Suppressing Lung Cancer Cell Migration and Invasion through Disruption of Rho GTPase Function by Diclofenac and Docosahexaenoic Acid

Abstract

Background Low survival rates of lung cancer patients and high recurrence rate in non-small cell lung cancer (NSCLC) patients have been attributed to metastasis. The aberrant signaling of GTPases, such as the Rho family of GTPases (Cdc42, Rac1, and RhoA) have been implicated in lung cancer metastasis. Several studies indicate that cyclooxygenases (COX) inhibitors and omega-3 polyunsaturated fatty acids (PUFAs) may help prevent cell migration and invasion, which are critical steps in cancer cell metastasis. We previously reported the inhibitory effect of co-treatment with docosahexaenoic acid (DHA) and diclofenac on the expression of hyperactive small GTPases. This study aimed to investigate the potential anti-metastatic effects of such co-treatment on NSCLC cancer cells as well as to investigate the related mechanisms. Methods We conducted western blotting to detect Rho GTPases (RhoA, Rac1, and CDC42) in A549, NCI-H1299, and NCI-H1975 cells after exposure to DHA and diclofenac. We also conducted immunostaining analyses of F-actin organization and Rho GTPases in A549 cells. We further examined the effect of co-treatment on NSCLC cell migration and invasion. Expression of marker proteins in EMT and MMP-2/9 were detected by western blot analysis. Results Co-treatment with DHA and diclofenac disrupted actin filament assembly as well as inhibited cell migration and invasion. Exposure of A549 cells to DHA (5 μM) and diclofenac (25 μM) resulted in suppression of both the distance of migration by 58.1 ± 2.8% as well as the number of cells that migrated into the wounded area by 62.2 ± 1.8%. In addition, the co-treatment resulted in altered expression of marker proteins involved in EMT by increasing expression of epithelial marker, E-cadherin, while downregulating the expressions of mesenchymal markers, N-cadherin, Fibronectin and Vimentin, ZEB1, and β-catenin. Conclusions Our findings indicate that co-treatment with diclofenac and DHA suppressed NSCLC cell motility and invasion, and is associated with disruption of the actin cytoskeleton and inhibition of the Rho GTPase activity. Our findings suggest that combination of diclofenac and DHA could potentially prevent or control NSCLC metastasis.