Two previously established primary cultures of human cervical cancer that were characterized as expressing cytokeratins and the E7 gene from human papilloma virus 16 by immunocytochemistry and PCR, respectively, were used in the current study and cultured as previously described (24). The human cervical cancer cell lines C33A, CaSki, HeLa, INBL and SiHa, and primary epidermal keratinocytes (normal human neonatal foreskin) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured according to the supplier's instructions.

Cell viability was measured by MTT assay. Cells were seeded at a density of 3.5×10³ cells into 96-well plates. After 24 h of incubation at 37°C, the cells were cultured with Dulbecco's modified Eagle's medium for cervical cancer cells (cat. no. 12430054; Gibco; Thermo Fisher Scientific, Inc., Waltham MA, USA) and Keratinocyte-SFM (cat. no. 17005042; Gibco; Thermo Fisher Scientific, Inc.) for epidermal keratinocytes)) and various concentrations (40–200 µM) of glibenclamide (cat. no. G0639-5G; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for different incubation times (24–72 h). Subsequently, MTT tetrazolium salt (0.5 mg/ml) was added to each well and cells were incubated for a further 4 h. Formazan crystals were then dissolved using SDS, and the absorbance was measured using a Multiskan FC microplate photometer (Thermo Fisher Scientific, Inc.).

HeLa and primary epidermal keratinocytes cells were incubated for 48 h at 37°C in culture medium as previously described, either with or without glibenclamide (100 or 150 µM) or vehicle (sterile water). Camptothecin (apoptosis inductor, 10 mg/ml) and methanol (necrosis inductor, 39.6 mg/ml) were used as positive controls. Apoptosis was determined using the Annexin V-FITC kit (Invitrogen; Thermo Fisher Scientific, Inc.) to measure binding to phosphatidylserine and DNA staining by propidium iodide (1 mg/ml), according to manufacturer's protocol. The experiments were performed using the CyAn ADP flow cytometer (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Quadrant analysis was performed using the Summit 4.3 software (Beckman Coulter, Inc., Fullerton, CA).

Human cervical cancer biopsies were obtained from the Instituto Nacional de Cancerología (Mexico City, Mexico). The protocol was approved by the corresponding Research and Ethics Committees [protocol no. 013/024/GII (CEI/846)] and written informed consent was obtained from all the patients. A total of 74 biopsies were studied and the age of the patients ranged from 25 to 82 years old. The present study only included samples from patients that had not received any anti-cancer therapy and excluded patients that had vaginal infections. Non-cancerous cervical tissues were obtained from hysterectomy patients with benign gynecologic pathology at the Hospital General ‘Dr Manuel Gea Gonzalez’ (Mexico City, Mexico). The protocol followed the local ethical considerations (protocol no. 11-84-2013) and was approved by the corresponding ethics committee. The non-cancerous cervical tissue group included patients whose cervixes were described as healthy, but excluded patients taking hormones. The tissues were either collected in TRIzol and placed in liquid nitrogen to obtain mRNA, or fixed in formaldehyde (10% w/v) and embedded in paraffin for subsequent immunohistochemical analysis. The samples were classified as adenocarcinoma (AC) or squamous cell carcinoma (SCC), according to the International Federation of Gynecology and Obstetrics (FIGO) staging system (25). A total of 24 cancer biopsies were analyzed for Kir6.2 mRNA expression, and 30 tumor tissues were analyzed for Kir6.2 and SUR protein expression.

