract was collected by centrifugation. This extraction procedure was repeated once more by adding 125 ml of water to the residue. Both extracts were pooled, representing 100 g of RAN in 250 ml distilled water, filtered and frozen at -80°C. The filtrate was lyophilized in a Scanlaf Coolsafe Lyophilizer (Lynge, Denmark). The lyophilized mass was kept at 4°C until use. The extract contained 0.9 g/100 g water-extractable material.
Animals maintenance and treatment
Swiss albino mice (25–30 gm) and aged 2–3 months were maintained in the laboratory in community cages and housed in the Animal Resource Facility of the university under the following conditions: 12-h dark/12-h light cycle, 20±2°C temperatures and 50±10% humidity. Standard mouse diet (NMC Oil Mills Ltd., Pune, India) and water ad libitum were used in all experiments. A total of six groups of mice (n=7 in each) were used 0, 60, 100, 180, 240 and 300 days for different experimental analysis. One group was treated with simple drinking water considered to be untreated control whereas other five groups were administered RAN extract ad libitum in the drinking water with slaked lime (calcium hydroxide; pH 9.8). Each mouse consumed 1 mg of extract per day. Such oral administeration was continued for 60 days after which the dose was increased from 1 mg to 2 mg per day till 120 days. This way every 60 days after, the dose was increased by 1 mg per day consumption. In the present study, the mice were fed till 300 days. The dose and the treatment pattern were similar to our earlier study where it was shown that continuous ad libitum administration of RAN extract with lime in drinking water for 220 days or more can induce stomach and esophageal cancer in mice [6].
Histopathological evaluation
Stomach tissues of mice were collected from untreated and treated with RAN + lime for 300 days and preserved in 10% formalin. Three mice were selected from the untreated group and none of them showed any indication of tumor externally. All the seven mice in the treated group for 300 days were selected for histological evaluation. Tissues were processed for histological sectioning as per standard protocol [30]. Formalin-fixed paraffin embedded tissue blocks were serially sectioned (5 μm) with a microtome (Leica Biosystems, Wetzlar, Germany) and stained with hematoxylin and eosin [31]. Sections were then observed under a light microscope and photographed (Carl Zeiss, Oberkochen, Germany).
Immunoblotting
Cells were collected from the inner layer of stomach from untreated (n=2) and RAN+lime treated mice for 300 days (n=3). The cells were washed with ice-cold 0.1 M phosphate-buffered saline (PBS; pH 7.4) and total protein was extracted with a lysis buffer containing 0.1% SDS, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 50 mM sodium fluoride, 100 U/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. After centrifugation, the cell lysate was collected and the protein concentration was determined using the bicinchoninic acid protein assay. Equal amount of protein (40 µg/well) was subjected to Novex Tris-Glycine 4–20% gradient gels and electrophoresis was performed in NuPAGE electrophoresis system (Invitrogen, California, USA). Then the proteins were transferred to a polyvinylidene difluoride membrane (Sigma) and probed with 1:1000 dilution of a mouse monoclonal antibody against Securin (DCS-280; ab3305; Abcam, California, USA) and β-actin (AC-15; ab6276; Abcam, USA). Alkaline–phosphatase conjugated anti-mouse IgG (Abcam, USA) used as secondary antibodies and immunodetection was performed by treating the blot with the substrate solution of BCIP/NBT (Bangalore Genei, India).
Immunohistochemistry (IHC) analysis
Stomach tissues of mice were collected from untreated and treated with RAN + lime for all the different time periods and preserved in 10% formalin. Four mice were selected from each group. Tissues samples were dehydrated, paraffin embedded and sectioned with a microtome. Briefly, after blocking for endogenous peroxidase activity, the sections of stomach tissues were incubated with anti-Securin (DCS-280; ab3305; Abcam, UK), anti-H3K4me3 (Histone H3 Lysine 4 trimethylation) primary antibody (ab8580; Abcam, UK), anti-H3K9me3 (Histone H3 Lysine 9 trimethylation) primary antibody (ab8898; Abcam, UK), anti-H3K9Ac (Histone H3 Lysine 9 acetylation) primary antibody (ab12179; Abcam, UK), anti-H3K18ac (H3 Lysine18 acetylation) primary antibody (ab1191; Abcam, USA), anti-Rb-phosphorylation primary antibody (SC-271930; Santa Cruz Biotechnology, USA) and anti-E2F1 primary antibody (SC-22820; Santa Cruz Biotechnology, USA). IHC analysis was performed with a Strept-Avidin Biotin Kit (Dako, Agilent Technologies Company, Denmark). The scoring of immunohisto-chemical stains in each specimen was determined using a histological score (H) [32] (please see Supplemental Information). Only Histone 3 antibody (ab1791; Abcam, UK) was used as an internal control.
Chromatin immunoprecipitation assay (ChIP)
ChIP assay for detection of posttranslational histone modifications patterns in the promoter region of Securin gene were performed in mouse stomach cells. Stomach epithelial cells from four different animals at each point were lysed, sonicated and incubated with antibodies specific to H3K4me3 (ab8580, AbCam, UK), H3K9Ac (ab12179), H3K9me3 (ab8898), H3K18Ac (ab1191) and Histone 3 (ab1791) with protein A/G beads (Pierce™ Protein A/G Agarose, Cat no. 20421) incubated for overnight at 4°C. The methodology of ChIP in details is mentioned in the Supplementary section.
Quantitative PCR (qPCR; BioRad CFX system) was used to quantitate amounts of DNA fragments in the immunoprecipitated samples from the ChIP analyses. qPCR was performed with the reagents containing SYBR green and specific two primer sets located within -478bp to -874bp of promoter of PTTG1 of mouse confirmed by sequencing (Science genome browser) of the qPCR products. Samples were heated to 95°C for 5 min and then amplified for 45 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. Immunoprecipitated DNA was detected by qPCR and normalized with input DNA. Enrichment was calculated relative to input. qPCR products were purified using SIGMA PCR clean up kit (NA 1020) and sent for sequencing to Agrigenome, Kochi, India.
RNA extraction and qRT-PCR
qRT-PCR was performed to assess the transcriptional levels of KMT2A (lysine methyltransferase2A), KAT2A (lysine acetyltransferase2A), EP300 (lysine acetyltransferase KAT3B), P300/CBP-associated factor (PCAF) also known as K(lysine) acetyltransferase 2B (KAT2B), HDAC3 (histone deacetylase 3), KDM4C (histone demethylase) and the reference gene GAPDH. Cell lysates were collected and the RNA was extracted and purified using the Rneasy® mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription of the RNA into cDNA was then performed using QuantiTect Reverse Transcription kit (Qiagen GmbH, Hilden, Germany). qRT-PCR was performed in a Bio-Rad CFX96 Real-Time PCR Detection System using SYBR Green PCR Master Mix (Life Technologies, Delhi, India). The primers used for qRT-PCR were listed in Supplementary Table S1.
Statistical analysis
For comparing the expression Securin between untreated and different duration of treated groups unpaired Student’s t-test was performed for statistical analysis. The statistical significance in the level of Histone3 K4-trimethylation, K9-acetylation, K9-trimethylation, K18-acetylation, pRb-phosphorylation, E2F1 activities and the expression of several epigenetic chromatin modification enzymes between treated and untreated groups, was determined by one-way ANOVA. The Turkey test was used for post hoc analysis. The results are shown as means ± SEM and P < 0.05 is considered as statistically significant. Statistical analysis was performed using GraphPad Prism 5.0.