Antibodies against PKD1 rabbit mAb (cat. no. 90039), phospho-PKD1 rabbit Ab (cat. no. 2051), p38 MAPK rabbit mAb (cat. no. 8690), phospho-p38 MAPK (Thr180/Tyr182) rabbit mAb (cat. no. 4511), LDHA rabbit mAb (cat. no. 3582) and β-actin rabbit mAb (cat. no. 8457) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against HIF-1α rabbit mAb (cat. no. ab51608), GLUT-1 rabbit pAb (cat. no. ab51608) and LC3 rabbit pAb (cat. no. ab51520) were obtained from Abcam (Cambridge, MA, USA). Annexin V-FITC apoptosis detection and lactate assay kits were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). We purchased 2-NBDG [2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose] from Cayman Chemicals (Ann Arbor, MI, USA). Human PKD1 shRNA and control shRNA plasmids were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Human pEZ-Lv105-PKD1-overexpressing plasmid vectors were purchased from GeneCopoeia, Inc. (Rockville, MD, USA). The Cell Counting Kit (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan).

Human oral squamous cell carcinoma (SCC25) cells were cultured in DME/F-12 (1:1) medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1% (vol/vol) penicillin/streptomycin mixture and 10μM hydrocortisone (both from Gibco, Thermo Fisher Scientific, Inc.). H1975, SCC25 and HSC-4 cells used in this study were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured at the State Key Laboratory of Oral Diseases in a humidified atmosphere of 5% CO₂ at 37°C. To induce hypoxia, SCC25 cells were placed in a chamber filled with a gas mixture of 1% O₂, 5% CO₂ and 94% N₂.

Human LVRH1GP-PKD1 shRNA (clone ID NM_002742.2) and LVRU6GP-control shRNA plasmids were obtained from Thermo Fisher Scientific, Inc. The PKD1-1-SH target sequences are as follows: gcaacaatat cccactcatga. PKD1-2-SH target sequences are as follows: gcaggtactacaaggaaattc. The shRNAs were transfected into the SCC25 cells using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Positive SCC25 clones were selected for at least two passages by using puromycin treatment at a concentration of 0.5 µg/ml. After two weeks of growth in selective medium, the cells were harvested and the expression level of PKD1 was determined by western blot analysis. Stable PKD1-overexpressing SCC25 cells were generated similarly.

The CCK-8 was used to detect the cell viability of the SCC25 cell group (as the normal control group), the PKD1-knockdown group (PKD1-SH), PKD1-overexpressing group (PKD1-OE) and SCC25 control groups (Con-SH and Con-OE). SCC25 cells were seeded at a density of 1×10³ cells/well in 96-well plates and cultured at 37°C in 1% O₂, 5% CO₂ and 94% N₂. Cell viability was assessed by measuring the absorbance of the converted dye at 450 nm.

SCC25 cells (wild-type SCC25, PKD1-SH, Con-SH, PKD1-OE and Con-OE) were seeded in 6-well plates at a density of 2×10⁵ cells/well and cultured at 37°C in 1% O₂, 5% CO₂ and 94% N₂. Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for flow cytometry was used to identify the percentage of apoptotic cells following the manufacturer's protocol [Annexin V-FITC apoptosis detection kit (Sigma-Aldrich; Merck KGaA)]. Annexin V⁺/PI⁻ apoptotic cells were identified via flow cytometry (FCM) using Summit 5.2 software (Beckman Coulter, Miami, FL, USA).

To analyze glucose uptake, the fluorescent glucose analog 2-NBDG was used for direct quantification of glucose incorporation in living cells by FCM (Beckman Coulter) (7,30). SCC25 cells were seeded in 6-well plates and subjected to hypoxia (1% O₂, 5% CO₂ and 94% N₂). After treatment for 24 h, the medium was removed and new medium containing 10 µM 2-NBDG was added. The cells were then incubated at 37°C in 1% O₂, 5% CO₂ and 94% N₂ for 30 min. Cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS; Invitrogen; Thermo Fisher Scientific, Inc.) then trypsinized, centrifuged 1,200 rpm for 10 min and resuspended in DPBS. A flow cytometer was used to analyze the uptake of 2-NBDG in the different cell groups.

SCC25 cells were seeded in 6-well plates at a density of 2×10⁵ cells/well and cultured at 37°C in 1% O₂, 5% CO₂ and 94% N₂ for 24 h. Lactate in the medium was measured using a lactate assay kit according to the manufacturer's instructions (Sigma-Aldrich; Merck KGaA).

