Introduction
Oral squamous cell carcinoma (OSCC) is the most common head and neck neoplasm affecting ~274,000 individuals worldwide (1). The 5-year survival rate of patients with OSCC is only 53% (1–3). The development of OSCC has been shown to be associated with oral habits including betel quid chewing, as well as tobacco and/or alcohol consumption, which lead to continuous generation of reactive oxygen species (ROS) (4–6). Excessive ROS generation due to imbalances in the redox status mediates DNA damage and/or lipid peroxidation, which are thought to subsequently promote the development of OSCC (7). A recent study showed that oxidative stress markers, 8-hydroxy-2′-deoxyguanosin and malondialdehyde, were higher in the saliva of patients with OSCC than levels in the saliva of healthy normal subjects, while the antioxidant vitamins C and E were lower (8). Thus, accumulating evidence has implicated a role for ROS generation in the pathogenesis of OSCC.
Recently, the NADPH oxidase (Nox/Duox) family, a family of enzymes that generate ROS, has been shown to play an important role in cancer development and progression (9,10). To date, the roles of Nox1, Nox2, Nox4 and Nox5 in cancer have been implicated, while those of Duox1, Duox2 and Nox3 in carcinogenesis are not well-reported (9). Upregulated Nox1 expression and subsequent ROS generation was found to promote cancer cell growth, as well as escape from cancer cell death through the redox-dependent activation of p38MAPK and AKT signaling pathways (11). Given the experimental evidence that the Nox family plays a pivotal role in cancer cell development, it would be interesting to examine the involvement of Nox/Duox family members in the pathogenesis of OSCC.
In the present study, we found that Nox1 and Nox4 mRNAs were highly expressed in a significant subset of OSCC cell lines. Knockdown of Nox1 significantly induced apoptosis, and enhanced the cisplatin-induced cytotoxic effect in OSCC cells. Additionally, we report the underlying molecular mechanism responsible for this signaling.
Materials and methods
Reagents
Dulbecco's modified Eagle's medium (DMEM) and penicillin-streptomycin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fetal bovine serum (FBS) was obtained from Nichirei Biosciences, Inc. (Tokyo, Japan). Diphenyleneiodonium (DPI), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), MTT and ammonium pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). AKT inhibitor, perifosine, was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). Propidium iodide (PI) was obtained from Merck Millipore (Billerica, MA, USA). Annexin V-FITC was obtained from MBL (Nagoya, Japan). Rabbit anti-NOX1 and anti-NOX4 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-AKT, anti-phosphorylated-AKT (Ser473), anti-β-actin and HRP-conjugated anti-rabbit IgG were purchased from Cell Signaling Technologies Inc. (Beverly, MA, USA).
Cell culture
Five OSCC cell lines (HSC-2, HSC-3, HSC-4, SAS and OSC-19) were obtained from the Japanese Collection of Research Bioresources Cell Bank. HSC-2, HSC-3 and HSC-4 cell lines were established from individual patients with OSCC (12). The cell lines were maintained in DMEM supplemented with 10% heat-inactivated FBS and penicillin-streptomycin at 37°C in a 5% CO2 humidified atmosphere. The cells were detached from 90-mm dishes using trypsin and seeded in either 96- or 6-well plates for experimental purposes.
Cell viability MTT assay
The OSCC cells were seeded in 96-well plates (5×103 cells/well) and incubated for 24 h at 37°C. Then, the cells were incubated with medium containing the indicated concentrations of DPI, PDTC and NAC. After 72 h of incubation, MTT solution was added into each well. Following 4 h of incubation, lysis buffer (10% SDS in 0.01 M of hydrogen chloride) was added and incubated overnight. Finally, the absorbance at 550 nm was measured using a SpectraMax M5 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
Annexin V assay
Apoptosis was evaluated using Annexin V (AxV)-FITC/PI double staining-based FACS analysis as previously described (13). Briefly, OSCC cells were seeded in 6-well plates (1×105 cells/well) and incubated for 24 h at 37°C. Then, the cells were incubated with the indicated concentrations of DPI followed by incubation in AxV-FITC and PI (10 µg/ml) at room temperature for 15 min. Finally, fluorescence intensities were determined by FACS using a FACSCanto II (BD, Franklin Lakes, NJ, USA).
