Synergistic Effect Of Eicosapentaenotic Acid And Cisplatin On A-549 Human Lung Cancer Cell Line

OBJECTIVE To observe the effects of anti-proliferation and inducing apoptosis on A-549 human lung cancer cell line induced by EPA and its synergistic effect with DDP,provide the experimental evidence for EPA as a new anti-tumor fungus drug.METHODS A-549 human lung cancer cell line was cultured in vitro.MTT assay was used to determine the effect of EPA on A-549 human lung cancer cells growth inhibitory.Morphological changes of the apoptosis of A-549 human lung cancer cells were observed by using inverted,light microscope.Apoptotic rate was calculated with TUNEL and apoptotic half-quantative method through AO/EB double fluorescent staining.FCM was used to detect cell cycle distribution of A-549 cells and apoptotic rate.RESULTSâ‘ The MTT test showed that A-549 cells exposure to EPA at final concentrations of 15μg/ml～60μg/ml were inhibited for 24h,48h,72h and 96h in dose and time dependent manner.EPA combined with Cisplatin shows significant synergistic anti-tumor effect on A-549 cells.â‘¡The A-549 human lung cancer cells in experimental group by HE staining showed morphological changes such as cellular volume decrease and round,nuclear chromatin condensation,plasma membrance bleb formation,also the apoptotic body formations were observed under light microscope. The A-549 human lung cancer cells treated with EPA showed cellular volume small and round,nuclear chromatin condensation,loose between the cells and adherent ability decrease under invert microscope.Human A-549 lung cancer cells stained by AO/EB double fluorescent staining were observed under fluorescent microscope,appearing as chromation orange-red condensation in nuclear,orange-red or orange-yellow fluorescence disappeared and light green fluorescent apoptotic bodies around cells after induction of EPA.â‘¢When A-549 lung cancer cells line exposure to EPA at final concentration of 60μg/ml for 12h,24h,48h,72h and 96h,using apoptotic half quantitative method through AO/EB double fluorescent staining,their apoptotic rate is 3.4%,9%,23%,13%and 6%,respectively,there was statistic significance compared with different time(p＜0.05 or p＜0.01).When the concentration of EPA altered from 15μg/ml to 60μg/ml,the apoptotic index(AI) of A-549 cells elevated accordingly by the method of terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL),the apoptotic index was 5.57±0.82%,11.68±1.26%,24.53±1.68%, respectively,there was statistic significance compared with different concentration(p＜0.01).â‘£A-549 apoptotic peak appeared after being treated with EPA at final concentration of 15μg/ml,30μg/ml,60μg/ml for 48h,the percentage of apoptosis rate was 10.50%,19.87%,30.12%,respectively,there was statistic significance compared with different concentration(p＜0.01),while the control group unappeared.A-549 lung cancer cells treated with EPA at final concentration of 15μg/ml,30μg/ml,60μg/ml for 48h were inhibited and arrested in S phase,the cell percentage of G₀/G₁ phase and G₂/M phase decrease and that of S phase increase was observed by FCM.CONCLUSIONS1.EPA inhibited growth of A-549 human lung cancer cell line in vitro in dose and time dependent manner.2.EPA combined with Cisplatin shows significant synergistic anti-tumor effect on A-549 human lung cancer cell line.3.Human lung cancer A-549 cell line exposure to EPA showed apoptotic changes through morphological,biochemical and FCM assay.4.EPA inhibited A-549 lung cancer cells growth through interfering with cell cycle,mainly arrested in S phase.EPA can inhibit the growth of A-549 cells, which may be one of the mechanisms of EPA on treating lung cancer.