ABSTRACT
BACKGROUND@#Fibroblast activation protein (FAP) is one of the surface markers of cancer-associated fibroblasts (CAFs) and is closely related to the malignant characterization of CAFs. SP13786 is a specific micromolecule inhibitor of FAP and this study is to investigate the effects and mechanism of SP13786 on the migration and invasion of A549 cells through regulating exosomes of CAFs.@*METHODS@#CAFs and paracancerous fibroblasts (PTFs) were isolated and subcultured from freshly resected lung adenocarcinoma tissues and paracancerous normal tissues separately. MTT assay was used to detect the proliferation of CAFs incubated by different concentrations of SP13786; PTFs-exo, CAFs-exo and CAFs+SP13786-exo were extracted by polymer precipitation method. The A549 cells were divided into Ctrl group, PTFs group, CAFs group and SP13786 group and each group was incubated with DMEM, PTFs-exo, CAFs-exo and CAFs+SP13786-exo separately. Laser confocal microscope was used to observe the endocytoses of exosomes by A549 cells. The expression of alpha-smooth muscle actin (α-SMA) and FAP in PTFs and CAFs and the expression of E-cadherin, N-cadherin, Slug, Stat3 and P-Stat3 in A549 cells were detected by immunofluorescence, immunohistochemistry and Western blot. The migration and invasion ability of A549 cells were detected by cell scratch and transwell methods.@*RESULTS@#α-SMA and FAP were expressed much higher in CAFs than that in PTFs which indicate that CAFs and PTFs were successfully obtained from lung adenocarcinoma and paracancerous tissues (P0.05). Finally, WP1066 (a specific inhibitor of Stat3) was used to comfirm whether SP13786 could influence EMT of A549 cells by inhibiting Stat3 phosphorylation via CAFs-Exo. The results showed that when the phosphorylation of Stat3 in CAFs group was inhibited by WP1066, SP13786 could not influence the P-Stat3 expression and EMT of A549 cells anymore (P>0.05).@*CONCLUSIONS@#As a specific micromolecule inhibitor of FAP, SP13786 indirectly inhibits the migration and invasion of A549 cells by affecting exosomes of CAFs. The possible mechanism is to inhibit the phosphorylation of Stat3 and thus affect the EMT of A549 cells.
ABSTRACT
BACKGROUND@#ANXA2 plays a very important role in cancer progression. chemokine ligand 18 (CCL18) is associated with the invasion, migration, metastasis and poor prognosis of lung adenocarcinoma (LUAD). In this study, we aimed to explore whether CCL18 promotes LUAD invasion through ANXA2, and its role and molecular mechanism in LUAD invasion.@*METHODS@#Western blot was used to detect ANXA2 expression in LUAD tissues and adjacent non-tumor tissues, the transfection efficiency of SiANXA2#2 in cells and the role of ANXA2 as an upstream regulator in the AKT/cofilin signaling pathway. In vitro cytological experiments such as chemotaxis experiment and transwell invasion test was used to explore the mechanism of ANXA2 on LUAD metastasis. F-actin polymerization experiment and Western blot were used to detect whether invasion ability alteration of SiANXA2#2 A549 cells are related to F-actin.@*RESULTS@#Western blot analysis showed that compared with adjacent non-tumor tissues, the protein expression level of ANXA2 in cancer tissues increased (P<0.05). In the chemotaxis experiment and invasion experiment, the chemotaxis and invasion ability induced by CCL18 decreased when ANXA2 knockdowned (P<0.05). Compared with the control group, F-actin polymerization was significantly lower in ANXA2 knockdown group, while phosphorylation of AKT at Ser473 and Thr308 and phosphorylation of Cofilin and LIMK were reduced in ANXA2 knockdown group (P<0.05).@*CONCLUSIONS@#ANXA2 knockdown can reduce the invasive effect of CCL18 on LUAD cells by reducing phosphorylation of AKT and downstream pathways.