Smokeless tobacco extract inhibits proliferation and promotes apoptosis in oral mucous fibroblasts

Abstract

The consumption of smokeless tobacco extract (STE) is growing rapidly, and it has been implicated in several human diseases including diabetes, inflammation and a number of types of cancer. The toxicity of STE requires evaluation, as it is known to induce numerous public health issues. To investigate whether STE serves a role in cultured human oral mucosa fibroblasts (hOMFs), the present study examined HOMF morphology with inverted microscopy and immunofluorescence staining. The cell viability was measured with MTT assays, which detected the cell apoptosis rate via flow cytometry. The activities of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured via flow cytometry and commercial kits, subsequent to exposing the cells to various concentrations of STE. Reverse transcription quantitative polymerase chain reaction and western blot analyses were used to demonstrate that the mRNA and the protein expression levels of cell cycle-associated genes (cyclin-dependent kinase inhibitor 1 and cyclin D1), apoptosis-associated genes [B cell lymphoma 2 (Bcl-2) and Bcl-2-associatied X protein], tumor protein (p53), nuclear factor kappa light chain enhancer of activated B cells (NF-κB)-transcription factor (p65) signaling pathways, NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H: quinoneoxidoreductase1 (NQO1). The results indicated that the hOMF cells were positive for cytokeratin staining. STE induced G1-S cell cycle progression and cell apoptosis by regulating the cell cycle or apoptosis-associated proteins. STE treatment increased the concentrations of ROS and MDA, and decreased the concentrations of SOD and CAT. STE unregulated phosphorylated-p53, NF-κB p65, Nrf2, HO-1, and NQO1 expression levels in the hOMF cells. The present study demonstrated that STE appears to promote oral disease.

Keywords: apoptosis; human oral mucosa fibroblast cells; nuclear factor kappa light chain enhancer of activated B cells; proliferation; smokeless tobacco extract; tumor protein 53.

Figure 1.

Morphology and cytokeratin of hOMF cells. (A) The cellular morphology of hOMF cells on the culture plates was observed and images were captured under the microscope after 48 h of cell culture. Left panel: Magnification, ×100, Scale bar, 100 µm; Right panel: Magnification, ×400, Scale bar, 20 µm. (B) Immunofluorescence staining indicated the expression level of cytokeratin in cultured hOMF and NF cells. The merged (Merge) image represents the superimposed images of cytokeratin in green and nuclei (DAPI) in blue. Scale bar, 50 µm. hOMF, human oral mucosa fibroblasts; NF, normal fibroblasts.

Figure 2.

Treatment with STE inhibits viability and induces cycle progression of hOMF cells. (A) The viability rate was detected by MTT assay in hOMF cells cultured with specified concentrations (0–800 µg/ml) of STE for 24, 48 and 72 h (*P<0.05, **P<0.01 and ***P<0.001 vs. 0 µg/ml). (B) The treated hOMF cells were collected at 48 h, stained with propidium iodide and measured by flow cytometry. (C) The cell cycle distribution (G1, G2 and S phases) was measured and analyzed by in surviving cell populations (*P<0.05 and ***P<0.001 vs. 0 µg/ml). (D) The mRNA expression levels of p21 and cyclin D1 were evaluated by reverse transcription-quantitative polymerase chain reaction (*P<0.05, **P<0.01 and ***P<0.001 vs. 0 µg/ml). (E) Proteins were extracted from hOMF cells treated with different concentrations of STE. p21, cyclin D1 and GAPDH protein levels were detected by western blot analysis. GAPDH was used as a loading control. The relative protein expression levels of p21 and cyclin D1 were analyzed according to the protein grey values (**P<0.01 and ***P<0.001 vs. 0 µg/ml). hOMF, human oral mucosa fibroblasts; STE, smokeless tobacco extracts; p21, cyclin-dependent kinase inhibitor 1.

Figure 3.

Treatment with STE promotes the apoptosis ability of hOMF cells. hOMF cells were treated with STE (0, 200, 400 and 800 µg/ml) for 48 h. (A) Flow cytometry with Annexin V-FITC/PI staining was performed to determine the apoptosis ratio of the treated hOMF cells. (B) The number of apoptotic cells in different groups was quantitatively analyzed (**P<0.01 and ***P<0.001 vs. 0 µg/ml). (C) Bax and Bcl-2 levels were detected by reverse transcription-quantitative polymerase chain reaction and normalized to GAPDH transcript levels (*P<0.05 and **P<0.01 vs. 0 µg/ml). (D) Bax, Bcl-2 and GAPDH protein levels were evaluated by western blot analysis using protein-specific antibodies and (E) quantified. The relative protein expression levels of Bax and Bcl-2 were analyzed according to the protein grey values (**P<0.01 and ***P<0.001 vs. 0 µg/ml). hOMF, human oral mucosa fibroblasts; STE, smokeless tobacco extracts; FITC, fluorescein isothiocyanate; PI, propidium iodide; Bcl-2, B cell lymphoma-2; Bax, Bcl-2-associated X protein.

Figure 4.

STE increases the concentrations of ROS and MDA and decreases the concentrations of SOD and CAT. Human oral mucosa fibroblasts cells were treated with STE (0, 200, 400 and 800 µg/ml) for 48 h. (A) Flow cytometry was used to analyze the concentration of ROS with DCFH-DA fluorescent probe. The representative results of DCF-derived fluorescence are presented. (B) The fluorescence intensity was quantitatively analyzed using the fold change (***P<0.001 vs. 0 µg/ml). (C) SOD activity was detected using a commercial kit (***P<0.001 vs. 0 µg/ml). (D) CAT activity was measured using a CAT activity assay kit (*P<0.05, **P<0.01 and ***P<0.001 vs. 0 µg/ml). (E) MDA activity was analyzed using a MDA activity assay kit (*P<0.05, **P<0.01 and ***P<0.001 vs. 0 µg/ml). STE, smokeless tobacco extracts; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase.

Figure 5.

STE upregulates p-p53, NF-κB p65, Nrf2, HO-1 and NQO1 expression levels in hOMF cells. hOMF cells were treated with STE (0, 200, 400 and 800 µg/ml) for 48 h respectively. (A) p53 expression was detected by RT-qPCR and western blot analysis and GAPDH was used as an internal reference (**P<0.01 and ***P<0.001 vs. 0 µg/ml). (B) RT-qPCR and western blot analysis of NF-κB p65 in hOMF cells (**P<0.01 and ***P<0.001 vs. 0 µg/ml). (C) Levels of Nrf2, HO-1 and NQO1 were measured by RT-qPCR and western blot analysis (*P<0.05, **P<0.01 and ***P<0.001 vs. 0 µg/ml). hOMF, human oral mucosa fibroblasts; STE, smokeless tobacco extracts; p53, tumor protein 53; p, phosphorylated p53; NF-κB, nuclear factor kappa light chain enhancer of activated B cells, p65, transcription factor p65; Nrf2, Nuclear factor (erythroid-derived 2)-like 2; HO-1, Hemeoxygenase 1; NQO1, NAD(P)H quinone dehydrogenase 1; RT-qPCR, reverse transcription quantitative polymerase chain reaction.