The Synergistic Antitumor Effect Of Dihydroartemisinin Combined With Cisplatin On Lung Adenocarcinoma A549Cells And Caspase-3Activity

Objectives: Taking lung adenocarcinoma A549cells as researching objects,observe the inhibitory effects of dihydroartemisinin(DHA) and cisplatin(DDP) on the cellproliferation, cycle regulation, apoptosis induction, caspase-3activity and preliminarilyinvestigate the mechanism of their synergistic effects, which for providing new methodsfor non-small cell lung cancer（NSCLC）chemotherapy and establishing theoretical andexperimental references for the future clinical application.Methods: Lung adenocarcinoma A549cells were cultured in vitro. MTT assay wasperformed to detect the effect of DHA, DDP and their combination on cell proliferationof24h,48h,72h, then used to evaluate joint effects of two medicines by the Q-valueformula; Using inverted phase contrast microscope to observe morphological changes ofdifferent treatments and to analyze the effect of cell cycle and apoptosis rate by flowcytometer; Caspase-3activity changes of different groups were measured by elisa.Results:1. DHA, DDP and the doublet could inhibit cell proliferation of lungadenocarcinoma A549cells dependent on concentration and time; During the sametreating time, the combination therapy of proliferation inhibition was superior to singlemedicine, but the synergistic and additive antitumor effect of the combination on theA549cells was respectively showed at48h,72h and24h.2. The morphology of apoptotic cells were observed under light microscope, suchas original spindle cell morphology instead of rounded, cell shrinkage and cytoplasmicconcentration,when the A549cells were treated with DHA (30μmol/L) and DDP(0.625mg/L) for48h. Then, using high magnification microscope to observe cellmembrane integrity, nuclear pyknosis and margination. At the same time, the apoptotic morphology of the combined group is more obvious than the single medicine.3. After treating the A549cell with different groups for48h, flow cytometer (FCM)results showed that the cell cycle distribution and apoptosis rate was basically the samewith no significant difference between Solvent control group (DMSO <0.1%) andnormal control group (p>0.05); DHA (30μmol/L) and DDP (0.625mg/L) couldrespectively arrest cell cycles in G0/G1and S phase (p<0.01), while the combinationcould arrest cell cycles in G0/G1, S phases (p<0.01); DHA、DDP could induce apoptosisof the A549cell and the apoptosis rate of the combination was was superior to singlemedicine (p<0.01).4. The activities of caspase-3were improved by different concentrations of DHAtreating the A549cell with48h and the effect was concentration dependent in a certainrange (7.5-60μ mol/L), but there was no statistic difference in treatment between DDPin dose of0.625mg/L and DHA in dose of7.5μmol/L (p>0.05).30μmol/LDHA combinedwith0.625mg/L DDP on the acttivity of caspase-3was more obviously increase thanbefore single medicine (p<0.01).Conclusion: Different concentrations of DHA, DDP have obvious inhibition onproliferation of lung adenocarcinoma A549cell in a time-dose dependent manner and thesynergistic effect was more obviously increased as the time of treatment wasextended.The DHA combined with DDP may arrest cell cycles in G0/G1, S phases, induceapoptosis and improve the activation of caspase-3.