Impact of red-lime and white-lime betel quid on oral cell lines: Cytotoxicity and effects on fibronectin and type I collagen expression

Abstract

Background/purpose: Chewing betel quid is linked to an increased risk of oral cancer. This study investigates the effects of red-lime and white-lime betel quid extracts on oral cell lines, focusing on cytotoxicity and their influence on fibronectin and Type I collagen expression, which were crucial for oral tissue integrity and cancer development.

Materials and methods: Four oral cell lines, human gingival fibroblasts, tongue squamous cell carcinoma cells, human periodontal ligament fibroblasts, and human fetal osteoblasts, were treated with red-lime and white-lime betel quid extracts. Cytotoxicity assays and Western blotting were used to assess cell viability and protein expression.

Results: Both red-lime and white-lime betel quid extracts exhibited dose-dependent effects on all tested cell lines, with variations in sensitivity observed among cell types. Notably, red-lime betel quid exerted stronger cytotoxic effects on human gingival fibroblasts and human fetal osteoblasts. Red-lime betel quid increased fibronectin and Type I collagen in periodontal ligament fibroblasts but reduced both proteins in fetal osteoblasts. White-lime betel quid extract generally elevated fibronectin and decreased Type I collagen across cell lines.

Conclusion: This study reveals a nuanced, concentration-dependent impact of betel quid extracts on oral cells, with significant variability in cytotoxicity and changes in fibronectin and Type I collagen expression. These findings suggest that abrupt cessation of betel quid chewing can lead to dental issues such as mobile teeth. Red-lime betel quid uniquely affects periodontal ligament fibroblasts by increasing both fibronectin and Type I collagen.

Keywords: Betel quid; Cytotoxicity; Periodontal ligament fibroblasts; Red-lime; Type I collagen.

Figure 1

Commercially available red-lime and white-lime betel quid in Taiwan: (A) The image on the left depicts a red-lime betel quid, while the image on the right shows a white-lime betel quid; (B) The left panel illustrates the ingredients of the red-lime betel quid, which include areca nut (AN), red-lime, and piper betle. The right panel shows the ingredients of the white-lime betel quid, consisting of areca nut (AN), white slaked lime, and betel leaf.

Figure 2

Effective concentrations of red-lime betel quid on various human cell lines. (Detailed data are shown in the Supplementary Figures) Eleven different concentrations of red-lime betel nut were administered to cultured cells for 1, 3, 5, and 7 days. Cell proliferation was analyzed using the CCK-8 assay to determine the effective concentration and treatment duration for subsequent experiments: (A) Human gingival fibroblast (HGnF) cells treated with 200 μg/ml red-lime betel quid for 7 days; (B) Squamous cell carcinoma (SCC-25) cells treated with 200 μg/ml red-lime betel quid; (C) Human periodontal ligament fibroblasts (HPLF) cells treated with 100 μg/ml red-lime betel quid; (D) Human fetal osteoblast cell line (hFOB1.19) cells treated with 100 μg/ml red-lime betel quid.

Figure 3

Effective concentrations of white-lime betel quid on various human cell lines. (Detailed data are shown in the Supplementary Figures) Eleven different concentrations of white-lime betel quid were administered to in vitro cultured cells for 1, 3, 5, and 7 days. Proliferation was analyzed using the CCK-8 assay to determine the effective concentration and duration for subsequent research: (A) Human gingival fibroblast (HGnF) cells treated with 400 μg/ml white-lime betel quid for 7 days; (B) Squamous cell carcinoma (SCC-25) cells treated with 200 μg/ml white-lime betel quid; (C) Human periodontal ligament fibroblast (HPLF) cells treated with 400 μg/ml white-lime betel quid; (D) Human fetal osteoblast (hFOB1.19) cells treated with 400 μg/ml white-lime betel quid. All cell images were captured using the JuLI FL cell history recorder at 4× magnification. There was a significant difference between the experimental and control groups (0 μg/ml). Data are expressed as mean ± SE. Statistical analysis was performed using one-way ANOVA. ∗ indicates P < 0.05, indicating statistical significance.

Figure 4

Effects of red-lime betel quid on protein expression of fibronectin and Type I collagen in various human cell lines. Western blotting was used to analyze the protein expression of fibronectin and Type I collagen in cells treated with red-lime betel quid (n = 4): (A) Red-lime betel quid stimulated fibronectin expression and inhibited Type I collagen expression in human gingival fibroblast (HGnF) cells; (B) Red-lime betel quid stimulated fibronectin expression and inhibited Type I collagen expression in squamous cell carcinoma (SCC-25) cells; (C) Red-lime betel quid enhanced the protein expression of both fibronectin and Type I collagen in human periodontal ligament fibroblast (HPLF) cells; and (D) Red-lime betel quid reduced the protein expression of fibronectin and Type I collagen in human fetal osteoblast (hFOB) cells. Data are presented as mean ± SE and were compared with the control group (0 μg/ml). Statistical analysis between the two groups was conducted using the unpaired t-test. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.001, indicating statistical significance.

Figure 5

Effects of white-lime betel quid on the protein expression of fibronectin and Type I collagen in various human cell lines. Western blotting was used to analyze the effects of white-lime betel quid on fibronectin and Type I collagen protein expression in cells (n = 4): (A) White-lime betel quid enhanced fibronectin expression and inhibited Type I collagen expression in human gingival fibroblast (HGnF) cells; (B) White-lime betel quid enhanced fibronectin expression and inhibited Type I collagen expression in squamous cell carcinoma (SCC-25) cells; (C) White-lime betel quid enhanced fibronectin expression and inhibited Type I collagen expression in human periodontal ligament fibroblast (HPLF) cells; and (D) White-lime betel quid reduced the protein expression of both fibronectin and Type I collagen in human fetal osteoblast (hFOB) cells. Data were presented as mean ± SE and were compared with the control group (0 μg/ml). Statistical analysis between the two groups was conducted using the unpaired t-test. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.001, indicating statistical significance.