Chelidonine inhibits melanoma cell malignancy by inactivating TLR4/NF-κB and PI3K/AKT signaling pathways

Abstract

Melanoma is a common and aggressive tumor, characterized by a high incidence rate and extensive metastasis. Chelidonine exhibits a broad range of biological properties including anti-inflammatory, antimicrobial, and anticancer effects. Our study is intended to explore the effects chelidonine of on melanoma cells. In detail, CCK-8 assay was used for detection of cell viability. The colony formation assay was carried out to measure cell proliferation. Wound healing assay and Transwell assay were employed to evaluate cell migration and invasion, respectively. Cell apoptosis was determined by flow cytometry analysis, and protein level was measured by Western blotting. The experimental results demonstrated that chelidonine treatment inhibited cell viability and cell proliferation but facilitated cell apoptosis of melanoma cells. Besides, chelidonine suppressed melanoma cancer cell migration and invasion by attenuating epithelial-mesenchymal transition process. Moreover, chelidonine inhibited the activation of TLR4/NF-κB and PI3K/AKT pathways by downregulation of the protein level of TLR4, phosphorylated p65, phosphorylated PI3K, and phosphorylated AKT in melanoma cells. Furthermore, TAK-242 or LY294002 further enhanced the inhibitory effects chelidonine of on malignant cell behavior. In conclusion, our findings demonstrate that chelidonine effectively suppresses the malignancy of melanoma cells through the inhibition of TLR4/NF-κB and PI3K/AKT signaling pathways, suggesting its potential as a promising therapeutic agent for melanoma treatment.

Keywords: Chelidonine; Melanoma; PI3K/AKT signaling; TLR4.

Fig. 1. The effect of chelidonine on cell viability of melanoma cells.

(A-D) The MEL270 and C918 cell viability was evaluated by CCK-8 assay under chelidonine (0.1, 0.2, 0.5, 1, 2, and 5 μM) treatment for 24 or 48 h. (E) The representative images of cell morphology of MEL270 and C918 cells. Values are presented as mean ± SD. CCK-8, cell counting kit-8. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. The effect of chelidonine on cell proliferation and apoptosis of melanoma cells.

(A, B) The representative images and quantification of colonies formed by MEL270 and C918 cells treated with chelidonine (0.5 or 1 μM). (C, D) The apoptotic MEL270 and C918 cells treated with chelidonine (0.5 or 1 μM) were determined by flow cytometry analysis. Values are presented as mean ± SD. PI, propidium iodide. ***p < 0.001.

Fig. 3. The effect of chelidonine on cell migration and invasion of melanoma cells.

(A–D) Cell migration and invasion of MEL270 and C918 cells treated with chelidonine (0.5 or 1 μM) were detected by wound healing and Transwell assays. (E, F) The E-cadherin and N-cadherin protein levels were analyzed by Western blot analysis in MEL270 and C918 cells treated with chelidonine (0.5 or 1 μM). Values are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. The effect of chelidonine on TLR4/NF-

κB and PI3K/AKT signaling pathways in melanoma cells. (A-F) The TLR4, phosphorylated and unphosphorylated p65, PI3K, and AKT protein levels were analyzed by Western blot analysis in MEL270 and C918 cells treated with chelidonine (0.5 or 1 μM). Values are presented as mean ± SD. ***p < 0.001.

Fig. 5. LY294002 or TAK-242 enhances the inhibitory effects of chelidonine on cell malignancy of melanoma cells.

(A, B) The protein level of TLR4, p65, PI3K, AKT, p-p65, p-PI3K, and p-AKT in MEL270 and C918 cells treated with chelidonine (0.5 μM) and LY294002 (50 μM) or TAK-242 (1 μM) was detected by Western blot. (C) Cell viability of MEL270 and C918 cells treated with chelidonine (0.5 μM) and/or LY294002 (50 μM) or TAK-242 (1 μM) was determined by CCK-8 assay. (D) The measurement of colony formation of MEL270 and C918 cells treated with chelidonine (0.5 μM) and/or LY294002 (50 μM) or TAK-242 (1 μM). (E, F) The apoptotic MEL270 and C918 cells treated with chelidonine (0.5 μM) and LY294002 (50 μM) or TAK-242 (1 μM) were determined by flow cytometry analysis. (G-J) Cell migration and invasion of MEL270 and C918 cells treated with chelidonine (0.5 μM) and/or LY294002 (50 μM) or TAK-242 (1 μM) were tested by wound healing assay and Transwell assay, respectively. (K) The measurement of E-cadherin and N-cadherin protein levels in MEL270 and C918 cells treated with chelidonine (0.5 μM) and/or LY294002 (50 μM) or TAK-242 (1 μM). Values are presented as mean ± SD. CCK-8, cell counting kit-8; PI, propidium iodide. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. ^###p < 0.001 vs. 0.5 μM group.