Lipoxin A4 inhibits the paracrine of Nodal in CAFs by suppressing FPRL1/ROS/NF-κB signaling to attenuate invasion and metastasis of gallbladder carcinoma
Authors: Y. Shi, Y. Fan, Y. Hu, J. Jing, E. Li
Journal: Annals of Oncology (2018)

Introduction: Gallbladder carcinoma is a lethal disease in part because of its potential for aggressive invasion and metastasis, and cancer associated fibroblasts (CAFs) in gallbladder carcinoma exerts crucial role in this process. Lipoxin A4 (LXA4), an anti-inflammatory lipid mediator derived from the endogenous arachidonic acid metabolite, has recently been demonstrated to exhibit anti-cancer effects. However, the role of LXA4 in gallbladder carcinoma remains to be elucidated. Methods: CAFs were isolated from tissues derived from patients diagnosed with gallbladder carcinoma, and treated with vehicle or LXA4. The levels of intracellular reactive oxygen species (ROS) was detected by 2'-7'-dichlorofluorescein diacetate, the expression of markers of CAFs, Nodal, IκBα and NF-κBp65 were determined by qRT-PCR, western blotting and immunofluorescence, and the paracrine of Nodal was detected by ELISA. Lentivirus carrier, si-RNA were used to overexpress p65 or silencing LXA4 receptor FPRL1. The conditioned medium (CM) of CAFs was collected after treated with or without LXA4 (400 nM) for 48 h, and indirect co-culture model was established to examine the effects of CAFs on the invasion of gallbladder carcinoma cells (GBC-SD cells). The subcutaneous xenograft model was applied to investigate the effect of LXA4 in vivo. The correlation between the Lipoxin effect score (LES) and the clinical-pathological features of gallbladder carcinoma was also analyzed. Results: We found that in patients with gallbladder carcinoma, low LES was correlated with aggressive metastatic potential. LXA4 could significantly inhibit expression of markers of CAFs, IκBα and NF-κBp65, and suppress the level of ROS. The immunofluorescence staining exhibited that LXA4 can restrain nuclear translocation of p65. ELISA assay also showed LXA4 could refrain the secretion of Nodal in a concentration-dependent manner. Furthermore, the LXA4 activity was mediated by the LXA4 receptor FPRL1 and ROS/NF-κB signaling to suppress the expression and paracrine of Nodal in CAFs. Silencing FPRL1 by si-RNA, adding exogenous H2O2 or overexpressing of p65 in CAFs can respectively reverse the paracrine of Nodal in the presence of LXA4. The results of indirect co-culture model showed that compared with control group, CM of CAFs with vehicle can significantly enhance the invasion of GBC-SD cells, increase the expression of N-cadherin and Vimentin and decrease the expression of E-cadherin, while CM of CAFs with LXA4 administration could dampen the effects of CM of CAFs with vehicle on SD cells. Interestingly, when anti-Nodal neutralizing antibody was administered in CM of CAFs with vehicle or rhNodal (10 ng/mL) was administered in CM of CAFs with LXA4, the expression of EMT-associated markers and the invasion capacity of GBC-SD cells were reversed respectively. In the subcutaneous xenograft model exhibited LXA4 could inhibit the growth and metastasis of GBC-SD cells. Conclusion: Our results demonstrated Lipoxin A4 could inhibit the paracrine of Nodal in CAFs by suppressing FPRL1/ROS/NF-κB signaling to attenuate metastasis of gallbladder carcinoma, which may provide a new strategy to prevent the metastasis of gallbladder carcinoma.