To Investigate The Proliferation Inhibition And Synergism Effect Of Aidi Combined With Low-dose Of DDP In Lung Adenocarcinoma A549Cell

Objectives:Use Addie injection of common traditional Chinese medicine and the chemotherapydrug cisplatin to observe the effects on cell cycle, inhibition of proliferation andapoptosis of lung adenocarcinoma A549cells while treat with single drug and2drugcombination at different concentration as well as administration time, to find an optimaldose of Addie injection combined with minimum dose of cisplatin to act on lung cancercells. Thus, we can explore the anti-cancer mechanisms of the drugs, providing theoriesas well as experimental evidences for better lung cancer treatments.Methods:We cultured lung adenocarcinoma A549cells in vitro until they entered thelogarithmic growth phase, and then divided them into blank group, control group,singledrug groups and drug combination group. By MTT, we detected the cell inhibition rate(IR) in different groups after intervening24h,48h and72h. In the meanwhile, weobserved morphological changes of these cells in different time periods as well asdifferent groups by inverted phase contrast microscope. After that, we used FACS todetect and analyze the change of cell cycle as well as apoptosis rate in different groupsafter intervening48h.Results:1. Inhibition of proliferation of lung adenocarcinoma A549cells in single drug groups Both Addie injection and cisplatin have the ability to inhibit the proliferation oflung adenocarcinoma A549cells by showing time-and concentration-dependence,which is the longer time period and higher drug concentration, the lower cellularactivity.2. Inhibition of proliferation of lung adenocarcinoma A549cells in drug combinationgroupDrug combination: After combining small dose of0.25mg/L cisplatin with1.875mg ml,3.75mg/ml,7.5mg/ml and15mg/ml Addie injection respectively, allthese groups showed time-and concentration-dependence, which is the longer timeperiod and higher drug concentration, the higher inhibition. Compared to the singledrug groups of the same concentration and time period,all the inhibition of drugcombination group was much higher, which showed significant difference(P<0.01)between them. However, the inhibition of7.5mg/ml Addie injection combined withcisplatin was lower than that of15mg/ml Addie injection combined with cisplatin.3. Morphological changes of these cells after given Addie injection and cisplatinIn single drug groups and drug combination group with small dose, the cellsdied by showing apoptosis, poor cell attachment, condensed cytoplasm, and theirstructure transformed from the original spindle shape to round shape. The cell activitydisplayed a significant decrease as the dose and time period were increasing. We wereable to observe a large amount of round-shaped cell debrises by inverted phasecontrast microscope.4. Effect of single drug use and drug combination on cell cycle and apoptosisAfter intervening48h, we used FACS to detect cells in single drug groups anddrug combination group. The results showed that given the regular dose of cisplatin,cell cycle was blocked at S phase, which means the cells at S phase were on the rise. However, compared to the control group, AIDI group cells at G2/M phase wasincreased while that at G0/G1and S phases was significantly lowered, whichindicated that AIDI had the ability to block cells at G2/M phase. We also found thatwhen used drug combination, the cells number in S phase was lower than givenregular dose of cisplatin while higher than the control group as well as the AIDIgroup, and its number at G2/M phase was lower than that in AIDI group while higherthan that in control group and cisplatin group, which demonstrated that drugcombination would result in blocking cells at S and G2/M phases by showingstatistically significant high apoptosis rate(P<0.01) when compared to the cisplatingroup as well as the AIDI group.Conclusions:Both Addie injection and cisplatin have the ability to block cell cycle progressionand promote apoptosis while display time-and concentration-dependence, of which themechanisms might be relevant to apoptosis induction. Based on the correlative references,combining with results from the preliminary experiment, we conclude that15mg/mlAddie injection combined with small dose of cisplatin could also reach or even surpassthe regular dose of0.5mg/L cisplatin, which reduced cytotoxicity while enhancing thesensitivity of chemotherapy drugs,improving therapeutic efficacy, thus provides a newpreparation to reduce side effects in clinical treatment. However, when concentration ofAddie injection exceeded15mg/ml, cells were almost killed, which indicates that largedoses of Addie injection would lead to a strong cytotoxicity in lung adenocarcinomaA549cells, so we set15mg/ml as the maximum concentration.