Human cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC; HeLa and SiHa) and 293T cell lines were obtained from American Type Culture Collection, which had characterized the cell lines by short tandem repeat profiling, cell morphology and karyotyping assay. The cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂. Oxamate was purchased from Sigma-Aldrich (Merck KGaA). SP600125 was purchased from Abcam. For JNK inhibitor assay, HeLa cells were treated with 10 µM SP600125 1 h at 37°C prior to oxamate treatment and cells were incubated for another 48 h at 37°C. LDHA-knockdown HeLa cells were treated with 10 µM SP600125 for 48 h at 37°C. The primary antibodies for LDHA, cleaved caspase-3, caspase-9, Cyclin A, Cyclin B1, p21, cyclin-dependent kinase (CDK)1, JNK, phosphorylated (p-)JNK, p38 and p-p38 were obtained from Cell Signaling Technology, Inc. The antibody for β-actin was purchased from Santa Cruz Biotechnology, Inc. The antibodies for cytochrome c and cytochrome c oxidase IV (COX IV) were obtained from Abcam.

Gene Expression Profiling Interactive Analysis (GEPIA; gepia.cancer-pku.cn/index.html) comprises open databases for analysis of gene expression data from The Cancer Genome Atlas (TCGA; cancer.gov/tcga) (30). GEPIA was used to analyze mRNA expression levels of LDHA in human tumor and normal samples, the correlation between LDHA and mitochondrial pyruvate carrier 1 (MPC1) and the potential prognostic value of LDHA expression levels. Data were extracted from TGGA.

HeLa and SiHa cells were seeded at 10,000 cells/well in triplicate in 96-well plates for 24 h at 37°C, then treated with 10, 20, 50, 80 and 100 mM oxamate for 44 h at 37°C. Cytotoxicity of oxamate was measured using MTT Cell Proliferation and Cytotoxicity Assay kit (cat. no. M1020; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol. MTT solution was added to each well and incubated for 4 h at 37°C in a humidified atmosphere. MTT formazan precipitate was dissolved in dimethyl sulfoxide. Absorbance was measured at 570 nm. In addition, viability of LDHA knockdown and control cells were also measured by MTT detection kit as aforementioned.

LDHA knockdown or oxamate-treated HeLa and SiHa cells were incubated with BrdU for 6 h at 37°C. BrdU uptake was analyzed by Cell Proliferation ELISA, BrdU (colorimetric) assay (cat. no. 11647229001; Roche Diagnostrics) according to the manufacturer's protocol.

For colony formation assay, 1,000 cells from LDHA knockdown or oxamate-treated HeLa and SiHa groups were plated in six-well plates and cultured for 7 days at 37°C. The cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature and stained with 0.1% crystal violet solution for 10 min at room temperature. Then, colonies (>50 cells/colony) were observed using a light microscope (magnification, ×40; Olympus Corporation), counted by using the ImageJ Lab software (version k1.7.9; National Institutes of Health), photographed by using a Canon EOS600D digital camera (Canon, Inc.).

ATP levels in HeLa and SiHa cells were determined using ATP Assay Kit (cat. no. S0026; Beyotime Institute of Biotechnology) according to the manufacturer's protocol and as previously described (31,32). In brief, samples were lysed with 200 µl lysis buffer, centrifuged at 6,000 g for 5 min at 4°C and the supernatant was collected. Then, the supernatant was mixed with 100 µl ATP detection working buffer and measured by a plate-reading luminometer.

Lactate production was determined by Lactic Acid assay kit (cat. no. A019-2-1; Nanjing Jiancheng Bioengineering Institute) as previously described (33). Briefly, HeLa and SiHa cells were harvested, washed twice with PBS and lysed in RIPA buffer (cat. no. 89900; Thermo Fisher Scientific) for 30 min on ice. Following centrifugation at 6,000 g for 10 min at 4°C, the supernatant was collected in microcentrifuge tubes, mixed with an equal volume of substrate and incubated at 37°C for 10 min. Following the addition of stop solution, each sample (volume, 200 µl) was aliquoted in the wells of a 96-well microtiter plate. Lactate concentration was evaluated by measuring absorbance using a microplate reader at 530 nm. The result was normalized to the total protein content, determined by BCA assay.

