Effects And Mechanisms Of Extract Of Laminaria Japonica,Fucoxanthin And Eckol On Sensitizing Non-Small Cell Lung Cancer Cells To Gefitinib

Objectives:To investigate the effects of extract of Laminaria japonica,FX and EC in combination with EGFR TKIs Gef on NSCLC cells PC9 and Gef acquired resistance cells PC9/G,as well as their potential mechanisms.Methods:NSCLC cells PC9 and Gefitinib resistant PC9(namely PC9/G)were cultured in vitro.Experiment was divided into six groups,namely Control,Gef,FX,EC,Gef+FX and Gef+EC groups.CCK8 was used to detect the inhibitory effects of indicated treatments on PC9 and PC9/G cells.PC9 xenograft-bearing models were established to investigate the effect of FX/EC alone and combined with Gef on tumor growth.Immunohistochemistry was used to detect the expressions of Ki67 in tumor tissues.Apoptotic rates of each group were detected by flow cytometry.After indicated treatments,MDC was used to stain cells and autophagic cells were observed under a fluorescent microscope.The expressions of autophagy-related proteins such as p62,BECN,LC3B in each group as well as potential signal pathway were measured by Western blotting.Results:The IC50 values of Gef in PC9 and PC9/G were 0.028±0.011 μM and 6.440 ±1.532 μM respectively.Compared with PC9 cells,relative resistance of PC9/G reached to 322,which was consistent to the characteristics of Gef resistant cell lines.FX,EC,FX+Gef and EC+Gef all exerted inhibitory effects on the proliferation of PC9 and PC9/G cells(P<0.05).In PC9 xenograft-bearing models,no significant weight loss of nude mice was observed in each group.Compared with Blank control group,tumor growth slowed in administration groups,especially in FX+Gef and EC+Gef groups.The expression of Ki67 decreased in FX,EC,FX+Gef and EC+Gef groups,especially in combination groups.Compared with Gef group,apoptotic rates of PC9 and PC9/G cells increased significantly in both FX+Gef and EC+Gef groups(P<0.05).MDC staining assay revealed that autophagic level increased in PC9/G cells compared with PC9 cells(P<0.05),while decreased after being treated with FX,EC,FX+Gef and EC+Gef respectively.Compared with Gef group,autophagic level decreased significantly in FX+Gef and EC+Gef groups(P<0.05).Western blotting revealed FX or EC in combination with Gef could regulate expressions of autophagyrelated proteins and inhibit EGFR/AXL-MEK-ERK signal pathway.Conclusions:FX or EC could sensitize NSCLC cells to Gef effectively in vivo and in vitro,which might exert their effects through inducing apoptosis,inhibiting autophagy,regulating EGFR/AXL-MEK-ERK axis.