Cells were collected and protein was collected using a Protein Extraction kit (Sigma-Aldrich; Merck KGaA). The protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Total protein (20 µg) was separated using 6 or 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies against PKD1, phospho-PKD1, HIF-1α, LC3, GLUT-1, LDHA, phospho-p38 MAPK, p38 MAPK or β-actin at a dilution of 1:1,000 at 4°C overnight. Membranes were washed with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at a dilution of 1:2,000 for 2 h at room temperature. Luminescent signals were detected by ECL Western Blotting Substrate (Millipore, Billerica, MA, USA). Band intensity was quantified using Quantity One Software (Bio-Rad Laboratories). Phospho-p38 MAPK/p38 MAPK was defined as the phosphorylated index (PI) of p38 MAPK.

All data are presented as mean ± standard deviation (SD). Significant differences between groups were analyzed by one-way analysis of variance (ANOVA), followed by a Bonferroni's post hoc test. A P-value <0.05 was considered to indicate a statistically significant difference. Graphs were plotted and analyses were performed using GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA).

The SCC25 cell line is the most common type of human oral squamous cell carcinoma. In our pre-experiment, we investigated the expression of PKD1 in H1975, SCC25 and HSC-4 cancer cell lines. The results showed that PKD1 was expressed in SCC25 cells at a high level (Fig. 1A and B); therefore, we selected SCC25 as the target cell line for further study.

Hypoxia is a common characteristic of the tumor microenvironment. Hypoxia induces cancer cells to express HIF-1α to initiate the expression of tumor growth factor and provide a growth signal for cancer cells. On the other hand, hypoxia promotes cancer cell switch to glycolysis for energy generation. To determine the role of PKD1 in regulating glycolytic metabolic progression of tumor cells, we first analyzed the expression and activation of PKD1 in tumor cells under a hypoxic microenvironment. SCC25 cells were cultured in a humidified atmosphere of 5% CO₂, 1% O₂ and 94% N₂ at 37°C to simulate a hypoxic tumor microenvironment. The expression of PKD1, p-PKD1 and HIF-1α were detected by western blot analysis. As shown in Fig. 1C, SCC25 cells exposed to 6 h of hypoxia exhibited enhanced expression of p-PKD1, PKD1 and HIF-1α. In particular, when SCC25 cells were subjected to 24 h of hypoxia, the expression of p-PKD1, PKD1 and HIF-1α reached the highest level (Fig. 1D). Moreover, the ratio of p-PKD1/PKD1 was significantly increased (Fig. 1D, left lower panel). Based on the above-mentioned research results, treatment with 24 h hypoxia was selected for the subsequent experiments. This result indicates that hypoxia not only induces the expression of PKD1, but also induces the phosphorylation and activation of PKD1.

Toleration of a hypoxic microenvironment is a characteristic of tumor cells. To investigate the role of PKD1 in the growth and apoptosis of tumor cells grown under hypoxia, SCC25 cells were transfected with PKD1 shRNA (PKD1-1-SH and PKD1-2-SH) to knockdown PKD1. Compared with the negative control (Con-SH), the expression of PKD1 was significantly reduced, as shown by western blot analysis (Fig. 2A and B). As shown in Fig. 2A and B, PKD1-2-SH was found to be the most effective shRNA plasmid with which to knockdown PKD1. Thus, we used PKD1-2-SH to knockdown PKD1 in the following experiments. Additionally, SCC25 cells were transfected with a pCDNA3.1/NT-PKD1 plasmid with a pre-inserted PKD1 gene for PKD1 overexpression (PKD1-OE) (Fig. 2C and D). PKD1-SH, PKD1-OE and wild-type SCC25 cells were cultured in a humidified atmosphere of 1% O₂, 5% CO₂ and 94% N₂ at 37°C. Live cells were analyzed using the CCK-8 cell counting kit; apoptotic cells were analyzed via FCM. As shown in Fig. 2E, the cell growth curve demonstrated a significantly highest cell death at 3 days in the PKD1-SH group when compared with the other 4 tested groups. Importantly, SCC25 cell apoptosis was significantly increased after PKD1 knockdown with PKD1-SH (Fig. 2F and G). However, the results revealed that there was no significant difference in growth between the PKD1-Con and PKD1-OE group, and growth of the PKD1-overexpressing cells was not markedly affected according to the cell growth curve (Fig. 2E). These results indicate that PKD1 plays an important role in maintaining the survival and growth of SCC25 cells in a hypoxic microenvironment.

