Journal: British Journal of Pharmacology
Article Title: Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma, et al. Direct inhibition of the TLR4/MyD88 pathway by geniposide suppresses HIF‐1α‐independent VEGF expression and angiogenesis in hepatocellular carcinoma
Figure Lengend Snippet: Direct inhibition of the TLR4/MyD88 pathway is involved in the anti‐angiogenic actions of geniposide in hepatocellular carcinoma (HCC). (a) 3D structure of binding profile between TLR4 and geniposide is visualized and analysed by in silico molecular docking approach. Magnified views are the potential binding clusters (left panel). The central figure is the sensorgram (red curves) for the binding affinity analysis of geniposide with various concentrations (0–12.8 μM) over a TLR4‐immobilized CM5 sensor chip, which is analysed by surface plasmon resonance (SPR) technology, while the right inset plot (black line) represents the response intensity of 0–12.8‐μM geniposide to TLR4. The linear regression is plotted by a four‐parameter logistic equation ( R 2 = 0.952, K D = 6.716e −6 M) by the BIAevaluation system (GE Healthcare, Sweden). (b) The interaction of MD2 and TLR4 was measured by co‐immunoprecipitation assay and immunoblotting (upper panel), while the lower panel is the mRNA expression of TLR4 in both MHCC‐97L and PLC/PRF/5 cells under geniposide treatment in normoxic condition. (c, d) Protein expressions of TLR4/MyD88 and its downstream factors p‐p38 MAPK, p65 and IκB‐α in both 24‐h geniposide‐treated MHCC‐97L and PLC/PRF/5 under either normoxic or hypoxic condition. (e) Protein levels of TLR4, MyD88, Sp1, STAT3, p‐STAT3, p‐p38 MAPK and p65 in MHCC‐97L and PLC/PRF/5 cells under co‐treatment of geniposide and LPS (100 ng·ml −1 ). (f, g) Secretion and mRNA activity of VEGF in both MHCC‐97L and PLC/PRF/5 cells upon the cooperative intervention of geniposide and LPS (100 ng·ml −1 ) under normoxic or hypoxic environment, analysed by elisa assay and RT‐PCR, respectively. (h) Determination of HCC‐derived tube formation of HUVECs via either single or co‐treatments as shown in the left panel, including geniposide (200 μg·ml −1 ), LPS (100 ng·ml −1 ) and recombinant VEGF (re‐VEGF; 20 ng·ml −1 ). Quantification of tubular networks is shown in the bar charts (lower panel). (i) The migration of HUVECs is significantly reduced when cultured in the supernatant derived from geniposide‐treated PLC/PRF/5 cells under normoxic conditions for 24 h. Note that the TLR4/MyD88 signalling pathway can be down‐regulated by geniposide treatment, which is caused by geniposide‐induced direct inhibition of TLR4 protein. Also, the addition of either LPS or recombinant VEGF leads to significant reversal of the anti‐angiogenic effect of geniposide. All data are presented as mean ± SD of five independent experiments with at least three replicates. * P
Article Snippet: After finishing the electro‐transfer, membranes were immersed in the blocking buffer containing 5% BSA for 2 h prior to the incubation with primary antibodies, including anti‐TLR4 (Cat: MAB6248, R & D Systems, USA), anti‐MD2 (Cat: ab24182, Abcam, USA), anti‐glypican‐3 (Cat: ab66596, Abcam, USA), anti‐Sp1 (Cat: 9389, CST, USA), anti‐STAT3 (Cat: 9139, CST, USA), anti‐phospho‐STAT3 (Cat: 9145, CST, USA), anti‐HIF‐1α (Cat: 36169, CST, USA), anti‐phospho‐p38 MAPK (Cat: 4511, CST, USA), anti‐IκB‐α (Cat: 4812, CST, USA), anti‐p65 (Cat: 8242, CST, USA) and anti‐MyD88 (Cat: 50010, CST, USA), respectively.
Techniques: Inhibition, Binding Assay, In Silico, Chromatin Immunoprecipitation, SPR Assay, Co-Immunoprecipitation Assay, Expressing, Planar Chromatography, Activity Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Recombinant, Migration, Cell Culture