Effects Of Lovastatin On Proliferation Of Human Lung Adnocarcinoma A549 Cell Line And Its Mechanisms

Objective:To study the effects of cholesterol inhibitor lovastatin on proliferate inhibitory effect and the apoptosis inducing activity on human lung adnocarcinoma A549 cell line. Meanwhile,to observe the effects of lovastatin combined with anti-cancer agents cisplatin(DDP) on A549 cell and elucidate its mechanism.Methods: 1 human lung adnocarcinoma A549 cells were incubated in culture medium in vitro.using MTT assay to detect the growth rate among different lovastatin concentration groups(0,2,5,10,20,32,50,64umol/L) and different time groups(24,48,72h),in order to choose proper drug concentration and action time. According to the result of MTT, establishing control and experimental group(2,5,10,20 umol/L).Apoptosis and distribution of cell cycle were examined with flow cytometry. The morphological alterations were confirmed by light microscopy. Extracting total RNA of each group cell, assessing the integrality and content of RNA, the level of PTEN,p27 mRNA expression was examined by semiquantitative Reverse transcription polymerase chain reaction(RT-PCR) technique in the A549 cells treated before and after with Lovastatin. The expression of PTEN,p27 protein was qualitationly studied by immunocytochemistry staining and was semi-qualititately examined by flow cytomitry in cell lines treated before and after with lovastin. 2 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining lovastatin and cisplatin on A549 cell. To determine whether the combination of lovastatin and cisplatin results in a synergistic cytostatic effect, Jin's formula was performed. Apoptosis induced by combining Lovastatin and cisplatin was examined by flow cytometry.Results: 1 MTT assay results: lovastatin (0,2,5,10,20,32,50,64umol/L) could inhibit the proliferation of A549 cells. After treated with lovastatin for 24h to 72h, compared with control group, the OD values of lovastatin-treated groups decreased, and there was statistically significant difference between control group and every treatment group(P<0.05). Furthermore, with the increasing concentration of lovasattin and prolonging of treatment time, the OD values decreased gradually, that was, lovastatin inhibited the proliferation of A549 cells significantly in dose-and time-dependent manner. When treated with lovastatin, A549 cells had significant morphological changes which were observed by light microscopy: The cells untreated with lovastatin were diamond or fusiform, and they were satiation. But the cells treated with lovastatin were minificated, nucleonic pycnosis, cell bodys refraction were weaken, intracytoplasm could see grana. With the increasing concentration of lovastatin and prolonging of treatment time, the cells rounded gradualy and more and more detached from the wall of culure flsak, and some cells were broke.When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the result were: After treatment with 0,2,5,10,20umol/L lovastatin for 48h, the number of cells in S phase and G₂/M phase decreased gradually. That was , lovastatin could induce an arrest of cell cycle in G₀/G₁ phase by a dose-dependent manner. In addition, after treated with 0,2,5,10,20umol/L lovastatin for 48h, the typical apoptotic peak which enhanced gradually with the increasing concentration of lovastatin was observed. Analysis on expression of PTEN and p27 by FCM showed that: Treating A549 cells with 0,2,5,10,20umol/L Lovastatin for 48h resulted in the FI values of PTEN and p27 increasing with the increasing concentration of lovastain together. To the FI values of every kind of protein, there was statistically significant difference between every Lovastatin-treated group and control group(P<0.05). RT-PCR detection results: Before being treated with lovastatin,PTEN and p27 were both expressed in A549 cells. Compared to the control group, the expression of PTEN mRNA and p27 mRNA were increased markedly in 4549 cells exposed to Lovastatin(P<0.05).The expression of p27 was positively correlated to PTEN in A549 cells.2 MTT colorimetric method showed: 2,5μmol/L lovastatin showed a synergistic cytostatic effect in A549 cells when combined with Cisplatin. lovastatin combined with cisplatin increased the inhibitory effect on the proliferation of A549 cell and q value is large than 0.85. the apoptotic percentage increased by combining 2,5umol/L lovastatin with 1.25,2.5mg/Lcisplatin, and there was statistically significant difference between the control group and the group of treating with Cisplatin alone (P<0.05).Conclusion: 1 lovastatin had the effect of anti-lung adnocarcinoma A549 Cell Line. 2 lovastatin can inhibit proliferation of A549 cells in vitro which depending on a dose-and time-dependent manner. 3 We confirm that lovastatin induced an arrest of cell cycle in G₀/G₁ phase as well as apoptosis in A549 cells. 4 Within a certain drug concentration, lovastatin can increase the expression of PTEN,p27 in A549 cells. We infer that lovastatin's effects of inhibiting proliferation and inducing apoptosis in A549 cells seem to due to up-regulation of the expression of PTEN and p27 mRNA and protein.5 lovastatin combined with Cisplatin increased the inhibitory effect on the proliferation of A549 cell 6 lovastatin combined with cisplatin had a synergistic effect on inducing the apoptosis of A549 cells, thus, we assumed that lovastatin could have increased the sensitivity to anti-cancer agents of A549 cells.