Human cervical carcinoma HeLa cells and cisplatin-resistant HeLa cells (HeLa/DDP) were obtained from Shenglong Biological Corporation (Shanghai, China). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml ampicillin and 50 µg/ml streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Cisplatin, ethyl pyruvate (EP) and MTT were obtained from Sigma Aldrich, Merck Millipore (Darmstadt, Germany). Recombinant human (rh) HMGB1/Fc was obtained from Sino Biological Inc. (Beijing, China; cat. no. 10326-H01H). The human HMGB1 short hairpin (sh) RNAs (cat. no. TG316576) and negative control (NC) shRNA (cat. no. TR30013) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

β-actin antibody was purchased from TransBionovo Co., Ltd. (Beijing, China; cat. no. HC201; 1:1,000). Antibodies for caspase-3 (cat. no. 9662), cleaved (c)-caspase-3 (cat. no. 9661), poly ADP ribose polymerase (PARP; cat. no. 9542), c-PARP (cat. no. 9541), extracellular signal-regulated kinase 1/2 (ERK1/2; cat. no. 9102) and phosphorylated (p)-ERK1/2 (cat. no. 8544) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA) and all diluted 1:1,000. β-Tubulin antibody (H-235), a rabbit polyclonal IgG provided at 200 µg/ml (cat. no. sc-9104; 1:1,000), was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-lamin B1 (cat. no. ab16048; 1:1,000) and anti-HMGB1 (cat. no. ab18256; 1:1,000), both rabbit polyclonal antibodies, were obtained from Abcam (Cambridge, UK). LC3B/MAP1LC3B antibody (cat. no. NB100-2220; 1:1,000) was purchased from Novus Biologicals, LLC (Littleton, CO, USA).

HeLa, SiHa and HeLa/DDP cells were plated into 48-well plates (5×10⁵ cells/well) and incubated for 6 h prior to transfection with HMGB1 shRNA or NC shRNA using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. The cells were then cultured for the indicated times before being subjected to MTT assays or western blotting analysis.

Annexin V-FITC apoptosis detection kit (catalog no. ab14085) was obtained from Abcam; 2×10⁵ cells were trypsinized into a single cell suspension. Resuspended cells were incubated with Annexin V-FITC (0.1 µg/µl) for 15 min in the dark on ice. Propidium iodide (0.05 µg/µl) was then added and used as a counterstain to discriminate between necrotic and apoptotic cells. Fluorescence-activated cell sorting (FACS) analysis was then performed and data was analyzed using FlowJo 10 software (FlowJo, LLC, Ashland, OR, USA).

HeLa cells and SiHa cells (2×10⁵ cells/well) were plated into 48-well plates. Six hours later, the cells were treated with 10 µg/ml of cisplatin for 24 and 48 h, respectively. Cytosolic extracts and nuclear extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction kit (catalog no. P0028; Beyotime, Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocols. Protein concentrations of the extracts were measured by bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts (15 µg) of the proteins were loaded and subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck Millipore) in Tris-buffered saline-0.1% Tween-20 (TBST) buffer for 40 min at room temperature. Membranes were then incubated with the indicated primary antibodies overnight at 4°C and secondary antibodies for 40 min at room temperature. The membranes were washed three times in each step for 5 min with TBST buffer. The bands were then visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.). The bands were captured and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) and the expression of HMGB1 was normalized to β-actin.

The tumor inhibition rates of cisplatin and EP on HeLa cells and HeLa/DPP cells were detected by MTT assay. The cervical cancer cells were plated into 96-well plates (2×10⁴ cells/well) and cultured for 6 h in DMEM. They were subsequently treated with the 10 µg/ml cisplatin and stimulated for 24, 48 and 72 h, respectively. MTT (5 mg/ml) was added into the medium and 150 µl DMSO was used to dissolve the formazan crystals. The 96-well plates were read on a microplate reader at a test wavelength of 490 nm (A₄₉₀).

The growth inhibition rate was calculated as follows: growth inhibition rate (%) = (average A₄₉₀ of the control group - average A₄₉₀ value of the experimental group)/average A₄₉₀ value of the control group × 100%.

All of the data were analyzed by SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) and are presented as the mean ± standard deviations. P<0.05 was considered to indicate a statistically significant difference.

HMGB1 is a nucleoprotein that is associated with cancer progression (22–24). To examine whether HMGB1 is involved in drug resistance in cervical cancer cells, two cisplatin-sensitive cell lines, HeLa and SiHa, were treated with 10 µg/ml of cisplatin for 24 and 48 h. Expression of HMGB1 was significantly increased compared with untreated cells in at 24 and 48 h in both HeLa (P=0.032 and P=0.014, respectively; Fig. 1A) and SiHa cells (P=0.038 and P=0.011, respectively; Fig. 1B). These findings suggest that cisplatin-induced HMGB1 expression in cervical cancers may be involved in drug resistance.

