Xiao Xianxiongtang Regulates Ca2+ Load and Inhibits Epithelial-mesenchymal Transition， Invasion， and Migration of MGC-803 Cells： Based on Wnt5a/ Ca2+/NFAT Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo explore the effect of Xiao Xianxiongtang （XXXT） on the transforming growth factor （TGF）-β1-induced invasion， metastasis， and epithelial-mesenchymal transition （EMT） of gastric cancer MGC-803 cells and the underlying mechanism. MethodThe molecular docking between XXXT and nuclear factor of activated T cells （NFAT） was performed by CB-DOCK （http：//clab.labshare.cn/cb-dock/）. The invasion and metastasis model of MGC-803 cells was established with 10 μg·L-1 TGF-β1. MGC-803 cells were classified into blank group， model group， 0.1 g·L-1 XXXT group， 0.2 g·L-1 XXXT group， and 0.4 g·L-1 XXXT group. For further clarifying the key role of Wnt5a/Ca2+/NFAT signaling pathway in the inhibition of XXXT on gastric cancer， MGC-803 cells were transfected with Wnt5a overexpression plasmid， and then the cells were classified into blank plasmid group， Wnt5a-OE group， blank plasmid + XXXT （0.4 g·L-1） group， and Wnt5a-OE + XXXT （0.4 g·L-1） group. Cell viability was determined by cell counting kit-8 （CCK-8） assay， cell invasion and migration ability by Transwell invasion assay and wound healing assay， expression of EMT-related proteins （E-cadherin， N-cadherin， Vimentin， Snail） and Wnt5a/Ca2+/NFAT signaling pathway-related key proteins ［Wnt5a， calcineurin （CaN）， NFAT1， and p-NFAT1］ by Western blot， and changes in intracellular Ca2+ concentration by immunofluorescence assay. ResultMolecular docking suggested that XXXT acted on Wnt5a/Ca2+/NFAT signaling pathway. XXXT （0.1， 0.2， 0.4 g·L-1） significantly promoted the loss of MGC-803 cell viability （P<0.05，P<0.01）. It inhibited cells from invading the transwell lower chamber and slowed down the healing of cell wounds in a dose-dependent manner （P<0.05， P<0.01）. Moreover， it promoted the expression of E-cadherin while suppressed the expression of N-cadherin， Vimentin， and Snail （P<0.05， P<0.01）. Further experiments showed that XXXT could inhibit the expression of Wnt5a， CaN， NFAT1， and p-NFAT1， and reduce the nuclear expression of NFAT1 and the transcription activity mediated by NFAT1， so as to reduce the cellular Ca2+ concentration （P<0.05， P<0.01）. XXXT can reverse the effect of Wnt5a （P<0.05， P<0.01）. ConclusionXXXT can attenuate the invasion， metastasis， and EMT of MGC-803 cells via the Wnt5a/Ca2+/NFAT pathway， thereby weakening the tumor-promoting effect of TGF-β1. In summary， XXXT may exert therapeutic effect on gastric cancer by regulating the invasion， metastasis， and EMT of gastric cancer cells.