PKD1 is a protease kinase that is involved in the bio-behavior of cancer cells. HIF-1α is an important regulator of the glucose metabolism in cancer cells. To reveal the role of PKD1 in the progression of hypoxia-induced HIF-1α expression, wild-type SCC25, PKD1-SH and PKD1-OESCC25 cells were cultured in a humidified atmosphere of 5% CO₂, 1% O₂ and 94% N₂ at 37°C for 24 h. SCC25 cells cultured in normal condition as a control, PKD1-knockdown or -overexpressing SCC25 cells did not exhibit a significant difference in the expression of HIF-1α under a normal condition (data not shown). PKD1 and HIF-1α expression was evaluated by western blot analysis. As shown in Fig. 3, the phosphorylation and activation of PKD1 and HIF-1α expression was significantly increased in the SCC25 cells under hypoxia. Moreover, the expression of HIF-1α was significantly inhibited after PKD1 knockdown by PKD1-SH (Fig. 3A and B). On the other hand, overexpression of PKD1 and phosphorylated activation significantly promoted hypoxia-induced HIF-1α expression (Fig. 3C and D). These results indicate that PKD1 is an important regulator of hypoxia-induced HIF-1α expression.

The Warburg effect is a unique characteristic of cancer cell glycometabolism. Cancer cells uptake glucose and release lactate through glycolysis under hypoxic conditions. Detection of the ability of cancer cells to uptake glucose and release lactate in culture media can reveal the glycolytic activity of cancer cells. Wild-type SCC25, PKD1-SH and PKD1-OESCC25 cells were cultured in medium with 10 nM 2-NBDG for 30 min. Glucose uptake and lactate release by cancer cells were analyzed via FCM and lactate assay kits, respectively. As shown in Fig. 4A and B, hypoxia stimulated SCC25 cells to uptake glucose and release lactate. When PKD1 was knocked down with PKD1-SH, the ability of SCC25 cells to uptake glucose was significantly reduced (Fig. 4A, left panel), as lactate release was also reduced (Fig. 4B, left panel). Conversely, when PKD1 was overexpressed, the ability of SCC25 cells to uptake glucose and release lactate was significantly increased (Fig. 4A and B, right panels). These results indicate that PKD1 is an important signaling molecule promoting cancer cell glycolysis.

We also detected the growth and the expression of autophagy proteins by SCC25 cells cultured under an acidic environment after knockdown of PKD1. As shown in Fig. 4C, the growth of SCC25 cells was significantly inhibited under an acidic condition. Fig. 4D shows that the expression of autophagy proteins was significantly decreased (LC3-II/LC-3I). These data indicated that PKD1 moderated autophagy to promote the adaptation of tumor cells to an acidic microenvironment.

GLUT1 and LDHA are two key enzymes involved in the process of glycolysis. Hypoxia induced both GLUT1 and LDHA expression in SCC25 cancer cells (Fig. 5). When PKD1 was knocked down with PKD1-SH, the expression of GLUT1 and LDHA was significantly reduced as compared to wild-type SCC25 or control-SHSCC25 cells under hypoxia (Fig. 5A and B). Conversely, PKD1-overexpressing SCC25 cells had significantly increased expression of GLUT1 and LDHA under hypoxic conditions (Fig. 5C and D). These results indicate that PKD1 promotes glycolysis in oral squamous cancer cells by inducing the expression of glycolytic enzymes under hypoxic microenvironments.

The p38 MAPKs are members of the MAPK family that are activated by a variety of environmental stresses and inflammatory cytokines. As shown in Fig. 6, hypoxia is an important environmental stress that activates p38 MAPK. SCC25 cells (wild-type SCC25, PKD1-SH and PKD1-OE) were cultured in a hypoxic environment for 24 h, and the expression of p-p38 MAPK and p38 MAPK was analyzed via western blot analysis. The phosphorylation level of p38 MAPK was increased under hypoxia (Fig. 6A and C), whereas the expression level of total p38 MAPK was not statistically significantly different. The phosphorylation level of p38 MAPK and phosphorylated index (PI) of p-p38 MAPK/p38 MAPK were significantly decreased when SCC25 cells were transfected with PKD1-SH in a hypoxic environment for 24 h (Fig. 6A and B). Moreover, the level of p-p38 MAPK and the PI of p-p38 MAPK/p38 MAPK were significantly increased when the PKD1-OE SCC25 cells were treated with hypoxia (Fig. 6C and D), compared with the expression level of total p38 MAPK.