Baicalein Binds to TLR4, Inhibits Colorectal Cancer Growth and Metastasis via the TLR4/HIF/VEGF Axis

Abstract

Background: Lipopolysaccharide(LPS) inserts into the hydrophobic binding pocket of the myeloid differentiation protein 2(MD-2) that bound to toll-like receptor 4(TLR4). TLR4 antagonists have been designed based on the structure of lipid A, the innermost regions of LPS. However, these antagonists have poor bioavailability and may be toxic. Furthermore, the presence of lipid A moiety is not necessary for driving TLR4 activation. Therefore, compound directly binds to TLR4 is a better strategy to inhibit TLR4 activity. \n \nMethods: Biophysical techniques were used to examine the direct binding between baicalein and TLR4. With colorectal cancer(CRC) as models, coupled with drug-target-disease network, protein-protein interaction network and functional studies, the effect and the associated underlying mechanism of action upon the binding baicalein to TLR4 were examined. \n \nFindings: We evidently show a direct physical binding of baicalein to TLR4 in molecular docking, surface plasmon resonance biosensor analysis and bio-layer interferometry analysis. With CRC cells as model, we demonstrate the binding of baicalein to TLR4 with cellular thermal shift assay; which interrupts the binding of LPS to TLR4-MD-2 in the cells. Baicalein inhibits TLR4 activity, reduces CRC cell viability and migration in TLR4-dependent manner. Drug–targets–disease network and protein-protein interaction network highlight hypoxia inducible factor(HIF-1α) and vascular endothelial growth factor(VEGF) as downstream of TLR4, which are further validated in\xa0 cell models. Involvement of TLR4/HIF-1α/VEGF axis in the anti-CRC and anti-angiogenic effect of baicalein is demonstrated in CRC-bearing mice and chick-chorioallantoic-membrane assay, respectively. Our study provides evidence to support the translation of baicalein into TLR4-targeting therapeutics. \n \nFunding Information: This work was partially supported by Research Grant Council of HKSAR HKBU-22103017-ECS, Innovation & Technology Commission #PRP/015/19FX, National Natural Science Foundation of China #SCM-2016-NSFC-003 and Natural Science Foundation of Guangdong Province #2018A0303130122 to HYK; the National Natural Science Foundation of China (82074019 and 81703705), Characteristic innovation projects of universities in Guangdong Province (2020KTSCX030), the Opening Project of Zhejiang Provincial Preponderant and Characteristic Subject of Key University (Traditional Chinese Pharmacology), Zhejiang Chinese Medical University (ZYAOX2018010) to ST. \n \nDeclaration of Interests: The authors declare that they have no conflict of interest. \n \nEthics Approval Statement: Animal experiments were approved by the Guangzhou University of Chinese Medicine Animal Care and Use Committee (Guangzhou, China), and conducted according to the ethical standards and national guidelines.