Reversal Effect Of Recombinant Human Endostatin On Cisplatin Resistance In A549/DDP Human Lung Adenocarcinoma Cells In Vitro

Background:Lung cancer is one of common human malignancies, and its incidence keeps rising. The dominant treatment for lung cancer at present is the comprehensive therapy, including chemotherapy which plays an important role. Cisplatin (DDP) is widely applied to chemotherapeutic regimens for lung cancer. However, its application is limited usually by the failed therapy due to resistance. Recombinant human endostatin (rh-endostatin) characterized by minor side effects and good safety acts as a mechanism of angiogenesis inhibition and nutrition blockage, and also improves the response when combined with chemotherapeutic drugs, however, in a way remaining elusive. The mechanism of the improved response to chemotherapy can be hardly explained simply by the premise of angiogenesis inhibition induced by rh-endostatin. The study was to explore the possible effect of rh-Endostatin to reverse DDP-resistance in A549/DDP cells in vitro and the mechanism.Objective:to explore the possible effect of rh-Endostatin to reverse DDP-resistance in A549/DDP cells in vitro and the mechanism.Methods:Lung adenocarcinoma cell line A549 and its DDP-resistant cell line A549/DDP were treated with DDP and/or recombinant human Endostatin. Difference in drug resistance was analyzed between different regimens (DDP, Endostatin and combination) and between different cell lines (A549 versus A549/DDP), after a 72h-treatment in vitro. MTT analyzing the survival influenced by rh-endostatin in A549 or A549/DDP was used to determine non-cytotoxic concentration of rh-endostatin, under which its possibility to reverse DDP-resistance in A549/DDP was evaluated. Cellular apoptotic rate and resistant proteins were analyzed with flow cytometry and western blot respectively for A549/DDP treated with endostatin and/or DDP, to discuss the possible mechanism of endostatin to reverse DDP-resistance in A549/DDP. We also established an A549 control and an A549/DDP control with their expression status of resistance proteins detected. The resistance protein detected in the study included P glycoprotein (P-gp) and topoisomerase II (TOPO-II).Results:1. MTT:rh-endostatin under 400ug/ml showed no cytotoxicity in either A549 or A549/DDP after 72h-treatment. The observed 50% inhibited concentration (IC50) for DDP was (0.79±0.05) ug/ml in A549 and (13.2±1.1) in A549/DDP respectively. The DDP-resistance index in A549/DDP was 16.7. IC50 was lowered to 2.57±0.05ug/ml in A549/DDP treated by rh-Endostatin under the non-cytotoxic concentrations in combination with DDP, with a reversal fold (RF) of 5.14 and a relative reversal rate of 85.6%, demonstrating Endostain enhanced the killing effect of DDP in A549/DDP through a potential to reverse the resistance.2. Flow cytometry:apoptotic rate were 2.01%,13.47%and 29.26% respectively for cells treated with Endostain, DDP, and combination. A rate of 0.99%was reported in the A549/DDP control.3. The apoptosis rate result of western blot:P-gp or TOPO-II was higher in A549/DDP cells than in A549 cells. An obvious drop in expression level of either P-gp or TOPO-II was noticed in A549/DDP treated with Endostatin-DDP combination with statistical significance (all Ps<0.05 in intergroup comparison).Conclusion:Rh-Endostatin may partially reverse DDP-resistance in A549/DDP cells in vitro, with a probable mechanism relating to lowered expression of P-gp and TOPO-II.