RNA was isolated using TRIzol reagent (cat. no. T9424; Sigma-Aldrich; Merck KGaA). The total RNA (5 µg) was treated with DNase I (Roche Diagnostics GmbH, Mannheim, Germany; 04716728001) and reverse transcribed using the M-MuLV reverse transcriptase (New England BioLabs, Inc., Ipswich, MA, USA). The temperature and time protocol was as follows: 65°C for 5 min, 37°C for 50 min, 70°C for 15 min and 4°C for 60 min. When small biopsies were obtained, the total RNA (518 ng) was processed using the Path-ID Multiplex One-Step RT-PCR kit (cat. no. 4388641; Ambion; Thermo Fisher Scientific, Inc.). The expression of Kir6.2 (Hs00265026_s1; Applied Biosystems; Thermo Fisher Scientific, Inc.) was assessed by RT-qPCR using the TaqMan™ Master-mix based detection system (cat. no. 4304437; Applied Biosystems; Thermo Fisher Scientific, Inc.). The temperature and cycling protocol were as follows: 95°C for 10 min, 95°C for 10 sec and 60°C for 1 min. The first two steps were repeated for 40 cycles. Expression of a housekeeping gene, hypoxanthine-guanine phosphoribosyltransferase (cat. no. 4326321E; Applied Biosystems; Thermo Fisher Scientific, Inc.), was used as an internal control. Relative expression levels of the gene of interest were determined by the 2^−∆∆Cq method according to the threshold cycle (26), and 2–3 experimental replicates were performed.

For immunohistochemistry, serial sections (5 µm) were mounted on charged glass slides and deparaffinized. Antigen retrieval and blocking were performed as previously described (24). The slides were then incubated for 2 h with the primary antibodies, anti-Kir6.2 (1:100; cat. no. NBP1-00900), anti-SUR1 (1:50; cat. no. NBP1-59778) or anti-SUR2 (1:300; cat. no. NBP1-84436; all from Novus Biologicals, LLC, Littleton, CO, USA), at 4°C. The antibodies were diluted in ImmunoDetector Protein Blocker/Antibody Diluent (cat. no. 0041; Bio SB, Santa Barbara, CA, USA). Subsequently, the slides were incubated at room temperature with a biotin-conjugated secondary antibody for 15 min, followed by 15 min with horseradish peroxidase (HRP)-conjugated streptavidin polymer (both provided as part of the Mouse/Rabbit ImmunoDetector DAB HRP Brown kit; cat. no. 0005; Bio SB). The staining reaction was completed in the presence of diaminobenzidine in a buffer reaction solution (#0005; Bio SB) and washed with distilled water. The slides were counterstained with hematoxylin (Sigma-Aldrich; Merck KGaA) and rinsed with water and ethanol as previously described (24). The slides were mounted with resin (EMD Millipore, Billerica, MA, USA) and observed using an Olympus IX51 light microscope and an Olympus DP70 camera (Olympus Corporation, Tokyo, Japan). Brown immunostaining indicated Kir6.2/SUR1/SUR2 expression.

For immunocytochemistry, cervical cancer cells were mounted on glass coverslips and fixed in ethanol. Subsequently, staining was performed as per the protocol previously described for the tissue samples.

Data are presented as the mean ± SD from three independent experiments with 6–8 replicates for cell proliferation assays and 2–3 replicates for PCR studies. Data were analyzed by a Student's t-test or by an analysis of variance followed by a Tukey-Kramer post hoc test using the GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Kir6.2 subunit expression was assessed in two primary cultures of cervical cancer (PC1 and PC2) and five human cervical cancer cell lines (C33A, CaSki, HeLa, INBL and SiHa). The expression of Kir6.2 subunit mRNA was evaluated by RT-qPCR and normalized relative to K_ATP channel expression in normal primary epidermal keratinocytes. K_ATP channel expression was present in each of the cell lines and primary cultures (Fig. 1A). However, the only statistically significant difference (P<0.05) compared with normal keratinocytes was K_ATP channel overexpression in HeLa cells. Therefore, HeLa cells were used in further analyses of K_ATP channel protein expression. Fig. 1B depicts the protein expression of the Kir6.2 and SUR2 subunits in HeLa cells. Human cerebral cortex and testis samples were used as positive controls for Kir6.2 and SUR2 protein expression, respectively (data not shown). SUR1 protein expression was not detected (data not shown), whereas the SUR2 subunit was positively stained. These results suggested that HeLa cells express K_ATP channels composed of Kir6.2 and SUR2 subunits, and that these channels may serve a function in cervical carcinogenesis.