Glucose Assay kit (cat. no. 361500; Shanghai Rongsheng Biotech Co., Ltd.) was used to detect the glucose uptake according to the manufacturer's instructions and quantified by measuring the absorption at 450 nm. HeLa and SiHa cells were cultured in 96-well plates seeded at 10,000 cells/well in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) for 24 h at 37°C, then treated with oxamate for 24 h. The cells were centrifuged at 6,000 g for 10 min at 4°C and the supernatant was collected. Glucose uptake was calculated as follows: original glucose concentration in medium-detected glucose concentration in culture supernatant.

shRNA sequences targeting LDHA were as follows: shRNA1, 5′-GATCCCCACCATGATTAAGGGTCTTTCTTCCTGTCAGAAAAGACCCTTAATCATGGTGGTTTTTG-3′ and shRNA2, 5′-GATCCGCAAACTCCAAGCTGGTCATTCTTCCTGTCAGAAATGACCAGCTTGGAGTTTGCTTTTTG-3′. The shRNA sequence of the negative control (PGreenpuro shRNA) was 5′-GATCCGTGCTCCACGGCATTTCATTACTTCCTGTCAGATAATGAAATGCCGTGGAGCACTTTTTG-3′. pGreenPuro-LDHA-shRNA1 and pGreenPuro-LDHA-shRNA 2 were prepared by inserting target sequences into PGreenpuro shRNA vectors. pGreenPuro-LDHA-shRNA1, pGreenPuro-LDHA-shRNA 2 and PGreenpuro shRNA were obtained from Chengdu Transvector Biotechnology. The concentration of extracted plasmid was >500 ng/µl with an A260/280 ratio of 1.8-2.0. A second generation lentivirus packaging system was used. The plasmid pGreenPuro-LDHA-shRNA (2 µg) was co-transfected into 293T cells together with packaging vectors psPAX2 (2 µg) and envelop plasmids pMD2.G (1.5 µg; both Addgene, Inc.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The viral supernatant fraction was collected at 48 h post-transfection and filtered through a 0.45 µm filter. Viral titers were determined by QuickTiter Quantitation kit (Cell Biolabs, Inc.) according to the manufacturer's instructions. Multiplicity of infection of 50 and 60 were used for HeLa and SiHa cells, respectively. HeLa and SiHa cells were co-cultured with viral supernatant and 6 µg/ml polybrene for 16 h at 37°C. Then, RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) was replaced with fresh medium. After 24 h, the medium was replaced with fresh RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 1 µg/ml puromycin (Thermo Fisher Scientific, Inc.). The cells were maintained in medium with 1 µg/ml puromycin for 2 weeks at 37°C.

For cell cycle analysis, HeLa and SiHa cells were collected following treatment with 20 mM oxamate for 48 h at 37°C, then fixed with 4% paraformaldehyde at 4°C for 30 min. Following washing with PBS three times, cells were stained with propidium iodine (PI; 50 µg/ml; cat. no. P2667; Sigma-Aldrich; Merck KGaA) at room temperature for 30 min in the dark according to the manufacturer's instructions. Cell cycle distribution was analyzed using 10,000 cells by flow cytometry analysis using a FACS Canto II flow cytometer (Becton, Dickinson and Company) and BD FACS DIVA software, version 6.1.3 (Becton-Dickinson and Company). For apoptosis assay, HeLa and SiHa cells were harvested, washed with PBS, stained with 5 µl Annexin V-FITC and 5 µl PI at room temperature for 15 min using FITC Annexin V Apoptosis Detection kit (cat. no. 556547; BD Biosciences) and analyzed by FACSCanto II flow cytometer as previously described (34). Apoptotic rate was calculated as follows: Late apoptosis cell number/total cell number ×100%.

HeLa and SiHa cells were harvested and washed with PBS. The protein was were extracted and western blot analysis was performed as previously described (35). Mitochondria were isolated from cells using Mitochondria Isolation Kit for Cultured Cells (TransGen Biotech Co., Ltd.) according to the manufacturer's protocol. Total protein was extracted from cells using Mammalian Total Protein Extraction kit (TransGen Biotech Co., Ltd.) according to the manufacturer's protocol. Enhanced BCA Protein Assay kit (cat. no. P0010; Beyotime Institute of Biotechnology) was used to determine protein concentration. Then, 12% separation and 5% stacking gels were prepared for electrophoresis. The proteins (20 µg/lane) were separated and transferred onto PVDF membranes (EMD Millipore) via wet transfer method. Next, 5% skimmed milk in 1X Tris-buffered saline-0.05% Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.), was used to block membranes at room temperature for 1 h. The membranes were incubated with primary antibodies against LDHA (cat. no. 3582; 1:1,000), Cyclin A (cat. no. 67955; 1:1,000), Cyclin B1 (cat. no. 12231; 1:1,000), p21 (cat. no. 2947; 1:1,000), CDK1 (cat. no. 28439; 1:500), p38 (cat. no. 8690; 1:1,000), p-p38 (cat. no. 4511; 1:1,000), JNK (cat. no. 9252; 1:1,000), p-JNK (cat. no. 9255; 1:2,000), cleaved-caspase-3 (cat. no. 9664; 1:1,000), caspase-3 (cat. no. 14220; 1:1,000; Cell Signaling Technology, Inc.), cleaved-caspase-9 (cat. no. 20750; 1:1,000; Cell Signaling Technology, Inc.), caspase-9 (cat. no. 9508; 1:1,000), β-actin (cat. no. 4778; 1:2,000), cytochrome c (cat. no. ab133504; 1:5,000) and COX–IV (cat. no. ab202554; 1:2,000) overnight at 4°C. The membranes were washed with 1X TBST for 5 min (3 times). Horseradish peroxidase-conjugated goat anti-rabbit (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) and anti-mouse antibody (cat. no. 7076; 1:2,000; Cell Signaling Technology, Inc.) were used as secondary antibodies. The membranes were incubated with secondary antibodies at room temperature for 1 h and washed with 1X TBST for 5 min (3 times). The membranes were visualized using Luminata™ Crescendo Western HRP Substrate (EMD Millipore). Protein expression was quantified using ImageJ Lab software (version k1.7.9; National Institutes of Health).

