Sodium hydrosulfide (NaHS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Freshly made NaHS solution was used as a hydrogen sulfide donor. Antibodies against cyclooxygenase 2 (COX2), phosphorylated ERK1/2 (p-ERK), total ERK1/2 (t-ERK), phosphorylated AKT (p-AKT), and total AKT (t-AKT), were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against GAPDH was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 and fetal bovine serum (FBS), trypsin-EDTA solution and 1% penicillin-streptomycin solution were purchased from Invitrogen (Grand Island, NY, USA). COX2 inhibitor niflumic acid (NA), ERK pathway inhibitor U0126 and AKT pathway inhibitor GSK690693 were purchased from Sigma-Aldrich. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Click-iT^® EdU HCS Assay kit and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA). GoScript™ reverse transcription system was purchased from Promega (Madison, WI, USA). SYBR Green PCR Master Mix was obtained from Roche Diagnostics (Indianapolis, IN, USA). RIPA buffer was a product of Applygen Technologies, Inc. (Beijing, China). Bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Polyvinylidene difluoride membranes were purchased from Millipore Corporation (Billerica, MA, USA).

Human head and neck squamous cell carcinoma cell lines WSU-HN6 and CAL27 were maintained in Dulbecco's minimum essential medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin solution. Human head and neck squamous cell carcinoma cell line Tca83 was maintained in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin solution in a humidified incubator at 37°C in an atmosphere of 5% CO₂.

To determine the optimal dose of hydrogen sulfide for the following experiments, we used NaHS, a commonly used donor of H₂S, to treat the oral cancer cells for 5 h in a T25 flask with 4 ml medium, and then detected the H₂S concentration in the atmosphere of a T25 flask by a halimeter. The optimal concentration of NaHS was based on the range of the H₂S concentration found in patients with halitosis.

Cell proliferation ability was evaluated using the CCK-8 assay which was performed as previously described (18). Briefly, cells were cultured in 96-well tissue culture plates (1×10⁴ cells/well) with 10% FBS overnight. Then, the cells were treated with 0–1,000 µM NaHS (different doses of NaHS were used in different plates) and sealed with parafilm for an additional 5 h. The cell proliferation was measured by the CCK-8 assay according to the manufacturer's instructions.

Cell proliferation ability was confirmed by crystal violet assay. Briefly, 1×10⁵ cells were plated in a T25 flask with 5 ml complete medium overnight. On the following day, the cells were treated with various doses of NaHS for 3 days. The cells were fixed with 95% alcohol, stained with crystal violet, and photographed using a digital camera. Then crystal violet was discolored from the stained cells using 33% glacial acetic acid. The optical density (OD)₅₇₀ of the crystal violet correlates with the number of stained cells.

Cell proliferation ability was also detected using the 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. Briefly, 5×10⁵ cells were plated in a T25 flask with 5 ml complete medium overnight, and then the cells were serum-starved. At 24 h post-starvation, the cells were treated with various doses of NaHS in the presence of EdU for an additional 5 h. The labeled cells were trypsinized for EdU incorporation assay using the Click-iT EdU HCS assay kit (Invitrogen). EdU-positive cells were detected by flow cytometry.

Cells in mid-logarithmic growth were used for real-time PCR. Total RNA was extracted from the tumor cells using TRIzol reagent, and cDNA was obtained by reverse transcription with the GoScript™ reverse transcription system. Relative quantitative real-time PCR reactions were performed with SYBR Green PCR Master Mix in an ABI Prism 7500 sequence detection system (Applied Biosystems Life Technologies, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. The sequences of the COX2 primers were 5′-CTGGCGCTCAGCCATACAG-3′ and 5′-ACACTCATACATACACCTCGGT-3′. The thermal cycling consisted of 10 min at 95°C, followed by 40 cycles at 95°C for 15 sec, and at 60°C for 1 min. All amplifications were performed in triplicate for each sample and repeated three times independently. Relative expression of the target genes was analyzed using the 2^−ΔΔCt method.

Cells were lysed on ice in RIPA buffer supplemented with protease inhibitors (Roche Diagnostics). The concentration of total protein was determined using the BCA protein assay kit. Equal quantities of protein (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% nonfat dry milk for 1 h and probed with primary antibodies against GAPDH, COX2, p-ERK, t-ERK, p-AKT, t-AKT, respectively, at 4°C overnight. After incubation with horseradish peroxidase (HRP)-linked secondary antibodies, the protein bands were visualized using an enhanced chemiluminescent substrate (Applygen Technologies).

To investigate the role of COX2, ERK and AKT pathways in hydrogen sulfide-induced cancer cell proliferation, pathway inhibition assays were carried out using an inhibitor for each molecule/pathway. Based on our preliminary data and previous studies (22–24), we found that COX2 could be markedly downregulated by 100 µM niflumic acid (NA) at 24 h post-treatment; p-ERK and p-AKT could be significantly blocked by 50 µM U0126 and 1 µM GSK690693 at 1 h post-treatment, respectively. Therefore, in the present study, WSU-HN6 cells were pretreated with either COX2 inhibitor NA for 24 h, ERK pathway inhibitor U0126 for 1 h, or AKT pathway inhibitor GSK690693 for 1 h, under serum-starvation condition, and then exposed to NaHS for 5 h for the real-time PCR and western blot assays or with EdU reagent for the EdU incorporation assay.

