Effect Of Melatonin On Human Lung Adenocarcinoma A549Cells

The incidence and mortality of lung cancer are very high while the rate ofearly diagnosis is low. The efficacy of various treatments and5years survivalrate are poor although there are variety of treatmens. In recent years, the studiesin vitro and in vivo found that melatonin has a clear anti-tumor effect. But thereis little research on the relationship between melatonin and lung cancer. NF-κB isan important cytokine, which translocates into the nucleus from cytoplasmic andcombines with the target DNA to regulate the transcription of the downstreamwhen activated. In tumor cells, NF-κB is usually abnormally active and closelyrelated with tumor proliferation, migration, invasion, and other biologicalprocesses. So inhibiting its activity has become one of the directions of cancertreatment.It was found that melatonin could enhance the efficacy of chemotherapy andimprove the survival rate of patients with advanced lung cancer in clinical studies,but no known mechanism. Some studies on other cells have reported thatmelatonin could inhibit the activity of NF-κB, but we did not find this reportabout lung cancer cells. We speculate that melatonin may play the role of anti-tumor effect through inhibiting activity of tumor nuclear factor κB. So wedesigned this experiment to investigate the function of melatonin on non-smallcell lung cancer. We observed proliferation, migration, invasion situation of thehuman lung adenocarcinoma A549cells by treating with different concentrationsof melatonin and infer the role of the transcription factor κB in the melatoninanti-tumor process through detecting the nuclear translocation of nucleartranscription factor NF-κBp65. Part Ⅰ: Effect of melatonin on human lung adenocarcinomaA549cell proliferation in vitroObjective: To investigate the effects and mechanisms of melatonin onhuman lung adenocarcinoma A549cell proliferation in vitro.METHODS: Cultured human lung adenocarcinoma A549cells, which weredivided into5groups, treated with5mmol/L,2.5mmol/L,1mmol/L,0.1mmol/Land0mmol/L melatonin respectively.1, MTT method was used to measure theproliferation in24h,48h and72h;2,DNA ends in situ labeling assay (TUNEL)was used to detect apoptosis of each group after48h;3, immunohistochemistrywas used to judge NF-κB nuclear translocation of cells preliminarily after48h ofintervening;4,Western-blot analysis was used for determing the expression ofanti-apoptotic protein Bcl2and apoptosis related protein Caspase3in each groupintervened after48h.Results:1, High concentrations of melatonin could directly inhibit theproliferation of human lung adenocarcinoma A549cells, however,no details oflow concentrations of melatonin on the proliferation and the inhibition increasingwith time.2, High concentrations of melatonin could raise the rate of apoptosisbut low concentration has no significant effects.3, immunohistochemistry resultsshowed that high concentrations of melatonin can reduce the positive rate of thenucleus transcription factor κBp65.4, Western blot results showed that afterbeing treated with melatonin(48h), Bcl2decreased significantly and Caspase3increased significantly in A549cells.Conclusion: Melatonin treatment can effectively inhibit the proliferation ofA549cells, the mechanisms of which are related to the activation of Caspase3and inactivation of Bcl2by inhibiting nuclear translocation rate of NF-κB. Part Ⅱ: Melatonin and doxorubicin synergy on lungadenocarcinoma A549cells and the effect of melatonin on thecell’s abilities of migration and invasionObjective: To investigate whether Melatonin has synergistic effects withDoxorubicin in the growth-inhibition of human lung adenocarcinoma A549cells and observe the effect of melatonin on the abilities of migration andinvasion.METHODS:1,Divided A549cells into nine groups, respectively treatedwith9combinations of0mmol/L,0.1mmol/L,1mmol/L melatonin and0ug/ml0.2ug/ml,2ug/ml doxorubicin for48h, then used MTT assay to determin theproliferation of cells;2, Used cell scratch assay to determin the change of theA549cell’s ability of migration treated with0mmol/L,0.1mmol/L and0.5mmol/L,1mmol/L melatonin;3, Used Transwell invasion experiment todetermin the effort of0mmol/L,0.1mmol/L,0.5mmol/L,1mmol/L of melatoninon the invasive ability of lung adenocarcinoma A549cells;4, Used Western-blotanalysis to detect0mmol/L,0.1mmol/L and0.5mmol/L,1mmol/L of melatoninon the A549nuclear transcription factor Bp65nuclear translocation.Results:1, When combined with Doxorubicin,0.1mmol/L and1mmol/Lmelatonin significantly increased the effects of cell growth inhibition.2,Scratchesexperimental results showed that melatonin could inhibit the migration in adose-dependent manner.3, Transwell invasion experiment showed that melatoninenhanced the inhibition of the invasive ability as the concentration increases.4,Western-blot results suggested that melatonin could reduce nucleardisplacement of nuclear transcription factor Bp65in dose-dependent manner.Conclusion: There are synergisms on A549cells between the melatonin and doxorubicin. Melatonin could inhibit the migration and invasion of the humanlung adenocarcinoma A549cells, of which the mechanism may be throughinhibiting nuclear translocation of transcription factor B.