Atractylenolide II inhibits the TLR4/EGFR/STAT3 pathway to exert anti-melanoma effects

Background: Malignant melanoma is aggressive and has a high mortality rate. Activation of signal transducer and activator of transcription 3 (STAT3) is a key event in toll like receptor 4 (TLR4) signaling-mediated melanoma progression. Epidermal growth factor receptor (EGFR) is an upstream kinase of STAT3 and linked to STAT3 activation. However, whether EGFR involves in TLR4/STAT3 signaling is unknown. Atractylenolide II (AT-II) is a sesquiterpene compound occurring in some medicinal herbs, e.g. Atractylodis Macrocephalae Rhizoma
(Baizhu in Chinese) and Swordlike Atracylodes Rhizome (Cangzhu in Chinese), and exerts anti-melanoma effects by inhibiting STAT3 activation. In this study, we established the TLR4/EGFR/STAT3 pathway in melanoma and determined the role of this pathway in the anti-melanoma effects of AT-II.

Methods: Specific siRNA-mediated TLR4 silencing was used to determine the role of TLR4 in EGFR and STAT3 activation in TLR4 ligand-stimulated A375 melanoma cells. Specific EGFR inhibitor gefitinib was used to determine the role of EGFR in STAT3 activation in A375TLR4-CA (overexpressing a constitutively activated variant of TLR4) cells. Molecular docking and surface plasmon resonance (SPR) analyses were used to determine the binding between AT-II and TLR4, and the binding between AT-II and EGFR. A375TLR4-CA and A375NC (harbouring the empty vector) stable cell lines were used to determine the role of the TLR4/EGFR/STAT3 pathway in the anti-melanoma effects of AT-II in cell and mouse models.

Results: Application of TLR4 ligands or overactivation of TLR4 activated EGFR/STAT3 signaling in melanoma cells; and pharmacological inhibition of EGFR downregulated STAT3 phosphorylation in A375TLR4-CA cells. AT-II bond TLR4 and EGFR, and inhibited their ligands-triggered EGFR/STAT3 signaling in melanoma cells. Moreover, constitutive activation of TLR4 in A375 cells diminished the effects of AT-II in inhibiting EGFR/STAT3 signaling and attenuated the anti-melanoma effects of AT-II in cell and mouse models.

Conclusions: Our findings indicate that inhibition of TLR4/EGFR/STAT3 signaling is a potential strategy for treating melanoma and contributes to the anti-melanoma effects of AT-II.