The effect of glibenclamide, a K_ATP channel inhibitor, on HeLa cell proliferation was investigated by assaying the metabolic activity. The HeLa cells were incubated with various glibenclamide concentrations (40–200 µM) for 48 h, which resulted in a concentration- and time-dependent decrease in cell viability of >60% at the highest concentration compared with the vehicle-treated cells (Fig. 1C and D). By contrast, glibenclamide (150 µM) decreased the proliferation of normal keratinocytes by 30% (Fig. 1D). The present study also studied the effect of glibenclamide on other cervical cancer cell lines that displayed varying mRNA expression levels of the Kir6.2 subunit. The results revealed that as the Kir6.2 mRNA expression levels increased, the inhibitory effect of glibenclamide also increased (Fig. 1E). Glibenclamide did not affect apoptosis (data not shown).

Kir6.2 subunit mRNA expression was assessed in 24 cervical cancer samples (22 SCC and 2 AC biopsies). The samples were classified as cervical cancer of FIGO stage I (4 samples from stage IA and 2 samples from stage IB), II (1 sample from stage IIA and 5 samples from stage IIB), III (7 samples from stage IIIB) or IV (3 samples from stage IVB), and included 1 AC in situ and 1 invasive AC. The mRNA expression of 10 non-cancerous cervical biopsies (controls) from other patients was normalized to 1 and used for comparison. Increased Kir6.2 mRNA expression was detected in the stage IV SCC biopsies (Fig. 2A), with some of these samples exhibiting mRNA levels ≤60× those of the controls. Differential Kir6.2 mRNA expression was also observed in invasive tumors; compared with non-invasive tumors or non-cancerous biopsies (control group; expression value normalized to 1), some of the invasive tumors displayed >50× the Kir6.2 mRNA expression (Fig. 2B). Non-invasive tumors were considered to be those where the invasive component was delimited to microscopic foci of only a few microns in length and depth, as observed in in situ carcinoma biopsies (microinvasive tumors, 4 samples from cervical cancer FIGO stage IAI and 1 AC in situ). The samples were also divided according to their differentiation grade into well, moderately or poorly differentiated groups. Increased Kir6.2 subunit expression was observed in samples from the moderate and poor differentiation grades and certain biopsies from these groups displayed ≤50× Kir6.2 mRNA levels compared with the controls (Fig. 2C). The present study also analyzed the potential association between Kir6.2 and lymphovascular space invasion. In total, 7 samples exhibited lymphovascular space invasion and 100% of these samples displayed increased Kir6.2 mRNA expression. However, 13/23 (56.6%) samples exhibiting no lymphovascular space invasion also displayed increased Kir6.2 mRNA expression. No significant association was identified between lymphovascular invasion and mRNA channel expression.

The present study determined the protein expression levels of the Kir6.2 and SUR2 subunits in human cervical biopsies by performing immunohistochemical analysis on 30 cervical cancer tissues (24 SCCs and 6 ACs) and 10 non-cancerous cervical tissues (controls). All of these samples were obtained from different patients from those that participated in the mRNA studies. Kir6.2 subunit immunostaining was not observed in the majority (9/10) of the non-cancerous cervical tissues (Fig. 3A), while 1 sample exhibited a weak signal. Similarly, SUR2 expression was not observed in any of these non-cancerous cervical tissues (Fig. 3B). By contrast, 10/30 cervical cancer samples revealed strong Kir6.2 staining (Fig. 3A). The positive biopsies corresponded to samples classified as cervical cancer FIGO stage I (1 sample from stage IA and 1 sample from stage IB), stage II (2 samples from stage IIB), III (2 samples from stage IIIB) and stage IV (1 sample from stage IVA and 2 samples from stage IVB), and 1 AC from stage IB. Stromal cells in the tumor tissues also exhibited Kir6.2 staining. SUR2 subunit expression was detected in 4 cancer tissues (Fig. 3B). Two samples in the immunochemical analysis were obtained from patients with lymphovascular space invasion, both of which displayed increased Kir6.2 protein expression in comparison with controls (data not shown).