Cells, Reagents, and Antibodies
In this study, the human cervical cancer cell lines, SiHa cells and HeLa cells, and the NK-92 cells were from a research laboratory of Foshan Maternity and Child Health Care Hospital, Guangdong Province, China. The SiHa and HeLa cells were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The NK-92 cells were cultured in alpha-minimum essential medium (ɑ-MEM) containing 100 U/mL interleukin (IL)-2. Dimethyl sulfoxide (DMSO), LY294002, and metformin hydrochloride were purchased from Sigma-Aldrich. Anti-MICA, anti-MICB, anti-ULBP-2/5/6 Phycoerythrin, anti-ULBP-1 Alexa Fluor 488, anti-ULBP-3 Allophycocyanin, anti-DDR-1, anti-HSF-1, anti-PI3K (110ɑ), anti-phospho-PI3Kp55 (Tyr199), anti-AKT, anti-phospho-AKT (ser473), anti- proliferating cell nuclear antigen (PCNA), anti-p53 primary antibodies, and their corresponding secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
Analysis Of Cell Proliferation
According to the methods described in a previous study[22], cells were trypsinized and seeded in 96-well plates (1 × 104 cells per well). After the cells were attached, metformin was added at various concentrations (0–400 µM) for 48 h. To analyze cell viability, the cell proliferation assay, cell counting kit-8 (CCK-8), was used to measure the absorbance of the samples using a microplate reader and at a wavelength of 450 nm.
Cell Apoptosis Assays
According to the methods described in a previous study[8], Cervical cancer cell lines (HeLa and SiHa) were seeded at a density of 2 × 105 cells/well in a 6-well culture plate. The cells were washed twice with cold PBS, and then resuspended in 1 × binding buffer. 100 µl of the cell suspension (1 × 105 cells) was transferred to a 5 ml culture tube and mixed with 5 µl of FITC anti-Annexin V and 5 µl PI. The mixture was gently vortexed and incubated for 15 min at RT (25 °C) in the dark. Then, 400 µl of 1 × binding buffer was added into each tube. The samples were analyzed by flow cytometry within 1 hour. The green fluorescence of Annexin V-FITC was measured at 530 nm, and the red fluorescence of PI was measured at 585 nm. The results were analyzed with FlowJo software.
Cell Cycle Assays
Cells were harvested in PBS and fixed by addition of ice-cold 70% ethanol with a Pasteur pipette during vortexing, as previously described[8]. Then, the cells were centrifuged at approximately 2,000 rpm for 5 minutes and washed twice with PBS. Finally, the cell were stained by PI staining solution (Invitrogen) and analyzed by flow cytometry collecting 25,000 events per sample. The results were analyzed with FlowJo software
Nuclear Protein Extraction
According to the methods described in a previous study[23], cells were washed three times with pre-cooled phosphate buffered saline (PBS) and subsequently scraped off with a cell scraper and collected in a 1.5 mL centrifuge tube. The cells were centrifuged at 1,000 rcf, 4 °C, for 3 min. A protease inhibitor, cell lysis buffer, and cell membrane rupture solution were added into the pellet to lyse the cells on ice for 1 h, followed by 1,000 rcf centrifugation at 4 °C for 20 min. The supernatant was discarded, and the pellet was then lysed with nuclear extraction lysis buffer on ice for 1 h, with vortexing every 5 h for complete lysis, which was followed by 12,000 rcf centrifugation at 4 °C for 15 min to collect the supernatant (i.e., nuclear protein extract).
Lactate Dehydrogenase (ldh) Cytotoxicity Assay
As described in the methods of a previous study[21, 23], the human cervical cancer HeLa or SiHa cells were seeded as target cells in 96-well plates (2 × 104 cells per well) in a total volume of 100 µL per well. The cells were then divided into five different groups as follows: drug treatment; effector cells (NK-92) with spontaneous LDH efflux; target cells with spontaneous LDH efflux; target cells with maximum LDH efflux; and volume correction (no cells) groups. Approximately 10 µL of lysis buffer (10×) was added to each well of cells and then incubated in a 37 °C incubator with 5% CO2 atmosphere. After 250 × g centrifugation for 3 min, 50 µL of a reaction solution were added to each well of the 96-well plates, followed by 50 µL stopping solution, with gentle mixing, and then the absorbance at wavelengths of 490 nm and 680 nm were read. The cytotoxicity of different target ratios (%) was calculated using the following formula: Cytotoxicity (%) = (Experimental group − Effector cells with spontaneous LDH efflux group − Target cells with maximum LDH efflux group)/(Target cells with maximum LDH efflux group − Target cells with spontaneous LDH efflux group) × 100.
