Microcystin-LR Promotes Melanoma Cell Invasion And Tumor Metastasis Through PI3K / AKT Signaling Pathway

Purpose:Accumulating evidences showed that microcystin-LR, a well-known cyanobacterial toxin produced by blue-green algae, has tumor-promoting activity in human. Our previous research reported that microcystin-LR increased invasive ability in melanoma MDA-MB-435cells. However, the molecular mechanisms is poorly understood. This study aimed to explore the molecular mechanisms behind the stimulation effects on invasion and metastasis in the human melanoma cells in vitro and in vivo.Methods:1. Western blot was performed to detect the expression levels of p-Akt/Akt and PTEN induced by microcystin-LR(0,5,12.5,25,50nM) on the melanoma cell.2. Luciference、Western blot and Matrigel chamber assays was conducted to investigate the effect of LY294002(PI3-K inhibitor) on the microcystin-LR-induced MMP-2/-9overexpression and cell invasion.3. To further investigate the role of PI3-K/AKT in microcystin-enhanced cell invasive ability, AKT siRNA was used to inhibit AKT expression in MDA-MB-435cells.4. In vivo, the efficacy of microcystin-LR to regulate melanoma tumor metastasis was evaluated using the MDA-MB-435xenograft model. Nude mice were exposed to microcystin-LR at three different dose levels:low (0.001mg/kg), medium (0.01mg/kg), high (0.1mg/kg) everyday by oral gavage. Body weights and tumor sizes were recorded once a week.5. Immunohistochemical analysis the expression of the MMP-2/-9and pAKT in the MDA-MB-435xenograft tumor.6. Lung colonization of tumor cells in nude mice were detected by hematoxylin-eosin (H&E).Results:1. Microcystin-LR treatment (25nM) was shown to increase the activity of phosphatidylinositol3-kinase (PI3-K)/AKT by increasing the expression of phosphorylation protein AKT. In addition, low concentration of microcystin-LR (25nM) was shown to decrease the expression of lipidphosphatase PTEN.2. Microcystin-LR-induced MMP-2/-9over-expression was reversed by the PI3-K/AKT chemical inhibitor, LY294002using western blot and reporter gene assay. Addition of the inhibitor LY294002could also suppress invasion induced by microcystin-LR using Matrigel chamber assays.3. Microcystin-LR-enhanced cell invasive ability in MDA-MB-435cells was suppressed by AKT interference.4. In vivo, the efficacy of microcystin-LR to regulate melanoma tumor metastasis was evaluated using the MDA-MB-435xenograft model. Microcystin-LR daily oral administration had no significant effect on tumor weight.5. Immunohistochemical analysis of tumors from microcystin-LR-fed mice showed increased expression of MMP-2/-9and the increased level of phosphorylation of AKT.6. Lung colonization of tumor cells in nude mice and histological examination of mice lung showed that more lung metastasis spots were found to be present in microcystin-LR-treated groups compared with control group, indicating that microcystin-LR has the ability to promotion of lung metastasis.Conclusion:Microcystin-LR exposure can promote melanoma cell MDA-MB-435invasion and metastasis by stimulating PI3-K/AKT and MMP-2/-9activities.