Effects of Extracellular Vesicles on the Drug Resistance of Lung Adenocarcinoma Cells by Modulating the ATP Binding Cassette Transporter G2

WANG Lei, MI Yuan, ZHANG Xin-fei, LI Xing, HE Cai-yi, LI Chao, LIU Liang

Abstract

To investigate the regulatory role of extracellular vesicles (EVs) carrying ATP binding cassette transporter G2 (ABCG2) on the drug resistance of lung adenocarcinoma cells and the relevant molecular mechanisms.  Methods  A549 cells, human lung adenocarcinoma cells, were used to form cisplatin (or cis-Diaminedichloroplatinum, CDDP)-resistant lung adenocarcinoma cells, i.e., A549/CDDP cells. EVs from A549 and A549/CDDP cells were extracted by gradient centrifugation method and were hence named EVs1 and EVs2, respectively. The A549 cells were treated with EVs1 and EVs2 for 48 hours, and the cells were named A549-EVs1 and A549-EVs2 cells, respectively. A549/ABCG2 cells were established by transfecting A549 cells with pCDNA3.1-ABCG2 recombinant plasmids. On the other hand, A549 cells transfected with empty vectors were named A549/pCDNA3.1 cells. MTT assay was conducted to calculate the 24-hour cell drug resistance index for CDDP. The ABCG2 gene expression in cells and EVs were assessed with real-time PCR. A549 and A549-EVs2 cells were transplanted subcutaneously into nude mice, which were labeled the control group and the experimental group accordingly. After tumor formation, 3 mg/kg CDDP was intraperitoneally injected once a week for two times. The ABCG2 gene expression of subcutaneous transplanted tumor cells was examined by real-time PCR. The cell apoptosis rate of subcutaneous transplanted tumor cells was examined by flow cytometry.  Results  Using the parental A549 cells as reference, the 24-h CDDP-resistance indexes of 549/CDDP, A549/ABCG 2, A549/pCDNA3.1, A549-EVs1, A549-EVs2 cells were 7.17, 10.06, 1.02, 1.19 and 5.40, respectively. When comparing the ABCG2 gene expression levels in all cells and EVs, the findings were higher in A549/CDDP cells than those inA549 cells, higher in A549/ABCG2 cells than those in A549/pCDNA3.1 or A549 cells, higher in EVs2 than those in EVs1, and higher in A549-EVs2 than those in A549-EVs1 cells (P<0.01) . The volume of transplanted tumor and the ABCG2 gene expression level in the experimental group were higher than those in the control group, while the apoptosis rate was lower than that in the control group (P<0.01).  Conclusion  EVs carrying ABCG2 gene can regulate the drug resistance of lung adenocarcinoma cells.

 

Keywords: Non-small cell lung cancer, Drug resistance, Extracellular vesicle, Nude mice, ATP binding cassette transporter G2

 

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