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ObjectiveTo observe the effect of Banxia Xiexintang （BXT）-containing intestinal absorption solution on the apoptosis of polymorphonuclear myeloid-derived suppressor cells （PMN-MDSCs） in gastric cancer microenvironment. MethodBXT-containing intestinal absorption solution was prepared， and gastric cancer cells and PMN-MDSCs were non-contact co-cultured in Transwell chamber to establish gastric cancer microenvironment. Cell counting kit-8 （CCK-8） assay was used to screen the optimal intervention concentration and time of 0-100% BXT-containing intestinal absorption solution prepared by 0.63 g·mL-1 reconstitution solution. Cells were classified into blank group， model group， oxaliplatin group （10 mg·L-1）， and BXT （26%， 18%， 10% BXT-containing intestinal absorption solution） group， and the apoptosis of PMN-MDSCs was detected by flow cytometry. The expression of apoptosis-related B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and cysteine-aspartic acid protease-3 （Caspase-3） in PMN-MDSCs was detected by Western blot. ResultAfter treatment for 24 h and 48 h， the PMN-MDSCs-inhibiting rate was increased by 5%， 50%， 75%， and 100% BXT-containing intestinal absorption solution compared with that in the blank group （P<0.05， P<0.01）. At 72 h， the PMN-MDSCs-inhibiting rate by 50% BXT-containing intestinal absorption solution was lower than that at 48 h （P<0.01）， and the PMN-MDSCs-inhibiting rate by 5%， 75%， and 100% BXT-containing intestinal absorption solution showed no significant difference from that at 48 h. Moreover， the half-maximal inhibitory concentration （IC50） at 48 h was 18.40%. Thus， 18% BXT-containing intestinal absorption solution and 48 h were the optimal intervention concentration and time. The survival rate of PMN-MDSCs in model group was higher than that in the blank group （P<0.05）， and the apoptosis rate was lower than that in the blank group （P<0.05）. Compared with model group， BXT containing intestinal absorption solution lowered the survival rate and raised apoptosis rate of PMN-MDSCs （P<0.05）， particularly the 26% BXT-containing intestinal absorption solution （P<0.05）. The expression of Bax and Caspase-3 in PMN-MDSCs was lower in the model group than in the blank group （P<0.05）， and the expression of Bcl-2 was higher in the model group than in the blank group （P<0.05）. The expression of Caspase-3 in PMN-MDSCs increased （P<0.05） and the expression of Bcl-2 decreased （P<0.05） in oxaliplatin group and BXT group compared with those in the model group. The expression of Bax rose in oxaliplatin group and BXT group （10% BXT-containing intestinal absorption solution） （P<0.05）. ConclusionBXT can induce the apoptosis of PMN-MDSCs by regulating the expression of apoptosis-related proteins Bax， Caspase-3， and Bcl-2 in gastric cancer microenvironment.
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ObjectiveTo observe the effect of Banxia Xiexintang containing intestinal absorption solution （BXCIAS） on migration and invasion of polymorphonuclear myeloid-derived suppressor cells （PMN-MDSCs） in gastric cancer microenvironment. MethodThe complex solution （containing 0.63 g·mL-1 crude drug） was prepared. Gastric cancer cells were subjected to non-contact co-culture with PMN-MDSCs in Transwell chamber to create gastric cancer microenvironment. Cell counting kit-8 （CCK-8） assay was used to screen the optimal intervention concentration and time of BXCIAS on PMN-MDSCs for subsequent experiment. The blank group， model group， FAK inhibitor group， and BXCIAS groups （26%， 18%， and 10%） were designed. Scratch assay and Transwell assay were employed to detect the migration and invasion ability of PMN-MDSCs， and enzyme-linked immunosorbent assay （ELISA） to measure the expression of vascular endothelial cell growth factor （VEGF） and matrix metalloproteinase-9 （MMP-9） in tumor microenvironment. The expression levels of PMN-MDSCs pathway-related proteins FAK， phosphorylated （p）-FAK， protein tyrosine kinase （Src）， and p-Src were detected by Western blot. ResultThe inhibition rates of PMN-MDSCs by 5%， 50%， 75%， and 100% BXCIAS at 48 h were higher than those at 24 h （P<0.05， P<0.01）. The inhibition rate of PMN-MDSCs by 50% BXCIAS at 72 h was lower than that at 48 h （P<0.01）， and the inhibition rates by 5% and 100% BXCIAS at 72 h were higher than those at 48 h （P<0.05， P<0.01）. There was no significant difference in the inhibition rate by other concentration levels at 48 h. The half-maximal inhibitory concentration （IC50） at 48 h was 18.09%， indicating that 18% BXCIAS and 48 h were the optimal concentration and time， respectively. The migration distance of PMN-MDSCs was large （P<0.01）， and the number of migrating and invading cells increased （P<0.01） in the mode group compared with those in the blank group. Compared with model group， FAK inhibitor and BXCIAS at different concentration decreased the migration distance of PMN-MDSCs （P<0.01）， and the number of migrating and invading cells （P<0.01）， especially the 26% BXCIAS （P<0.01）. The expression of PMN-MDSCs pathway-related proteins FAK， p-FAK， Src and p-Src （P<0.01） and the expression of VEGF and MMP-9 （P<0.01） were higher in the model group than in the blank group. Compared with model group， FAK inhibitor and BXCIAS （26%， 18%， 10%） decreased the expression of FAK， p-FAK， and Src （P<0.01）， and FAK inhibitor and 18% BXCIAS reduced the expression of p-Src （P<0.01）， and the expression of VEGF and MMP-9 （P<0.01）. ConclusionBXCIAS can inhibit the migration and invasion of PMN-MDSCs by down-regulating the expression of FAK， p-FAK， Src， and p-Src proteins in the FAK signaling pathway of PMN-MDSCs in gastric cancer microenvironment.
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Background The gastric cancer (GC) microenvironment has important effects on biological behaviors, such as tumor cell invasion and metastasis. However, the mechanism by which the GC microenvironment promotes GC cell invasion and metastasis is unknown. The present study aimed to clarify the effects and mechanism of galectin-1 (GAL-1, encoded by LGALS1) on GC invasion and metastasis in the GC microenvironment. Methods The expression of GAL-1/ LGALS1 was determined using western blotting, immunohistochemistry, and quantitative real-time reverse transcription PCR in GC tissues. Besides, methods including stable transfection, Matrigel invasion and migration assays, and wound-healing assays in vitro; and metastasis assays in vivo, were also conducted. Results GAL-1 from cancer-associated fibroblasts (CAFs) induced the epithelial-mesenchymal transition (EMT) of GC cells though the transforming growth factor beta (TGF-ß1)/ Sma- and mad-related protein (Smad) pathway, and affected the prognosis of patients with GC. The level of GAL-1 was high in CAFs, and treating MGC-803 and SGC -7901 cell line with the conditioned medium from CAFs promoted their invasion and metastasis abilities. Overexpression of LGALS1 promoted the expression of TGF-ß1 and induced EMT of GC cell lines. A TGF-ß1 antagonist inhibited the invasion and migration of GC cells. In vivo, overexpression of LGALS1 promoted GC growth and metastasis, and the TGF-ß1 antagonist dramatically reversed these events. Conclusions These findings suggested that high expression of GAL-1 in the GC microenvironment predicts a poor prognosis in patients with GC by promoting the migration and invasion of GC cells via EMT through the TGF-ß1/Smad signaling pathway. The results might provide new therapeutic targets to treat GC.