Rna Extraction And Quantitative Real-time Pcr
According to the methods described in a previous study[20], After transfection for 48 h, total RNA was isolated from SiHa and HeLa using TRIzol reagent (TAKARA, Dalian, China) according to the manufacturer’s instructions. For mRNA quantification, RNA was reverse transcribed into cDNA using the PrimeScript™ RTreagent Kit (Takara, Japan) and then quantified on the CFX96 touch q-PCR system (BIO-RAD, USA) with SYBR Premix Ex Taq Kit (Takara, Japan) according to the manufacturer’s protocols. In this study, GAPDH was used as an internal control for determining the levels of HSP70 and MICA. The primers used for quantitative real-time polymerase chain reaction (qRT-PCR) are listed in Table 1. Reactions were performed using a SYBR Green kit (TAKARA, Dalian, China), according to the manufacturer’s instructions. Each 20-µl reaction mixture included 2 µl of cDNA, 10 µl of SYBR Green Mix, 0.4 µl of forward primer, 0.4 µl of reverse primer, 0.4 µl of RoxReference Dye, and 6.8 µl of RNase-free water. Then, the PCR reactions were performed in the CFX96 touch q-PCR system (BIO-RAD, USA) under the following conditions: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 30 sec, 95°C for 15 sec, and 60°Cfor 60 sec. Relative gene expression was determined by using the ΔΔCt method. Significance was defined according to P values from the two-tailed t-test.All of the reactions were performed in triplicate.
Western Blotting
Western blotting was performed as previously described[24]. Briefly, the cells were harvested and lysed with RIPA lysis buffer, and the concentration of the collected proteins was determined. Then, 100 µg of the extracted protein was separated in 10%, 8%, or 5% SDS-PAGE gel based on the molecular weight of the target protein. The separated protein gel with a pre-stained protein marker was transferred onto a PVDF membrane. Subsequently, the membrane was blocked in a 5% skim milk solution at room temperature for 2 h, followed by incubating with the corresponding primary and secondary antibodies and washing with Tris-buffered saline, 0.1% Tween 20 (TBST) in between. The PVDF membrane was developed using an enhanced chemiluminescence solution (Pierce) and subsequently photographed in a Bio-Rad gel imaging system. The exposure time was adjusted according to the protein bands and background. After selecting the clear protein bands in the image, the gray value of each protein band was analyzed by software and statistical analysis was conducted.
Tumor Xenograft Modeling and In Vivo Experiments
BALB/c nude mice of 4 weeks old (weighing approximately 15–17 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangdong Province, China). All mice were housed and bred in a specific-pathogen-free (SPF) grade animal facility, with 22–25 °C temperature, 40–60% humidity, and 12 h/12 h light/dark cycle. To generate tumor xenograft, 20 mice were used. The skin of the left forelimb near the armpit was disinfected and 0.1 mL SiHa cells suspended in serum-free medium (containing approximately 5 × 106 cells) were injected. After inoculation of the cervical cancer cells, the nude mice were continuously housed under the same conditions. Once the subcutaneous nodules grown to a rice grain size (required approximately a week), the subcutaneous xenograft model of cervical cancer in nude mice was successfully constructed. The subcutaneous tumor size in each nude mouse was measured using a digital vernier caliper. Once the tumor diameter reached approximately 0.3–0.5 cm, the nude mice were numbered, randomly divided into four groups (with five mice per group), namely, control, model, 50 mg/kg/d metformin, and 250 mg/kg/d metformin groups. Metformin was given by gavage. All nude mice were closely monitored for tumor growth, skin condition, and behavior daily and any tumor ulceration or irritation was noted. The longest (A) and the shortest (B) diameters of the subcutaneous tumors were measured with a digital vernier caliper before each metformin administration to calculate the tumor volume (V) using the following formula: V = 0.5 × A × B2. In addition, all nude mice were weighed daily, and their daily food intake was also measured. After the completion of the 23-day metformin administration, all nude mice were sacrificed and placed on ice, their skin was immediately cut open, and the subcutaneous tumor xenografts were collected. After weighing each tumor xenograft on a digital scale, one part of the tumor tissue was dissected and frozen in liquid nitrogen for western blotting. All experimental procedures were approved by the Institutional Animal Care and Use Committee of South Medical University.
Statistical Analysis
The SPSS16.0 software (IBM SPSS, Chicago, IL, USA) was used for data analysis in this study. An independent T test was used to compare the results between the two groups. Multivariate ANOVA was used to compare differences between multiple groups, followed by multiple corrections using the Bonferroni’s test. P < 0.05 was considered statistically significant.