Chemicals and reagents
Azoxymethane (AOM; #A5486-100MG) of 98% purity was obtained from Sigma-Aldrich Co. (MO, USA), whilst both active VD3 (Calcitriol; #HY-10002) and metformin (#HY-17471A) were from MedChemExpress LLC (NJ, USA). Moreover, 5-FU was purchased from Hospira Australia Ltd. (Melbourne, Australia), and all drugs were freshly prepared as per the manufacturers’ instructions prior to their use. DMEM media (#10566032), foetal bovine serum (FBS; #A3160802), antibiotic-antimycotic solution (#15240062) and all the utilised cell culture materials were from Thermo Fisher scientific (CA, USA).
Experimental animal studies and treatment protocols
Ninety male BALB/c mice weighing 25-30 gm each and of 12 weeks of age were equally divided into nine groups (10 mice/group) as follow: the negative (NC) and positive (PC) controls, 5-FU (5-FU), calcitriol (VD3) and metformin (Met) monotherapy groups, the dual therapy groups that simultaneously received VD3/5-FU (VF), Met/5-FU (MF) or VD3/Met (VM), and the triple therapy group (VMF) that was treated with all drugs. All animal experiments were conducted in compliance with the European guidelines for the care and use of laboratory animals and the study was approved by the Committee for the Care and Use of Laboratory Animals at Umm Al-Qura University (AMSEC 19/05-10-20).
AOM was injected for two consecutive weeks (10 mg/Kg/week) in all groups, expect the negative control mice, to induce CRC as previously reported . The animals were observed for another 20 weeks with no intervention and received standard laboratory chow with water ad libitum. All treatment protocols were initiated at week-21 post-AOM and continued for four weeks. Freshly prepared calcitriol (0.07 µg/Kg/day; five times/week) and metformin (430 mg/Kg/day; five times/week) were administrated orally to the designated groups, and the delivered amounts were equivalent to the highest daily recommended doses for a 60 Kg body weight adult human for calcitriol (0.25 µg/day; 0.0042 µg/Kg/day)  and metformin (1500 mg/day; 25 mg/Kg/day)  as per the dose conversion formula between human and mice . 5-FU was administrated to the designated groups for four successive cycles as a single weekly intraperitoneal injection (50 mg/Kg/week) as previously described .
Types of animal samples
Euthanasia was performed on the first day of week-25 post-AOM by cervical dislocation under anaesthesia a described earlier . The colon from each animal was removed, flushed with cold phosphate buffer saline (PBS), cut longitudinally, and soaked in 10% formalin overnight between layers of filter papers .
Gross examination and tumour counting
The numbers of tumours/colon were grossly counted in the next morning by two researchers followed by cutting each colon into three equal parts representing proximal, middle, and distal segments to the caecum . All parts were stained for 10 minutes by 1% Alcian blue solution (#sc-214517; Santa-Cruz Biotechnology Inc.; CA, USA) in 3% acetic acid (pH 2.5) and then examined under a dissecting microscope (Human Diagnostics; Wiesbaden, Germany) to identify mucin depleted foci (MDF) as well as count the small tumours that were not detected by naked eye .
Each colonic segment was halved longitudinally, and a specimen was Swiss-rolled and processed by conventional histopathology techniques. Total RNA was extracted from the residual colonic samples with tumours by PureLinkTM RNA Mini Kit (#12183025; Thermo Fisher Scientific), whilst total proteins were extracted by homogenising colonic specimens in RIPA lysis buffer (#89900) with protease inhibitors (#78429; Thermo Fisher Scientific). While the RNA quality and quantities were measured with a Qubit4 Fluorometer (Thermo Fisher Scientific), the concentrations of extracted total protein were measured by a Pierce™ Rapid Gold BCA Protein Kit (#A53225; Thermo Fisher Scientific). The total protein samples were then diluted with deionized water (2000 µg/ml) to be used for Enzyme Linked Immunosorbent Assay (ELISA).
Histopathological studies of colon tissues
Tissue sections of 5 μm thickness from each colon were stained by H&E and examined on a Leica DMi8 microscope (Leica Microsystems, Wetzlar, Germany) at 100× and 200× magnifications by two expert researchers who were blind to the source group. Both examiners reported the histological features of adenocarcinomas in five random fields from each section using a set of well-established criteria . In case of a wide disagreement between both examiners, an independent expert histopathologist re-examined the sections. The captured images were analysed by the ImageJ software (https://imagej.nih.gov/ij/) to measure the surface areas (μm2) of adenocarcinomas, as reported earlier [21,37].