ROS assay
ROS generation was detected using DCFH-DA as previously described (14). Briefly, HSC-2 and HSC-3 cells were incubated with DPI (5 or 10 µmol/l). The cells were further incubated with the DCFH-DA fluorescent probe in the presence of DPI for 0.5 h. After 48 h incubation, the cells were examined using FACSCanto II, which analyzed 10,000 events (determined by forward and side scatter). Data are presented as mean fluorescence intensity of triplicate determinations ± SE.
RT-PCR analysis
The OSCC cells (1×105 cells/well) were seeded in 6-well plates. Following incubation for 48 h, total RNA was extracted from the cells using NucleoSpin® RNA (Takara Bio, Inc., Shiga, Japan), and 2 µg total RNA was reverse-transcribed with High-Capacity cDNA Reverse Transcription kit (Life Technologies, Inc., Tokyo, Japan). mRNA expression levels of seven Nox/Duox family members (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2) were examined using Veriti® Thermal Cycler (Applied Biosystems, Carlsbad, CA, USA) with specific primer sets as listed in Table I. mRNA expression of GAPDH was used as an internal control.
Quantitative RT-PCR (qRT-PCR) analysis
HSC-2 and HSC-3 cells (1×105 cells/well) were seeded in 6-well plates and incubated for 24 h. The following day, real-time qRT-PCR analysis was performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). qRT-PCR analysis using TaqMan probes was performed according to the manufacturer's instructions. Gene specific TaqMan probes used in this study are listed in Table 1.
Western Blot analysis
HSC-2 and HSC-3 cells (2×105 cells/well) were incubated with DPI as described above. The cells were washed with ice-cold PBS and lysed in loading buffer [125 mmol/l Tris (pH 6.8), 4% SDS, 10% β-mercaptoethanol, 20% glycerol and 0.02% bromophenol blue]. Western blot analysis was performed as previously described (15). The relative protein levels were calculated after normalization to an internal control β-actin.
RNA interference
HSC-2 and HSC-3 cells (1×105 cells/well) were plated in 6-well plates. On the following day, the cells were transfected using Lipofectamine RNAi/MAX (Life Technologies Inc.) to deliver the indicated concentrations of Nox1 or Nox4 siRNA (Santa Cruz Biotechnology, Inc.) according to the manufacturer's protocol. siGENOME RISC-Free siRNA (Dharmacon-Thermo Fisher Scientific, Tokyo, Japan) was used as a negative control.
Statistical analysis
At least three independent experiments and three replications per experiment were performed. The results are expressed as the mean ± SE. Statistical significance between groups was determined using Student's t-tests. Statistical analyses were performed using SPSS 23.0 (SPSS, Inc., Chicago, IL, USA). Statistical significance was defined as P<0.05.
Results
ROS scavengers inhibit cellular growth in a panel of OSCC cell lines
To investigate the involvement of ROS in OSCC cell growth, we first examined whether a Nox inhibitor, DPI, as well as ROS scavengers, including PDTC and NAC, affect the cell survival in five OSCC cell lines, HSC-2, HSC-3, HSC-4, SAS and OSC-19. The MTT assay revealed that the cell viability was dose-dependently suppressed by treatment with DPI (Fig. 1A), PDTC (Fig. 1B) or NAC (Fig. 1C). The IC50 values ranged from 0.07 µM (SAS cells) to 1.59 µM (OSC-19 cells) for DPI, 40 µM (HSC-4 cells) to >100 µM (SAS cells) for PDTC and 2.53 mM (HSC-2 cells) to 14.0 mM (HSC-4 cells) for NAC among the OSCC cell lines tested (Table II). These results suggest that scavenging of ROS may suppress cell growth in OSCC cells.
Expression levels of the Nox/Duox family in OSCC cell lines
Since DPI reduced the cell viability in OSCC cells, we assumed that the Nox/Duox family plays an important role in cell survival. To clarify this issue, we first examined Nox/Duox mRNAs in five OSCC cell lines using RT-PCR analysis. As shown in Fig. 2A, while Nox1 and Nox4 mRNA expression was readily detected in four cell lines, except for OSC-19, Nox2 and Nox3 mRNA expression was primarily detected only in HSC-4 cells. Nox5 mRNA expression was detected in both SAS and OSC-19 cells (Fig. 2A). Additionally, Duox1 mRNA expression was detectable in all of the cell lines examined, while that of Duox2 was readily detected in the HSC-4 cells but diminished in the OSC-19, HSC-2 and HSC-3 cells (Fig. 2A). These results are summarized in Table III. We also examined protein expression of Nox1 and Nox4; Nox1 protein expression was elevated in the HSC-2 and HSC-3 cells, while Nox4 was slightly expressed in the five OSCC cell lines examined (Fig. 2B).