All data were presented as the as mean ± SD of ≥3 independent experimental repeats. The overall survival curve was generated and assessed via Kaplan-Meier and log-rank tests. Correlation analysis was performed using Pearson correlation analysis. Data containing two groups were analyzed by unpaired Student's t-test and two-tailed distribution. Data containing >2 groups was analyzed by one-way ANOVA followed by Tukey's post-hoc test. Data were analyzed using SPSS 18.0 (SPSS, Inc.) and GraphPad Prism 5.0 software (GraphPad Software, Inc.). *P<0.05 was considered to indicate a statistically significant difference.

To determine the gene expression of LDHA in human tumor and normal samples, TCGA datasets were analyzed via GEPIA. LDHA gene expression levels were upregulated in most types of tumor, including CESC, compared with normal tissue (Fig. 1A). Furthermore, analysis of TCGA data using GEPIA showed a significantly higher LDHA gene expression in CESC compared with normal cervical tissue (Fig. 1B). In addition, tumor samples were separated into low and high groups based on median expression level of LDHA. LDHA overexpression was significantly associated with shorter overall survival in patients with cervical cancer (HR=1.7; Fig. 1C), suggesting that high LDHA expression was a risk factor for poor prognosis in cervical cancer. These results suggesting that LDHA may play a critical role in cerivical cancer progression. Because of the key role of MPC1 in pyruvate metabolism, the association between LDHA and MPC1 was evaluated using GEPIA. Correlation analysis (Fig. 1D) showed that LDHA levels were negatively correlated with MPC1 (R=−0.14), supporting the hypothesis that cervical cancer was aerobic glycolysis-dependent. Taken together, these results suggested that LDHA was upregulated in cervical cancer and dysregulation of LDHA served a key role in cervical cancer.

As LDHA overexpression were negatively correlated with overall survival in patients with cervical cancer, the effect of LDHA inhibition on glycolysis in CESC cells was investigated. To determine the effect of LDHA inhibition on energy metabolism in CESC cells, oxamate, a small-molecule inhibitor of LDHA (36), was used. According to the concentrations of oxamate used in previous reports (18,20,37), we selected 10 and 20 mM oxamate for the energy metabolism study. Meanwhile, four stable LDHA-depleted CESC cell lines (HeLa and SiHa sh1 and sh2) were constructed to investigate the effect of knockdown on protein expression levels (Fig. 2A). The results indicated that sh1 and sh2 efficiently knocked down expression of LDHA compared with the control. Considering that the knockdown effect of sh1 was significantly greater compared with sh2 in both HeLa and SiHa cells, HeLa and SiHa sh1 cells were selected for further study. Intracellular biochemical indicators were detected in both HeLa and SiHa cells. Following treatment with oxamate for 24 h, glucose uptake, lactate production and ATP levels decreased significantly in both HeLa and SiHa cells (Fig. 2B-D). Similarly, knockdown of LDHA in CESC cells decreased glucose uptake, lactate production and ATP levels (Fig. 2E-G). These results demonstrated that LDHA inhibition disrupted energy metabolism and suppressed the Warburg effect in CESC cells.

As increased glycolysis enables cancer cells to suppress apoptotic signaling and promotes viability and proliferation (38), the effect of LDHA on viability of CESC cells was assessed. Firstly, MTT assay was performed to investigate the cytotoxic effect of oxamate on CESC cells. HeLa and SiHa cells were treated with different doses of oxamate for 48 h. Oxamate exhibited significant cytotoxicity toward these two CESC cancer cell lines in a dose-dependent manner (Fig. 3A and B). The IC₅₀ value of oxamate for HeLa cells was 59.05 mM, while the IC₅₀ value for SiHa cells was 70.19 mM (Fig. 3C). Cell proliferation was quantified by measuring BrdU incorporation (39); oxamate significantly inhibited proliferation of HeLa and SiHa cells (Fig. 3D and E). Clone formation assay showed that HeLa and SiHa cells formed significantly fewer clones following treatment with oxamate (Fig. 3F), suggesting that oxamate inhibited clone formation capacity in both HeLa and SiHa cells. Collectively, these results suggested that LDHA inhibition by oxamate in HeLa and SiHa cells inhibited proliferation.

