Study On Mechanism Of N-3 Polyunsaturated Fatty Acids Against Lung Metastasis Of Melanoma

Objective:To investigate the effects of n-3 polyunsaturated fatty acids(n-3 PUFAs)on melanoma metastasis.Control and treatment group were treated with safflower oil and algal oil by intragastric administration,respectively.Cell growth pathway,tumor microenvironment,apoptosis pathway,autophagy and oxidative stress were studied to clarify the anti-melanoma mechanism of n-3 PUFAs.The present study will provide a theoretical basis for the prevention and treatment of melanoma or other highly metastatic malignant tumors with n-3 PUFAs supplements.Methods:1.Control and treatment group were treated with safflower oil at the dose of2.5ml/kg BW and algal oil at the dose of 5ml/kg BW by intragastric administration,respectively.The frequency of intragastric administration was once every other day and the duration of intragastric administration was two months.2.Cultured B16F10 melanoma cells were harvested and resuspended at a concentration of1×10~6 cells/ml.A volume of 0.2 mL of suspended cells was injected into the tail veins of 10 control mice and 10 treatment mice.All mice were sacrificed by exsanguination under ether anesthesia 5weeks after injection and the lungs,livers,spleens,kidneys and intestines were dissected out and photographed.3.For histological investigations,the tumor tissues were fixed in 4%formaldehyde in phosphate buffer overnight and then embedded in paraffin.Histological sections were stained with haematoxylin and eosin(HE).4.Fatty acid profiles of plasma were detected by gas chromatography.5.The expressions of adhesion protein,inflammatory cytokines,apoptosis pathway proteins,autophagy-associtated proteins and oxidative stress pathway proteins were detected by western blot.6.The level of malondialdehyde(MDA)in mouse urine was checked by lipid peroxidation kit.Results:1.5 weeks after injection,reduced food intake,slower activity,and slower response appeared in control group.Compared with control group,the symptoms were less severe in treatment group.2.Tumor foci were found in lungs in control and treatment group.Compared with control group,tumor tissues showed decreased cell density,less cell morphologic polymorphisms,slighter hyperchromatosis,less polynuclear cells and reduced cell mitosis in treatment group.3.Compared with control group mice,treated group mice exhibited significantly increased levels of n-3 PUFAs,including EPA,DPA,DHA and total n-3 in the plasma.The ratio of n-6/n-3PUFAs of the experimental group was significantly lower than that of control group.4.Compared with control group,E-cadherin,cleaved caspase 3,p-AKT and p-JNK was up-regulated,whereas Snail,Slug,N-cadherin,claudin-1,S-TNF-?,cleaved IL-1?,IL-6,p-c-Raf,p-p38 and HO-1 were down-regulated in treatment group;At the same time,the ratio of LC3 II/?-actin increased and the ratio of phosphorylated mTOR(p-mTOR)/total mTOR was decreased in treatment group.5.Compared with control group,MDA level of the urine in treatment group was significantly increased.Conclusion:Exogenous n-3 PUFAs inhibit metastasis and growth of metastatic melanoma tumor,the anticancer mechanisms of n-3 PUFAs are as follows:(1)To inhibit metastatic tumor growth by increasing E-cadherin level through reducing expression of its transcription repressors such as Snail and Slug;(2)To inhibit the expression of cytokines secreted tumor necrosis factor(S-TNF-?),IL-1?and IL-6 in the tumor inflammatory microenvironment;(3)To induce apoptosis of melanoma cells mediated by cleaved caspase 3 and p-c-Raf;(4)To trigger autophagy through regulating JNK and p38 MAPK signaling pathway;(5)Enhanced oxidative stress.In conclusion,the present study provided a scientific rationale for dietary n-3 PUFAs to prevent and treat melanoma.