Reverse Effect Of Recombinant Human Endostatin On Drug-resistance Of A549/DDP Cells To Cisplatin

Background: Lung cancer has become the most common malignant tumor in the world and is the No 1 cause for malignant related deaths. At present, the main method to treat lung cancer is synthesized treatment. Chemotherapy also plays an important role in treatment of lung cancer. However, one of main reasons for chemotherapy failures is the medicine resistance. One of approaches to overcome this medicine resistance is to use reversal agents. DDP is major chemotherapeutic drugs for lung cancer treatment. Unfortunately, it is prone to drug resistance, which undermines treatment effect. Recombinant human endostatin (Endostar), as an endothelial vessel inhibitor, has direct functionalities, less side-effects and no medicine resistance. Better treatment performance is expected when combining it with chemotherapy. This is probably due to recombinant human endostatin's (Endostar) ability to reverse drug resistance by tumor cancer cells. This study mainly focuses on investigating recombinant human endostatin's (Endostar) resistance-reverse effect and mechanisms, when used in conjunction with DDP, and further explores the mechanism of Endostar to improve the efficiency of chemotherapy .Objective: To study the recombinant human endostatin (Endostar) to reverse A549/DDP cells and reverse the incidence of drug resistance mechanisms.Methods: this experiment used human lung adenocarcinoma cell lines A549 and drug resistant cell lines A549/DDP as studied objects. The main process includes: one is to use DDP and Endostar of drug delivery to human lung cancer cell lines A549 and drug resistant cell lines A549/DDP respectively, two is to combine DDP with Endostar of drug delivery to the human lung cell lines A549 and drug resistant cell lines A549/DDP. Then give then 72 hours to observe the differences between different ways of drug delibery on same cell and different ways of drug delivery on two different cell lines. The effect of Endostar to reversal of drug resistance on A549/DDP cells could be detected by using MTT method. Relative Fluorescence Intensity among A549/DDP group, DDP group and Endostar combined with DDP group affected on A549/DDP respectively could be measure by using flow cytometry. The expression of the drug resistant gene of mRNA could be detected by DDP group, Endostar with DDP group and Endostar group affecting on A549/DDP cell by Using RT-PCR method. And it could establish A549 cell control group, A549/DDP cell control group. And the expression drug resistant gene of mRNA in the control group could be detected. Detection of drug resistance genes include: glutathione-S- transferase (GST), topoisomeraseⅡ(TOPO-Ⅱ) and lung resistance protein (LRP).Results:1. The 50% inhibitory concentration (IC50) of DDP to A549cells was (0.72±0.05) ug/ml, the 50% inhibitory concentration (IC50) of DDP to A549/DDP cells was (11.54±0.64) ug/ml. so, the resistance index of A549/DDP cells to DDP was 16.After infection of Endostar ,the 50% inhibitory concentration (IC50) of DDP to A549/DDP cells reduced from (11.54±0.64) ug/ml to (2.0±0.1) ug / ml by 5.77 folds, The relative reversal rate (RRR%) was 88.2℅. Endostar could reverse A549/DDP cells resistance. Endostar combine with DDP, the 50 % inhibitory concentration (IC50) of DDP to A549 was(0.25±0.06)ug/ml, there was a clear synergy in vitro .2. Flow cytometry results showed the fluorescence intensity of DDP: DDP group, A549/DDP group, Endostar combine with DDP group. Their relative fluorescence intensity was1618±82.31, 974±13.45, 2958±63.96, statistical analysis P <0.05 ,it is suggest that Endostar can increase intracellular platinum content, increase the treatment efficacy.3. RT-PCR test results showed that: The GST, TOPO-Ⅱgene mRNA expression of A549/DDP cells were higher than A549 cells , the GST, TOPO-Ⅱgene mRNA expression of Endostar combined with DDP has decreased obviously. P <0.05, statistical significance. LRP expression exist in cells of every group ,and was higher in A549/DDP cells than A549 cells .The LRP expression of A549/DDP cells slightly decreased in the Endostar combined with DDP group,P> 0.05, no statistical significance.Conclusion:Endostar may reverse drug-resistance of A549/DDP cells to DDP through down-regulating GST and TOPO-Ⅱand increasing intracellular cumulation of platinum.