Nox inhibitor DPI suppresses ROS generation and induces apoptosis
To investigate the involvement of the Nox/Duox family in ROS generation, we further examined the effect of DPI on ROS generation in HSC-2 and HSC-3 cells. As expected, RO generation was significantly suppressed by DPI treatment (Fig. 3A and B). This result prompted us to further examine the effect of DPI on the induction of apoptosis in HSC-2 and HSC-3 cells. As shown in Fig. 3C, the percentages of AxV+/PI+ cells was significantly increased 48 h after DPI treatment in both the HSC-2 and HSC-3 cell lines, suggesting that the Nox family may play a role in the anti-apoptotic effect as well as ROS generation.
Knockdown of Nox1, but not Nox4, suppresses cell survival
Since Nox1 and Nox4 mRNA expression was readily detectable in the HSC-2 and HSC-3 cells, we next examined the effect of Nox1 or Nox4 knockdown on cell viability using MTT assay. We verified that transfection of Nox1 or Nox4 siRNAs significantly reduced their endogenous mRNA levels (65–70% in Nox1 or 50–55% in Nox4), respectively, in the cells compared to those transfected with control siRNA (Fig. 4A and B). Knockdown of Nox1, but not Nox4, significantly suppressed the cell viability in both the HSC-2 and HSC-3 cell lines (Fig. 4C). In addition, the percentages of AxV+/PI+ cells were significantly increased after the transfection of Nox1 siRNAs (Fig. 4D), strongly suggesting that Nox1 contributes to anti-apoptosis in OSCC cells. Unexpectedly, under Nox1 knockdown, no significant reduction in intracellular ROS was detected (Fig. 4E), suggesting that Nox1 may contribute to cell survival through a mechanism other than ROS generation.
Nox1 knockdown, as well as perifosine treatment, prevents phosphorylation of AKT
AKT (protein kinase B) is a regulator of cell survival in response to growth factors. AKT is activated through its phosphorylation, and this kinase inhibits apoptosis-inducing proteins, thereby promoting cell survival. Thus, we investigated phosphorylated AKT levels in OSCC cell lines using western blot analysis. As shown in Fig. 5A, phosphorylated forms of AKT were readily detected in the HSC-2, HSC-3 and HSC-4 cells (Fig. 5A). We therefore examined the effect of a specific AKT inhibitor perifosine on cell survival using MTT assay. As shown in Fig. 5B, perifosine treatment dose-dependently reduced cell viability in the OSCC cell lines (Fig. 5B), suggesting that AKT plays a pivotal role in OSCC cell survival. Since the Nox family has been shown to enhance phosphorylation of AKT (9), we then examined the effect of Nox1 knockdown on AKT phosphorylation using western blot analysis. As shown in Fig. 5C, Nox1 knockdown significantly reduced the phosphorylation level of AKT. These results suggest that Nox1 contributes to cell survival through activation of the AKT signaling pathway.
Nox1 knockdown and cisplatin treatment act cooperatively to suppress cell survival and induce apoptosis
Cisplatin is one of the most frequently used anticancer drugs for OSCC treatment. Therefore, we sought to examine whether inhibition of AKT activity or Nox1 knockdown influences the cytotoxic effect of cisplatin. As shown in Fig. 6A, the perifosine and cisplatin combination treatment significantly suppressed cell viability in both te HSC-2 and HSC-3 cell lines, compared to the cisplatin monotherapy. Similarly, combined perifosinecisplatin treatment decreased cell viability in the SAS and OSC-19 cells (data not shown). Notably, the Nox1 knockdown and cisplatin combination treatment significantly suppressed cell viability, compared to the cisplatin monotherapy (Fig. 6B). This novel finding prompted us to examine the effect of Nox1 knockdown and cisplatin combination treatment on induction of apoptosis. As expected, the treatment significantly induced apoptosis (Fig. 6C), indicating that Nox1 knockdown enhances the cytotoxic effect of cisplatin in OSCC cells.
