OECM-1 and SAS human oral squamous cell carcinoma (OSCC) cell lines, were purchased from ATCC (Rockefeller, MD, USA). Briefly, OECM-1 and SAS cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Paisley, UK) containing 10% fetal bovine serum (FBS; HyClone, VT, USA), at 37°C in a humid atmosphere with 5% CO₂. Hypoxic conditions were induced by incubation of the cells in a sealed Bug-Box anaerobic workstation (Ruskinn Technology, Ltd., Bridgend, UK). For the hypoxia experiments, the oxygen levels were maintained at 2%, with the residual gas mixture being 94.0 to 94.9% N₂ and 5% CO_2, as compared with the normoxia experiments, in which the oxygen levels were maintained at 21%.

A rabbit monoclonal antibody against PDK1 was used at a 1:1,000 dilution(#3820; Cell Signaling Technology, Danvers, MD, USA); a β-Actin antibody was used as a loading control, at a 1:2,000 dilution (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA, USA). DCA and Taxol were purchased from Sigma-Aldrich (St. Louis, MO, USA).

RNA was extracted from oral cancer cells using TRIzol™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The cDNA synthesis was performed using a SuperScript First-Standard Synthesis System for RT-PCR (Invitrogen Life Technologies), according to the manufacturer’s instructions. qPCR analyses were performed using Assay-on-Demand primers and the TaqMan Universal PCR Master Mix reagent (Applied Biosystems, Foster City, NJ, USA). The samples were analyzed using an ABI Prism^® 7700 Sequence Detection System (Applied Biosystems). The primers for the qPCR were: PDK1 forward, 5′-GCTTATCAGAAACTCCAAAGACTGC-3′ and reverse, 5′-GCCGGAAAGGTGGGACA-3′. The expression levels of β-actin were used to normalize the relative expression levels. All experiments were performed in triplicate.

Cells were harvested and lysed in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton, 1 mM phenylmethanesulfonyl fluoride and Protease Inhibitor Cocktail (Sigma-Aldrich) for 20 min on ice. Lysates were centrifuged at 16,000 × 6 at 4°C for 10 min. The supernatants were collected and the protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA). The proteins were then separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). Following blocking of the membranes with phosphate-buffered saline (PBS) with 5% non-fat dry milk for 1 h, the membranes were incubated with the primary antibodies, in PBS with 5% non-fat dry milk, overnight at 4–8°C. The membranes were extensively washed with PBS and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody or anti-rabbit antibody (1:2,000, Bio-Rad). Following additional washes with PBS, the antigen-antibody complexes were visualized using an Enhanced Chemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA).

Oral cancer cells were seeded in a 6-well plate, at a density of 10,000 cells/well in 2 ml DMEM containing 10% FBS. Following overnight incubation under the same culturing conditions, each well was refreshed with 2 ml serum-free medium (SFM) for another day. Following this, the cells were then treated with 2 ml SFM containing various concentrations of Taxol or DCA. The drug-containing SFM was refreshed after 2 days, and incubated under the same conditions for a further 2 days. The cell viability was assessed using MTT reagent (Sigma-Aldrich), and by measuring the absorbance at 570 nm using a plate reader. The relative viability was obtained from the absorbance at 570 nm of both the drug-treated and untreated OECM-1 cells. The experiment was repeated three times.

OECM-1 Taxol-resistant cells were obtained by gradually increasing the concentration of Taxol in the cell culture medium, followed by selection of Taxol-resistant cells. After successive Taxol treatments for a duration of 3 months, several resistant cell clones were generated.

The data was analyzed using the unpaired Student’s t-test. All the data are expressed as the means ± standard error. A P<0.05 was considered to indicate a statistically significant difference.

Since the switch from mitochondrial oxidation to anaerobic glycolysis renders cancer cells resistant to chemotherapy (23), OECM-1 and SAS human oral cancer cell lines were cultured under hypoxic conditions for 24 h followed by measurements of Taxol sensitivity. Under hypoxic conditions, both cell lines acquired resistance to Taxol at multiple concentrations, as compared with cells cultured in normoxic conditions (Fig. 1A and B). It has previously been reported that low oxygen stabilizes hypoxia inducible factor-1 α (HIF-1α) which promotes the transcriptional expression of enzymes that are required for glycolysis (24). The results of the present study indicated that HIF-1α-mediated upregulation of glycolysis may contribute to Taxol resistance in oral cancer cells.

The protein and mRNA expression levels of pyruvate dehydrogenase kinase 1 were measured. Both PDK1 mRNA and protein expression was upregulated by hypoxia (Fig. 2A), suggesting that PDK1 may be involved in Taxol sensitivity in oral cancer cells.

It has been reported that treatment with Taxol can upregulate and increase the activity of lactate dehydrogenase A (LDHA) expression, which contributes towards Taxol resistance in breast cancer cells (25). Since both LDHA and PDK1 are key enzymes of glycolysis, it was hypothesized that PDK1 may participate in Taxol resistance. PDK1 protein and mRNA expression was measured in oral cancer cells in response to Taxol treatments. PDK1 was significantly upregulated by Taxol treatment at multiple concentrations (Fig. 2B), indicating that PDK1 may be induced by Taxol treatment and hypoxia. These results may provide a potential mechanism for Taxol resistance.

OECM-1 Taxol-resistant cells were generated by gradually increasing concentrations of Taxol in the cell culture medium, followed by selection of Taxol-resistant cells. Several resistant cell clones were developed after successive Taxol treatments for 3 months. OECM-1 Taxol-resistant cells had ~70% viability under 5 and 10 μM Taxol treatments, which was much higher as compared with Taxol-sensitive cells (Fig. 3A). The mRNA and protein expression of PDK1 was measured in both Taxol-sensitive and Taxol-resistant cells. Fig. 3B and C showed that both the PDK1 mRNA and protein expression levels were increased in Taxol-resistant cells, as compared with Taxol-sensitive cells. These results suggest that PDK1 is important in cancer cell Taxol resistance.

DCA, an inhibitor of PDK, has been demonstrated to inhibit glycolysis and promote cancer cell apoptosis. OECM-1 cells were treated with 5 and 10 mM concentrations of DCA for 24 h, after which, the PDK1 protein and mRNA expression levels were measured. Figure 4A shows protein expression levels of PDK1 were significantly suppressed by DCA at 5 and 10 mM, as compared with the controls. To further explore the possible biological significance of DCA in Taxol resistance, oral cancer cells were treated with Taxol alone, DCA alone, or a combination of Taxol and DCA under hypoxia. The combination of Taxol and DCA demonstrated a synergistic inhibitory effect on both Taxol-sensitive and resistant cells under hypoxia (Fig. 4B). However, a combination of Taxol and DCA did not exhibit a synergistically inhibitory effect in Taxol-sensitive cells under normoxia. (Fig. 4B). A possible explanation for this may be because the PDK1 expression in Taxol-sensitive cells was not altered when the cells were conditioned under the normal oxygen conditions, resulting in a non-synergistic effect.