Cigarette smoking induces the activation of RIP2/caspase-12/NF-κB axis in oral squamous cell carcinoma

Patients and tissue specimens

The OSCC tissue samples were collected in Nanjing Stomatological Hospital, Medical School of Nanjing University, and the Ethics Committee of Nanjing Stomatological Hospital, Medical School of Nanjing University approved our study (IRB Approval Number: 2018NL-008 (KS)). Written informed consent was obtained from all the enrolled patients as requested by the Institutional Ethics Committees. Surgical specimens of 40 patients were diagnosed with OSCC and were collected for our study. Pregnant women, lactating women, patients with systemic diseases and other habits, such as alcohol consumption, were excluded from our study. Cigarette smokers included in our study were OSCC patients who had smoked more than 10 cigarettes per day for at least 10 years, while non-smokers were considered patients who had never smoked among OSCC patients.

Our study included 40 patients with OSCC in the groups of smokers and non-smokers, consisting of 24 men and 16 women, with an age range from 30 to 81 years old (median 58 years). The average number of cigarettes among the group of smokers was 20 pack years. Tissue specimens were either immediately processed for immunohistochemistry or stored at −80 °C for western blotting analysis.

Western blotting

Tissues were rinsed with chilled PBS twice, homogenized by using a high-efficiency tissue grinder, and then lysed in chilled RIPA buffer supplemented with protease inhibitor. The lysates were incubated on ice for 30 min and centrifuged at 14,000 × g for 10 min at 4 °C. The nuclear fractions were separated using a nuclear and cytoplasmic protein extraction kit (KeyGEN BioTECH, Jiangsu, China) according to the manufacturer’s instructions. Total proteins and nuclear proteins were collected and separated by a 10% gradient gel and electrophoretically transferred to polyvinylidene difluoride membranes. The membrane was shaken and sealed for 1 h in 5% BSA at room temperature. The primary antibodies used in our study included RIP2, caspase-12 and NF-κB p65 (1:1000 dilution; Abcam, Cambridge, UK), GAPDH and histone (1:5000 dilution; Cell Signaling, Danvers, MA, USA). The membrane was incubated with the primary antibodies overnight at 4 °C, followed by each corresponding secondary antibody at room temperature for 1 h at 37 °C. Finally, using ECL kits (Pierce, Rockford, IL, USA), the data of the target protein were first normalized to GAPDH or histone.

Immunohistochemistry

Serial tissue sections (4 µm thick) were sliced from paraffin-embedded formalin-fixed tissues, and immunohistochemical staining was performed. Briefly, tissue sections were stained using specific primary antibodies, biotin-conjugated secondary antibodies, and horseradish peroxidase (HRP)-conjugated avidin. Subsequently, specific antibody interactions were detected with the HRP substrate 3,3′-diaminobenzidine (DAB). Finally, sections were rinsed and counterstained with hematoxylin. The primary antibodies used were mouse monoclonal anti-RIP2 (1:50 dilution), rabbit polyclonal anti-caspase-12 (1:800 dilution), and rabbit polyclonal anti-NF-κB p65 (1:400 dilution).

Densitometry image analysis was performed using the procedure described in a previous study (Qian et al., 2015). The densitometry analysis of immunohistochemical results was performed by a blinded investigator using Image-Pro Plus analysis software (version 6.0; Dallas, TX, USA). Ten discontinuous fields (400 ×) were randomly selected from each section for evaluation, and the color segmentation algorithm was then used to separate the contributions of the DAB and hematoxylin dyes. The average value of the optical densities of all selected pixels was the mean optical density (integrated option density/area).

Preparation of CSE

The cigarettes used in the study (3R4F) were manufactured by the Tobacco Research Institute, University of Kentucky (Lexington, KY, USA). CSE was prepared using a protocol similar to that previously described (Giebe et al., 2021; Ye et al., 2021; Zeller et al., 2019). Briefly, 3R4F was lit and suctioned with a miniature desktop vacuum pump to 10 ml of cell growth medium at a speed of 50 ml/min. After filtration with a 0.22-µm pinhead filter and adjusting the pH to 7.45, the filtered aliquot was used as the 100% CSE original solution. Then, the CSE original solution was stored at −20 °C for use in in vitro assays, to avoid repeated freeze-thaw.

We measured the contents of CSE after thawing, including nicotine, using an Agilent 1200 series rapid resolution LC system (Agilent Technologies Inc., Santa Clara, CA, USA). The chromatographic separation was achieved on an Ultimate XB-C18 column, 150  × 4.6 mm i.d. with 5-µm particles (Welch Materials, Shanghai, China). The separation was performed at a flow rate of 1.0 ml/min, and the column temperature was 25 °C. The quantitation of nicotine was performed using calibration curves of peak area versus nicotine concentration. The concentration of nicotine within 100% CSE ranged from 1.65 to 1.86 µg /ml.

