HeLa, SiHa and CaSki cervical cells were obtained from American Type Culture Collection (ATCC), cells were maintained in Dulbecco’s Modified Eagle´s Medium (DMEM) supplemented with 5% of fetal bovine serum (FBS), 37 oC and 5% of CO2. HeLa cell line was also used in the “in vivo” studies. All experiments were prepared under sterility into a laminar flow hood. All compounds used were purchased from recognized commercial houses.
Effect of insulin on cell proliferation
Cell proliferation was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma) and crystal violet assays. 1X103 cells/well was seed and expose with normoglycemic (5 mM) or hyperglycemic (25 mM) medium by 7 days alone or in combination with insulin (0, 0.01, 0.1 or 1 ug/mL). SiHa and CaSki cells were only expose to the highest concentration of insulin. All experiments were made by triplicate. Time of exposition was stablished as a result of previous experiments made in the laboratory and by reports in the literature using different cancer cell line. Insulin concentration was selected because high physiological levels has been reported as inductors of proliferative effects (up 10 nM) [16, 17].
The assay is based on the conversion of MTT to formazan, this compound is developed by viable cells with active metabolism. The measured absorbance is proportional to the number of viable cells. Briefly, 5 mg/mL MTT was dissolved in DMEM (1:9), the solution was added in each well and was incubated during 1 to 3 h. Formazan dye was dissolved with 2-propanol acid, optical density was measured using an ELISA reader at λ= 570 nm. The results were expressed as the percentage of MTT reduction in relation to cero insulin concentration groups.
Apoptosis was evaluated by flow cytometry. Briefly, once the cells were expose to hyperglycemia and/or insulin, they were washed with PBS and harvested with PBS-EDTA-Tripsin, followed by centrifugation and resuspended in DMEM, finally they were exposed to Guava Nexin Reagent, in the dark at room temperature for 20 minutes. 10,000 events were acquired in the Guava easyCyte equipment (Merck Millipore, Darmstadt, DE) per sample. Early and late apoptosis were determined, and the results were expressed as the total percentage of apoptosis. Experiments were carried out independently and by triplicate.
Cell cycle assay
Determination of cell cycle was performed by flow cytometry. Briefly, once the cells were expose whit hyperglycemia and/or insulin, the cells were washed with PBS and harvested with PBS-EDTA-Tripsin, followed by a centrifugation. After that were fixed in 70% ethanol and incubate at 4 oC for 24 h. Thereafter, cells were washed with PBS and incubated with Guava Cell Cycle Reagent at room temperature for 30 minutes in the dark. 10,000 events were acquired in the cellCycle software (Merck Millipore, Darmstadt, DE) per sample and cell cycle phases were analyzed. Experiments were carried out independently and by triplicate.
In vivo experiments
Female mice BALB/c (nu/nu) of 20-25 g body weight were purchased from laboratory animal production and experimentation unit (UPEAL-Bioterio) of Universidad Autonoma Metropolitana Unidad Xochimico. The mice had free access to sterile water and food, they were housing into microisolator (Allentown Inc.) under 25oC, 70% of humidity and 12 h light-12 h dark cycle. The sample size was calculate by the comparison of two means, where the sample size corresponds to the value of the standard deviation, with Zα (Z of alpha) which refers to the type I error (confidence level α = 0.05 corresponding to a value of Z = 1.96), and Zβ (beta Z) with a power of 80% (value of Z = 0.84) . All the mice were handling in accord to Mexican Guide (NOM-062-ZOO-1999) and the Committee for the Update of the Guide for the Care and Use of Laboratory Animals and Biological hazard residues Guide (NOM-087-ECOL-SSA1-2001) [19-21]. This protocol was approved for the Instituto Nacional de Cancerología (011/045/IBI and CB714/11).
Thirty-nine female mice (BALB/c (nu/nu) of 20-25 g body weight) were used. Random way, one part of the mice was hyperglycemia induced using streptozotocin (100 mg/Kg/week) until the mice became hyperglycemic. Once the mice present hyperglycemia (≥ 125 mg/dL), then were inoculated subcutaneously into the back with 5X106 HeLa cervical cancer cells. Once the tumor reached around 170 mm3 (week 4), mice were divided randomly in four groups: A) control (no hyperglycemic), B) insulin (1IU/Kg i.p. daily until the end of experiment, no hyperglycemic), C) hyperglycemia and D) hyperglycemia+insulin. The animals were weekly weight, and glucose blood concentration as well as tumor size were measured. The tumor xenografts were measured with a caliper in two dimensions; tumor volume was calculated by following equation: V=((width)2*length*π)/4) ; time of duplicated rate of tumor was calculate using the following equation: T=((days of treatment)/(((Log(final volume)-Log(initial volume))/Log 2)) . When the tumor reaches 2000 mm3, the mouse was sacrificed. At the end of experiments the mice were sacrificed under anesthesia overdoses using a mixture of isoflurane/oxygen (3%); plasma and pancreas were obtained; samples were store in formaldehyde at 10% for histological studies.
Glucose, cholesterol, triglycerides, creatinine and BUN were quantified in serum at the end of the experiments using a Beckman Coulter laboratory analyzer AU680 Chemistry System. Also, blood glucose concentration was measured with glucometer (Accu-Chek Performa, Roche) each week for hyperglycemia follow-up. Plasma insulin concentration was determined using an ELISA kit (Abnova).
Pancreas was perfused through pancreatic conduct catheter with saline solution following by 10% formalin until fixation was completed. After that, the tissue was including in paraffin, sectioned at 3 µm thickness and stained with hematoxylin-eosin method. The slides were analyzed blindly. Langerhans islets by mm2 were count in all groups.
All the results were present as mean ± SEM (standard error of the mean). The tumor volume was analyzed by ANOVA for repeated measures followed by the Student Newman-Keuls comparison test. Overall survival was calculated with Kaplan-Meier analysis. The other parameters were evaluated using one-way ANOVA followed by Student Newman-Keuls multiple comparison test, GraphPad Prism 4 software (San Diego, CA, USA) and SPSSv.21.0.0 (Chicago, IL, USA) were used. Statistical significance was defined when p value was ≤ 0.05.