Mechanism of Modified Tongyou Decoction and Its Separated Formulas Inhibiting Vasculogenic Mimicry in Esophageal Cancer TE-1 Cells via NF-κB/HIF-1α Axis

Objective: To explore mechanisms of Modified Tongyou Decoction (MTD) and its separated formulas inhibiting vasculogenic mimicry (VM) in esophageal cancer (EC) TE-1 cells and improving the hypoxic environment via NF-κB/HIF-1α axis on the basis of the hypoxia stimulating the cells’ VM formation. Methods: The extract of MTD was prepared by water extraction and alcohol precipitation-whirl evaporation, and amygdalin, the content of the antitumor active component, was determined by chromatography. TE-1 cells were divided into normoxia control (NC) group, hypoxia control (HC) group, MTD group, activating blood (AB), promoting Qi (PQ) and removing toxin (RT) separated formula groups. The optimal concentration and time dependent manner of MTD and the 3 separated formulas were screened by CCK-8 assay. Migration and invasion ability was detected by wound healing and invasion assay. Vasculogenesis experiment was performed to investigate TE-1 cells’ vasculogenic capacity. Protein expression of NF-κB/HIF-1α axis and VM related molecules were detected by Western blot. Interaction between HIF-1α and NF-κB was detected by double immunofluorescence labeling assay. mRNA expression of HIF-1α and NF-κB was detected by qRT-PCR. Protein secretion of HIF-1α and E-cadherin in supernatant of culture media was determined by ELISA assay. Results: Extract of MTD contained amygdalin, an active anti-cancer component, the concentration of it being 104.397 μg/mL. Effect-time dependent manner was achieved when cells were treated with MTD and the 3 separated formulas for 24 hours with IC50 concentration, MTD, 2550 μg/mL; PQ, 3384 μg/mL; AB, 3979 μg/mL; RT, 3832 μg/mL. Cell migration area rate of NC group was 43% while that of HC group was 56% after 12 hours; the rate of the 4 medications decreased differently, showing statistical significance with that of NC group ( P < .05). Cell invasion area in HC group was 80% within invasion field while it was 55% in NC group after 48 hours; Cell invasion ability was inhibited differently by the 4 medications, invasion area showing statistical significance with that of NC group ( P < .05). Hypoxia stimulation promoted formation of reticular structure of cells after 6 hours. The structure disappeared after the 4 medications’ activity. Fluorescent signals of HIF-1α and NF-κB were enhanced in the hypoxia, compared with those in the normoxia, showing statistical significance ( P < .05). The 2 fluorescent signals were notably inhibited by the medications, among which MTD and the AB formula were much stronger. Proteins of the NF-κB/HIF-1α axis and VM related molecules were over-expressed in the hypoxia except E-cadherin was down-regulated. After intervention with the medications, the protein expression was remarkably inhibited, while E-cadherin was over-expressed. HIF-1α mRNA expression significantly increased after hypoxia stimulation, however, the expression in AB and RT groups was notably inhibited. NF-κB mRNA expression didn’t change notably in the hypoxia, but was inhibited in all medication groups with no statistical significance. ELISA assay showed that HIF-1α protein from supernatant was increased and E-cadherin reduced after hypoxia stimulation. The 4 medications inhibited HIF-1α protein secretion, among which MTD and the AB formula showed stronger function. However, the 4 medications showed no obvious function on E-cadherin protein secretion. Conclusions: Hypoxia is an important condition that promotes VM generation in EC TE-1 cells and the mechanism relates to NF-κB/HIF-1α axis over-expression. Based on separated formulas being in harmony with each other, MTD shows explicit effect on EC VM regulated by NF-κB/HIF-1α axis.