Cell culture

The OSCC cell line (HSC-3) was purchased from ATCC and used in our study. The cell culture medium contained Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA), penicillin (100 U/L), streptomycin (100 U/L) and 10% (v/v) fetal bovine serum (Gibco). Fresh medium was supplemented every 2–3 days. Cells were then treated with different concentrations of CSE (0, 0.5%, 1%, 2%, 4%) for 24 h in a 37 °C constant temperature incubator containing 5% CO₂.

siRNA interference

To inhibit RIP2 expression, small interfering RNA (siRNA) against RIP2 was used. The RIP2 siRNA (∼50 nM in each well of 6-well-plate) was added to Opti-MEM reduced serum medium at a concentration of 100 pmol/µl, mixed with 5 µl of Lipofectamine 3000, and maintained at room temperature for 20 min. For the transfection assay, 5 × 10⁶ cells were seeded in 6-well plates and incubated overnight at 37 °C in 5% CO₂ until 60%–80% confluence was achieved and were then transfected with siRNA. RIP2 siRNA sense: CACCAATCCTTTGCAGATAAT.

Statistical analysis

Statistical analyses were performed using SPSS 22.0 statistical software (IBM Corporation, USA) and GraphPad Prism 6.0 (La Jolla, CA, USA). All continuous data are reported as the mean ± SD. Data normality tests were carried out by the Shapiro - Wilk statistical test. The student t-test and One-way ANOVA were used for normally distributed data, and comparisons between each group were performed using Dunnett’s test. P < 0.05 was considered statistically significant.

Cigarette smoking induced RIP2-mediated NF-κB activation in OSCC patients

To explore the possible role of RIP2/NF-κB in OSCC patients with cigarette smoking, we first detected the levels of RIP2 and NF-κB expression by using western blotting and immunohistochemical assays. Interestingly, compared with the non-smoker group, RIP2 was markedly increased in the smoker group (Fig. 1A). Immunohistochemistry analysis showed that RIP2 staining was negative in non-smokers, and stronger in the OSCC tissues of smokers. The positive expression of RIP2 was mainly located in the cytoplasm of tumor cells (Fig. 1B). Consistently, results also indicated that NF-κB p65 expression was increased in the OSCC tissues of the smokers compared with those of the non-smokers (Fig. 1C). Moreover, we observed positive staining for NF-κB p65 in the cytoplasm and nucleus of tumor cells in the OSCC tissues of smokers (Fig. 1D). As NF- κB is a nucleus-cytoplasm shuttling protein, we further analyzed whether its cellular localization changes after stimulation with cigarette smoking. The results indicated that cigarette smoking promoted nuclear translocation of NF-κB p65 in OSCC tissues (Figs. 1E and 1F). Together, these data indicated that cigarette smoking induced RIP2-mediated NF- κB activation in the OSCC tissues of smokers.

RIP2 mediated NF-κB activation correlated with the upregulation of caspase-12

We further detected the expression of caspase-12 in the OSCC tissues of smokers. As shown in Fig. 2A, caspase-12 protein was obviously higher in the OSCC tissues of smokers than in those of non-smokers. Moreover, immunohistochemical assays also revealed that a significantly stronger staining intensity of caspase-12 in the tumor cells and stroma was observed in the OSCC tissues of smokers than in those of non-smokers (Fig. 2B). The above results indicated that the expression of RIP2/NF-κB was positively correlated with that of caspase-12 in OSCC tissues.

CSE promoted activation of RIP2/NF-κB and upregulated caspase-12 in HSC-3 cells

To further confirm the effect of cigarette smoking on the RIP2/NF-κB signaling pathway and the expression of caspase-12, we examined the levels of RIP2, NF-κB and caspase-12 expression in HSC-3 cells by using western blotting. The results in Fig. 3A indicated that CSE induced activation of the RIP2/NF-κB pathway in a dose-dependent manner (Fig. 3A). To further identify the effect of CSE on NF-κB-mediated transcriptional activity, the nuclear protein NF-κB p65 in HSC-3 cells was measured. Our results clearly showed that CSE treatment induced the activation of the NF-κB signaling pathway (Fig. 3B). In addition, the protein level of caspase-12 was obviously increased after stimulation with CSE (Fig. 3C). Our in vitro results were similar to the results obtained in OSCC tissues. These results indicated that the RIP2/NF-κB pathway was activated and caspase-12 expression was increased after CSE treatment.

RIP2 positively regulated caspase-12 expression in CSE-treated HSC-3 cells

To further confirm the relationship between RIP2 and caspase-12 in the presence of CSE treatment, we used siRNA to reduce the expression of RIP2 in HSC-3 cells. We found that the knockdown of RIP2 significantly reduced the levels of caspase-12 and total NF- κB p65 expression in CSE-treated HSC-3 cells (Figs. 4A–4D). The effect of RIP2 knockdown also resulted in a reduction in the accumulation of nuclear NF-κB p65 after treatment with CSE (Figs. 4E and 4F). These results indicated that RIP2 positively regulates the NF-κB pathway and caspase-12 expression, which may be involved in the development of OSCC induced by cigarette smoking.