Discussion
Nox/Duox family members are regarded as major sources of ROS generation, which plays an important role in cancer development (9,16). It has been reported that inhibition of Nox activity induces apoptosis in a number of cancerous cells including pancreatic cancer, mesothelioma and sarcoma (17–19). In the present study, we demonstrated that knockdown of Nox1 but not Nox4 significantly reduced the cell viability in a subset of OSCC cell lines. We also found that Nox1 knockdown decreased phosphorylated AKT levels accompanied by induction of apoptosis. Our observation that the specific AKT inhibitor perifosine reduced the cell viability suggested that Nox1-mediated AKT phosphorylation contributes to cell survival in OSCC cells.
The Nox/Duox family consists of seven members. Our RT-PCR analysis revealed that Nox1 and Nox4 mRNAs were highly expressed in a significant subset of OSCC cell lines. Knockdown of Nox1, but not Nox4, significantly suppressed the cell viability by 50% in OSCC cells. Additionally, Nox1 knockdown significantly induced the apoptosis of OSCC cells. Notably, Nox1 knockdown markedly enhanced cisplatin-induced apoptosis. This novel finding raises the possibility that Nox1 is a potential therapeutic target for a significant subset of oral cancer cells. We unexpectedly observed that Nox1 knockdown had almost no suppressive effect on ROS generation. It may be possible that Nox1 exerts anti-apoptotic effects through a mechanism other than ROS generation. Puca et al reported that Nox1 inhibits acetylation of p53 (K382), which is a target of SIRT1 deacetylase, and impaired p53 pro-apoptotic transcriptional activity (20). Thus, it may be interesting to investigate the role of p53 in Nox1-mediated anti-apoptotic signaling in OSCC cells.
It was previously reported that Nox4-mediated induction of pro-inflammatory cytokines after epidermal growth factor receptor inhibition (21) and erlotinib-induced cytotoxicity is associated with hydrogen peroxide production thorough Nox4 signaling in head and neck squamous cell carcinoma (HNSCC) cells (22). We recently reported that Nox4 knockdown induced apoptosis in mesothelioma (19). However, in the present study, Nox4 knockdown did not induce apoptosis in OSCC cells. Thus, it would also be interesting to pursue the mechanism underlying the discrepancy in the effect of Nox4 knockdown between OSCC and mesothelioma cells.
AKT, a regulator of cell survival, is activated through its phosphorylation and inhibits apoptosis-inducing proteins, thereby preventing cell death and prolonging cell survival. Recent studies have also implicated the use of a selective inhibitor for phosphatidylinositol 3-kinases (PI3K)/AKT as treatment for patients with non-small cell lung cancer, hematologic malignancies and HNSCC (23–25). We observed that Nox1 knockdown attenuated the phosphorylation level of AKT, raising the possibility that inhibition of Nox1 activity may suppress cell survival by inhibiting the AKT signaling pathway. Kozaki et al reported that a missense mutation of PIK3CA, which encodes the 110-kDa catalytic subunit of PI3K, was detected in OSCC cell lines, including HSC-2 and HSC-3, as well as OSCC patients tumors (26). In the present study, Nox1 knockdown significantly attenuated the phosphorylation of AKT and reduced cell viability in the OSCC cells. This suggested that inhibition of Nox1 potentially suppresses activation of AKT, thereby promoting apoptosis in OSCC cells harboring a mutation of the PIK3CA gene.
In conclusion, we demonstrated that Nox1 likely contributes to cell survival through its anti-apoptotic effects in a significant subset of OSCC cells. Based on our experimental data, we hypothesized that Nox1 contributes to cell survival through the AKT signaling pathway without initiating ROS generation. It would be of particular interest to investigate the molecular mechanism by which Nox1 mediates the AKT signaling pathway in OSCC cells. Although we only performed in vitro experiments using a subset of OSCC cell lines in the present study, our novel finding that Nox1 knockdown enhanced cisplatin-induced cytotoxic effects provides an attractive option for the combined therapy of Nox1 knockdown and cisplatin chemotherapy in OSCC treatment. Additional studies such as in vivo experiments and/or Nox1 expression status in patients with OSCC are warranted to further understand the molecular mechanisms underlying the pathogenesis related to cell survival in OSCC and the associated clinical applications.