All the primary antibodies were from Cell Signaling Technology Inc. (MA, USA). Rabbit monoclonal antibodies were used to detect CCND1 (#55506), CDK inhibitor-1A (p21; #37543) and mTOR (#2983), whereas PI3K-p85α (#13666) was detected by mouse monoclonal IgG antibodies. An avidin-biotin horseradish peroxidase technique was applied on 5μm tissue sections to detect the targeted proteins as previously described . Briefly, BLOXALL® Solution (#SP-6000-100; Vector Laboratories Inc., CA, USA) was used to block endogenous peroxidases. The primary antibodies (1:200 concentration for all) were added and the slides were incubated at 4°C overnight. In the following morning, ImmPRESS® HRP Horse Anti-mouse (#MP-7402) or anti-rabbit (#MP-7401) IgG Plus Polymer Peroxidase Kits were used as per the manufacturer’s guidelines (Vector Laboratories Inc.). An identical protocol was applied for the negative control slides, but primary isotype mouse (#sc-2025) or rabbit (#sc-2027) IgG antibodies (Santa-Cruz Biotechnology Inc) were used to control for non-specific staining. The sections were counterstained with haematoxylin and examined with 20× objective on a Leica DMi8 microscope. Digital images were acquired from 10 different fields/section and the protein expression was analysed by ImageJ software (https://imagej.nih.gov/ij/) using the IHC Image Analysis Toolbox as previously reported [38,39].
Terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) Assay
Cell apoptosis was detected in colonic tissues with a Click-iT™ Plus TUNEL Assay (#C10617; Thermo Fisher Scientific) and by using the manufacturer’s protocol. The apoptotic bodies were co-localised with cleaved Casp-3 by applying a sequential protocol as reported earlier [40,41]. After completing the TUNEL protocol, anti-cleaved Casp-3 rabbit IgG monoclonal antibodies (#9661; Cell Signaling Technology Inc.) were added at 1:400 concentration and the slides were incubated for 3h. Donkey anti-rabbit IgG antibodies tagged with Alexa Fluor™ 555 (#A-31572; Thermo Fisher Scientific) were then added for 30 min followed by DAPI counterstaining (#D3571; Thermo Fisher Scientific). The slides were cover-slipped with a permanent fluorescence mounting medium (#S3023; Dako, CA, USA) and observed on a Leica DMi8 microscope at 400× magnification. Images were acquired from 15 non-overlapping fields/section, and the apoptotic cells’ numbers and cleaved Casp-3 staining intensity were measured by ImageJ software as previously reported [39,42].
The colonic tissue concentrations of survivin (#SEC045Mu), cytochrome C (#SEA594Mu), Akt1 (#SEC231Mu), and PTEN (#SEF822Mu) were measured by specific mouse ELISA kits (Cloud-Clone Corp.; TX, USA). The samples were processed in duplicate on an automated ELISA machine (Human Diagnostics) according to the manufacturer’s protocols.
Cell culture and treatment protocols
The human SW480 primary and SW620 metastatic colon cancer cell lines were purchased from the American Type Culture Collection (ATCC; MA, USA). The SW480 and SW620 cells were cultured in DMEM that contained 10% FBS and 1% antibiotic-antimycotic solution. All cells were grown in a humified incubator at 37°C and 5% CO2.
The 5-FU (50 µM), VD3 (25 µM) and metformin (39.8 mM) concentrations (IC50) were determined by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) cytotoxicity assay at 24h (Supplementary Figure 1). For cell cycle analysis, the SW480 (2x105) and SW620 (3x105) cells were seeded in 6-well plates for 24h and then treated for 12h with 5-FU, VD3 and/or metformin single, dual, and triple therapies, resulting in the following groups: untreated control (Ctr), 5-FU, VD3 and Met monotherapies, VF, MF and VM co-therapies, and VMF triple therapy. The 12h time-point was applied to assure that any effects of combination therapies could be accurately analysed by cell cycle, apoptosis, gene, and protein expression techniques.
Cell cycle analysis
Cell cycle analysis was performed following the different treatment regimens in the SW480 and SW620 cells as previously described . Briefly, cells were washed twice with PBS (500× g for 5 min) following trypsinisation and fixed in ice-cold 70% ethanol for 24h at 4°C. The cells were washed twice in PBS (600× g for 5 min) and treated for 15 min with RNase A (20 µg/ml; #12091021; Thermo Fisher), and 2 µg/ml propidium iodide (PI; #P1304MP; Thermo Fisher) was added. Following staining, cell cycle analysis was immediately performed with a Novocyte 3000 flow cytometer (Agilent Technologies, CA, USA). The percentages of cells in different phases of cell cycle (Sub-G1, G0/G1, S, G2/M) were determined for 20,000 acquired events using the NovoExpress software cell cycle algorithm, and data are shown as mean ± SD (n = 3).