To confirm the role of LDHA on proliferation of CESC cells, proliferation and viability of LDHA-knockdown CESC and control cells were investigated. Consistent with the aforementioned results, MTT assay showed that viability was significantly inhibited in all LDH-knockdown CESC cells (Fig. 4A and B). Furthermore, lower levels of BrdU incorporation were detected in HeLa and SiHa #h1 cells compared with control (Fig. 4C and D). The clone formation assay demonstrated that LDHA knockdown inhibited clone formation capacity of HeLa and SiHa cells (Fig. 4E). In summary, similar to LDHA inhibition by oxamate, sh-induced LDHA knockdown suppressed viability, proliferation and colony forming ability of CESC cells.

As LDHA inhibition suppressed proliferation of CESC cells, cell cycle distribution was investigated. The aforementioned data demonstrated that 20 mM oxamate had a more significant effect than 10 mM oxamate on cell survival, proliferation and metabolism; therefore, 20 mM oxamate was used for cell cycle analysis. Cells were exposed to 20 mM oxamate for 48 h and flow cytometry was used to analyze the cell cycle distribution following PI staining. There was an increase in the number of HeLa and SiHa cells in G₂/M phase following treatment with oxamate (Fig. 5A and B). To verify the G₂/M arrest induced by oxamate and determine the underlying mechanisms, western blot analysis was used to investigate changes in expression levels of proteins associated with G₂/M transition (Fig. 5C and D). Protein levels of Cyclin B1, Cyclin A and CDK1 were significantly decreased in both SiHa and HeLa cells following treatment with oxamate, suggesting that oxamate induced G₂/M arrest by modulating expression of Cyclin B1, Cyclin A and CDK1. Expression levels of proteins in upstream signaling pathways affecting cell cycle were also assessed; expression levels of p21 significantly increased following treatment with oxamate. These findings were consistent with inhibition of cell proliferation. The same results were found in both SiHa and HeLa cells following knockdown of LDHA (Fig. 5E and F). Collectively, these data indicated that LDHA inhibition in CESC cells induced cell cycle arrest, suggesting a tumor-suppressive role of LDHA in CESC cells.

The present study demonstrated that LDH inhibition impaired proliferation and increased the G₂/M fraction in CESC cells. To determine whether LDHA inhibition induces apoptosis, cells were exposed to oxamate for 48 h, then Annexin V/PI staining and flow cytometric analysis were performed. Following 48 h treatment with oxamate, the percentages of apoptotic cells significantly increased (Fig. 6A and B). Next, expression of apoptosis-associated proteins were detected by western blot analysis. Expression of cleaved-caspase-3 and −9 was significantly enhanced following treatment with oxamate for 48 h (Fig. 6C and D). Apoptosis-associated protein expression levels were compared between LDHA-knockdown CESC and control cells (Fig. 6E and F). Western blot results showed that higher expression of cleaved-caspase-3 and −9 was detected following knockdown of LDHA, supporting the results of oxamate treatment experiments. Protein levels of cytochrome c in the cytoplasm and mitochondria were detected; cytochrome c was released from mitochondria into the cytoplasm following LDHA inhibition (Fig. 6G and H). Taken together, these results indicated that LDHA inhibition induced apoptosis via the mitochondrial pathway in CESC cells.

Multiple studies have indicated that mitogen-activated protein kinases (MAPKs), such as p38 and JNK, pathways are associated with cell proliferation, apoptosis and cell cycle progression (40,41). As HeLa cells are the most commonly used cell model for cervical cancer, the present study investigated whether p38 or JNK pathways were involved in LDHA inhibition-mediated cell cycle arrest and apoptosis in HeLa cells. Western blot analysis (Fig. 7A) showed a significant increase in JNK phosphorylation in HeLa cells following treatment with oxamate, while there was no notable change in phosphorylation level of p38. The same results were observed following knockdown of LDHA (Fig. 7B).

To verify whether JNK signaling was required for inhibition of cell proliferation following LDHA knockdown or oxamate treatment, SP600125, a JNK inhibitor (42), was used to suppress JNK signaling in HeLa cells. SP600125 led to a significant decrease in JNK phosphorylation induced by oxamate treatment and LDHA knockdown (Fig. 7C and E). The present study investigated the regulatory effect of SP600125 on cell viability. SP600125 treatment significantly reversed the inhibitory effect on cell viability caused by LDHA knockdown and oxamate treatment (Fig. 7D and F). These findings indicated that the JNK pathway was involved in regulating apoptosis and cell cycle arrest induced by LDHA inhibition in HeLa cells.