The cervical cancer cell line HeLa and its cisplatin-resistant subline, HeLa/DDP were used as models. MTT assays were performed to examine the levels of cisplatin resistance in these cell lines. The cisplatin-sensitive HeLa and cisplatin-resistant HeLa/DDP cells were treated with increasing concentrations of cisplatin for 48 h, and half maximal inhibitory concentration (IC₅₀) values were calculated as 4.27 and 22.70 µg/ml, respectively (Fig. 2A). The IC₅₀ value in the HeLa/DDP cell line was ~5.3-fold that in the HeLa cell line, confirming that HeLa/DDP had higher resistance to cisplatin than HeLa cells (Fig. 2A).

HMGB1 protein expression levels were detected by western blotting analysis in both HeLa and HeLa/DDP cells. HeLa/DDP cells exhibited higher protein expression levels of HMGB1 than cisplatin-sensitive HeLa cells (Fig. 2B), which suggested that increased HMGB1 expression was associated with cisplatin drug resistance in HeLa cervical cancer cells.

Under normal conditions, HMGB1 is a non-histone nuclear protein (25,26). To establish whether the location of HMGB1 was altered in drug resistant cancer cells, protein in the cytoplasm and nucleus was extracted from cisplatin-treated HeLa cells, and HMGB1 expression was detected in cytoplasmic and nuclear fractions by western blotting (Fig. 3). Protein expression levels of HMGB1 in HeLa cells increased in a time dependent manner over the course of 48 h (Fig. 3A). In addition, cytoplasmic HMGB1 increased with time when treated with cisplatin (Fig. 3B), while the levels of HMGB1 in the nucleus decreased (Fig. 3C). This suggests that HMGB1 translocation from the nucleus to cytoplasm may be associated with cisplatin resistance.

To further investigate the involvement of HMGB1 translocation in drug resistance a pharmacological inhibitor of HMGB1 cytoplasmic translocation, EP, was used to treat HeLa cells. EP treatment alongside cisplatin reduced the levels of cytoplasmic HMGB1 in cisplatin-treated HeLa cells at 24 and 48 h (Fig. 4A). Notably, the growth inhibition rate in EP-treated HeLa and HeLa/DDP cells was significantly increased compared with cells without EP treatment (Fig. 4B), suggesting EP may significantly inhibit translocation of HMGB1 into the cytoplasm and suppressed the proliferation of cervical cancer cells.

RNA interference technology was used to interfere with the expression of endogenous HMGB1. As demonstrated in Fig. 5A, the expression of HMGB1 was reduced in both the cytoplasm and nucleus of HeLa cells transfected with HMGB1 shRNA compared with NC shRNA. MTT assays were then used to detect the effects of interference with HMGB1 on the drug resistance of cisplatin in HeLa cells. As demonstrated in Fig. 5B, the growth inhibition rate was significantly higher in HMGB1 shRNA transfected, cisplatin-treated HeLa cells, compared with NC shRNA transfected, cisplatin-treated HeLa cells at 48 and 72 h (P=0.009 and P=0.009; Fig. 5B, HeLa cells). This effect was also observed in HeLa/DPP cells at 48 and 72 h (P=0.009 and P=0.007; Fig. 5B; HeLa/DPP cells). Therefore, HeLa and HeLa/DPP cells transfected with HMGB1 shRNA demonstrated reduced proliferation.

The cell apoptosis rate was determined by FACS analysis in HeLa cells transfected with HMGB1 shRNA and NC shRNA, and then treated with 10 µg/ml cisplatin for 48 h. FACS analysis revealed that the apoptosis rate in HMGB1 shRNA transfected HeLa cells was significantly greater than in NC shRNA transfected HeLa cells (P=0.003; Fig. 6A). In addition, the presence of caspase-3, the executor of cell apoptosis, and its substrate PARP, were detected by western blotting analysis in cisplatin-treated HMGB1 shRNA/NC shRNA transfected HeLa cells, and in untreated/untransfected cells (Fig. 6B). The levels of cleaved, and therefore activated, caspase-3 and PARP were higher in HMGB1 shRNA transfected HeLa cells than in untreated/untransfected cells and cisplatin-treated NC shRNA cells (Fig. 6B).

Compared with FBS-treated cells, the levels of LC3-II in HeLa cells treated with rhHMGB1 were increased, while nucleoporin p62 levels were reduced (Fig. 7A). This suggests that administration of rhHMGB1 resulted in degradation of p62 and induction of cell autophagy. Furthermore, treatment with rhHMGB1 increased the phosphorylation of ERK1/2 (Fig. 7A), which results in activation of the MEK/ERK1/2 signaling pathway. By contrast, interference with endogenous expression of HMGB1 using HMGB1 shRNA, did not affect levels of p-ERK1/2, LC3-II and p62 compared with NC shRNA transfected cells (Fig. 7B).