The Annexin V-FITC/PI Apoptosis Assay Kit (#V13245; Thermo Fisher Scientific) was used according to the manufacturer’s protocol. The cells were harvested after the different treatment protocols, washed twice with cold-ice PBS, and re-suspended in 100 µl of 1× Annexin V (AV) binding buffer. For cell staining, the mixture of AV-FITC (5 µl) and PI (1 µl) were added to each 100 µl of the SW480 and SW620 cell suspensions followed by incubation in dark for 15 minutes at room temperature. Subsequently, the AV binding buffer (400 µl) was added, and the cells were placed on ice and immediately analysed using the NovoCyte 3000 flow cytometry. The experiments were processed in triplicate and the data represents percentage (mean ± SD) of the different apoptosis stages as follows: live (unstained), early (AV+/PI-) and late apoptotic (AV+/PI+), and dead (AV-/PI+) cells.
Quantitative reverse-transcription polymerase chain reaction
Following 12h treatment with the different therapies, the SW480 and SW620 cells were trypsinised and washed in PBS. Total RNA was extracted by a PureLinkTM RNA Mini Kit (Thermo Fisher Scientific) and a high-capacity Reverse Transcription Kit was used for cDNA synthesis (#4368814; Thermo Fisher Scientific). PCR was performed in triplicate wells/sample and consisted of 40 amplification cycles (95°C/15s and 60°C/1min) that were processed with a QuantStudio™ 3 System. Each well contained 5µl SYBR Green, 3µl nuclease-free water, 5 pmol (1µl) of each set of primers (Supplementary Table 1) and 25 ng cDNA (1µl). A minus-reverse transcription control from the prior RT step and a separate minus-template PCR, in which the cDNA was replaced by nuclease-free water, were used as negative controls. The relative expression of CCND1, CCND3, p21, p27, BCL2, BAX, Cyto-C, Caspase-3, PIK3CA, PTEN, AKT1 and mTOR genes was calculated by the 2-∆∆Ct method .
All primary antibodies were from Cell Signaling Technology Inc. (MA, USA). While mouse monoclonal IgG antibodies were used to detect CCND3 (#2936), BCL2 (#15071) and PI3K-p85α, rabbit monoclonal antibodies were used to detect CCND1, p21, p27 (#3686), BAX (#2772), Cyto-C (#4272), cleaved Casp-3, PTEN (#9188), Akt1 (#75692) and mTOR, proteins by Western blotting. GAPDH loading control mouse monoclonal antibodies (#MA5-15738-1MG; Thermo Fisher Scientific) were used for normalisation.
Briefly, total proteins were extracted from each cell pellet and 50 µg total protein from each sample were loaded on gradient 4–20% Mini-PROTEAN® TGX Stain-Free™ SDS-PAGE gels (#4568096; Bio-Rad Laboratories Inc.; CA, USA). The resolved proteins were then transferred to 0.2 µm Trans-Blot® TurboTM PVDF membranes using a Trans-Blot® TurboTM Transfer System (Bio-Rad Laboratories Inc). The membranes were blocked for 15 min with SuperBlockTM T20 buffer (TBS-T; #37543; Thermo Fisher Scientific), and incubated with primary antibodies (1:1000 for all antibodies) overnight at 4°C. Next, the membranes were washed with TBS-T and incubated for 1h with WestVision™ secondary anti-mouse (#WB-2000-.8) or anti-rabbit (#WB-1000-.8) IgG antibodies (1:10,000) conjugated with peroxidase micropolymer (Vector Laboratories Inc.). The membranes were washed, and the signals developed by SignalFire™ Plus ECL Reagent (#12630; Cell Signaling Technology Inc.). The images were acquired by a ChemiDocTM XRS+ (Bio-Rad Laboratories Inc.) and the density of each protein band was quantified and then normalised against the densitometry of the GAPDH band by ImageJ software (https://imagej.nih.gov/ij/) as previously described . Data are shown as mean ± SD of three blots/cell line for each targeted protein.
SPSS statistical analysis software version 25 was used to analyse the data, and all variables were assessed for normality and homogeneity by the Kolmogorov and Smirnov’s test and the Levene test, respectively. One-way analysis of variance (ANOVA) followed by Tukey’s HSD or Games-Howell post-hoc tests were performed to compare between the study groups based on variance equality. Significance was considered when P value was < 0.05.