The results are expressed as mean ± SD. Differences between groups were analyzed by one-way ANOVA. Significance was established at the P<0.05 level.

The NaHS dose finding assay showed that the concentration of H₂S in the atmosphere of 1,000 µM NaHS-treated cells was 698.33±18.93 ppb, which is equal to 1073.34±29.09 µg/l. Based on previous studies, the concentration of H₂S in patients with halitosis is 810.30±204.09 µg/l (25). Therefore, the H₂S concentration released from 250, 500 and 1,000 µM NaHS was comparable with the H₂S concentration found in the oral cavity of patients with halitosis, and 1,000 µM NaHS was in the range of the highest concentration of H₂S in the oral cavity of halitosis patients (Fig. 1A).

To evaluate the effect of H₂S on oral cancer cell proliferation, the CCK-8, crystal violet staining and EdU incorporation assays were performed. The CCK-8 assay showed that H₂S significantly promoted the proliferation of the WSU-HN6 and CAL27 cells in a dose-dependent manner. The WSU-HN6, CAL27 and Tca83 cells treated with 1,000 µM NaHS exhibited a 22.94, 33.79 and 32.20% increase in proliferation, respectively (Fig. 1B, P<0.05). Crystal violet staining assay showed that the cell growth of WSU-HN6 and CAL27 cells was accelerated by NaHS in a dose-dependent manner (Fig. 1C–F). The CCK-8 results were further confirmed in the WSU-HN6 cells using the EdU incorporation assay, which showed a 16.19±1.89% increase in the proliferation of the WSU-HN6 cells following treatment with 1,000 µM NaHS (Fig. 1G, P<0.05).

To analyze the molecular changes during NaHS-induced cancer cell proliferation, we detected the changes in cancer proliferation-related molecule COX2 by real-time PCR (Fig. 2A) and western blotting (Fig. 2B), and pathways including ERK and AKT by western blotting (Fig. 2C). The results showed that compared with the corresponding controls, hydrogen sulfide markedly upregulated COX2 at the mRNA and protein levels in a dose-dependent manner (Fig. 2A and B). Meanwhile, H₂S significantly activated p-ERK and p-AKT expression in a dose-dependent manner (Fig. 2C).

To investigate the role of COX2, the ERK and AKT pathways in H₂S-induced oral cancer cell proliferation, we uses the CCK-8 and EdU cellular incorporation assays to detect the change in proliferation. The proliferation of WSU-HN6 cells caused by H₂S was decreased by inhibition of COX2, ERK and AKT pathways. CCK-8 assay showed that in the WSU-HN6 cells, treatment of NA (COX2 inhibitor) resulted in a nearly 50% decrease in growth caused by H₂S. Meanwhile, U0126 (ERK pathway inhibitor) and GSK690693 (AKT pathway inhibitor) achieved a less significant decrease in proliferation as determined by the CCK-8 assay (Fig. 3A).

The EdU incorporation assay further confirmed the CCK-8 results. Compared with the untreated control, all these inhibitors alone decreased the percentage of EdU-positive cells while NaHS alone increased the percentage of EdU-positive cells. NA alone caused a 11.07±3.71% increase, while U0126 and GSK690693 caused a 3.9±0.98 and 3.22±1.00% decrease in the WSU-HN6 cells. Compared with NaHS treatment alone, NaHS in combination with NA yielded a 31.24±6.39% decrease; NaHS combined with U0126 yielded a 51.50±1.95% decrease; and NaHS plus GSK690693 yielded a 50.35±1.89% decrease in Edu-positive cells. The results above indicated that H₂S-induced upregulation of COX2, p-ERK and p-AKT plays a positive role in the process of H₂S-induced proliferation of oral cancer cells (Fig. 3B).

To further verify the role of COX2 in the promotion of proliferation, we detected the changes in the ERK and AKT pathway folowing the treatment of NA. NA not only decreased COX2 expression caused by H₂S at the mRNA and protein levels (Fig. 4A and B), but also decreased p-ERK and p-AKT expression when the cells were treated with 1,000 µM of NaHS (Fig. 4C).

To investigate the role of the ERK pathway in proliferation, U0126 was used to inhibit p-ERK. Western blotting showed that U0126 completely blocked the activation of the ERK pathway by eliminating p-ERK expression (Fig. 5C). Real-time PCR and western blotting showed that inhibition of the ERK pathway by U0126 did not change COX2 expression at the mRNA and protein levels (Fig. 5A and B). Furthermore, U0126 increased the activation of the H₂S-induced p-AKT pathway (Fig. 5C).

To verify the role of the AKT pathway in H₂S-induced cell proliferation, GSK690693 was used for inhibition of p-AKT. Western blotting showed that GSK690693 decreased the expression of p-AKT (Fig. 6C). Real-time PCR results showed that GSK690693 did not change H₂S-induced COX2 expression at the mRNA and protein levels (Fig. 6A and B), although GSK690693 alone induced an increase in COX2 at the mRNA level. In addition, inhibition of the AKT pathway decreased the expression level of p-ERK (Fig. 6C).