7,8-dihydroxy Coumairn Inhibits A549Huamn Lung Adenocarcinoma Cells

Backgrounds:7,8-hydroxycoumarin as the main active ingredient oftraditional Chinese medicine flavescens, is mainly used for clinicalanalgesic, antibacterial, anti-virus, and prevention of pulmonary fibrosis.Recent studies have shown that7,8-hydroxy coumarin has an appearentanti-tumor effect. DT-αMSH has the MC1-R-mediated biologicalfunctions in lung adenocarcinoma cells.Objective: To explore the role of7,8-hydroxycoumarin in inhibitinggrowth of A549human lung adenocarcinoma cell lines and the molecularmechanism to induce apoptosis of A549. At the same time, conjugatediphtheria toxin(DT) and alpha melanocyte-stimulating hormone(αMSH)protein, forming DT-αMSH protein for the treatment of lungadenocarcinoma. To compare the action of7,8-dihydroxy coumarin andDT-αMSH to inhibit A549human lung adenocarcinoma cells.Methods:(1) DT-αMSH was used to co-incubated with A549humanlung adenocarcinoma cells, which expressed α-MSH receptor (MC1-R),and BGC human gastric cancer cells (Negative control), followed bydetection of cell proliferation by MTT assay. DT-αMSH was also injected into tumor-bearing mice, followed by detetion of the tumor weight.(2)7,8-dihydroxy coumarinwas also used to treat A549cells andtumor-bearing mice (inject0.2ml containing0,5,10and20g of7,8-dihydroxy coumarins, once every other day for10days), forcomparing the anti-tumor effects with DT-αMSH protein.(3)7,8-dihydroxy coumarin (final concentration of0,25,50,100μg/ml) wasused to treat A549cells, followed by real-time PCR and Western blottingto detect AKT/NF-kappaB pathways.Results:(1) The DT-αMSH targeted well to A549lung adenocarcinomacells. By receptor-mediated endocytosis, the DT-αMSH presentedreceptor specific toxicity to A549cells, not to BGC-823cells, which didnot express α-MSH receptor.7,8-dihydroxy coumarin showed strongercytotoxicity to A549cells, when compared to DT-αMSH protein.DT-αMSH protein also significantly inhibited the growth of solid tumorsin nude mice. A549cells detected MC1-R overexpression, BGC-823cellsnot. αMSH bind to MC1-R of A549cells, DT-αMSH entered the cells viathe MC1-R-mediated endosytosis, then DT functioned the cytotoxiceffects. By comparison, BGC-823cells did not bear MC1-R, not bind toαMSH. There was no αMSH-mediated endocytosis of DT-αMSH, andtherefore did not show toxicity of DT.(2) The cytotoxic effect of7,8-dihydroxy coumarin on A549human lung adenocarcinoma cells wasstronger than the DT-αMSH protein in vitro and in vivo.(3) 7,8-dihydroxy coumarin inhibited the AKT/NF-kappaB pathways inA549human lung adenocarcinoma cells to induce the apoptosis. Theanti-apoptotic signaling phospho-Akt1, phospho-IκBα, NF-κBp65andBcl-2were inhibited and the pro-apoptotic signaling Caspase-3upregulated in a dose-dependant manner. At100μmol/L strength, theanti-apoptotic NF-κBp65and Bcl-2mRNA decreased down to0.12(5.82/48.5)-fold and0.17(6.7/39.4)-fold, respectively. Thepro-apoptotic Caspase-3mRNA upregulated to4.43(39.4/8.9)-fold. Theanti-apoptotic pAkt1, pIκBα, NF-κBp65and Bcl-2proteins weredownregulated to7.3(blot grayscales vs.52.4control),4.3(vs.42.2),5.08(vs.44.5) and5.92(vs.38.5). The pro-apoptotic Caspase-3wasupregulated to27.8(vs.5.8). The proliferative activity of A549cellslowered significantly when compared to control cells (83.7,27.2,9.5vs.100%, respectively, P <0.05for each).Conclusions:(1) The DT-αMSH protein can recognize the specific α-MSH receptor(MC1-R) carried on A549human lung adenocarcinoma cells, and kill thetarget A549cells. MC1-R is essential for DT-αMSH to be cytotoxic, andthe lack of MC1-R results in the insensitivity of BGC-823cells to DTtoxins.(2)7,8-dihydroxy coumarin inhibits human lung adenocarcinoma A549cells more than DT-αMSH in vitro and in vivo in dose range. (3)7,8-dihydroxy coumarin can inhibit cell proliferation of A549cells,and induces the apoptosis of A549cells by inhibiting AKT/NF-κBpathways in a concentration-dependant manner.(4)7,8-dihydroxy-coumarin as a natural extract from a plant, has a lowtoxicity and a hign efficiency in the treatment of lung tumors, pendingfurther studies in the clinical treatment of lung tumors. The plant resoucesare rich in China. It is of important significance for expanding clinicalapplications and even the development of new drugs to strengthen themechanism of action of the active ingredient.