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[Establishment of a Doxorubicin-Resistant Acute Myeloid Leukemia Cell Line and Study on Its Drug Resistance
Lu-Lu Feng1, Yu-Ting Wang2, Chao-Fan Huang1
1Department of Central Laboratory,The Affiliated Huai 's an No.1 People's Hospital of Nanjing Medical University, Huai 's an 223300, Jiangsu Province, China.
Objective:
To establish a doxorubicin (DOX)-resistant acute myeloid leukemia (AML) cell line and explore the mechanisms of its drug resistance.
Methods:
A DOX-resistant THP1 cell line (THP1-Rdox) was established using a low-dose, concentration-gradient intermittent induction method. The resistance effect was evaluated by calculating the resistance index (RI). The expression levels of resistance-related proteins ［P-glycoprotein (P-gp) and lung resistance-related protein (LRP)］ and cell cycle-related proteins (Cyclin A2, Cyclin B1 and Cyclin D1) were examined by Western blot. Intracellular DOX accumulation was observed using confocal laser scanning microscopy. Apoptosis rate and cell cycle distribution were analyzed by flow cytometry. RNA sequencing (RNA-seq) was performed to compare the differential gene expression profiles between DOX-sensitive parental THP1 cells and DOX-resistant THP1-Rdox cells.
Results:
The DOX-resistant cell line THP1-Rdox was successfully established. The THP1-Rdox cells could stably proliferate at a DOX concentration of 500 ng/ml with a resistance index as high as 198.7. The THP1-Rdox cells exhibited cross-resistance to homoharringtonine (HHT) and paclitaxel (PTX) but no significant resistance to cytarabine (Ara-C). Compared with parental THP1 cells, the expression levels of drug resistance-related proteins P-gp and LRP in THP1-Rdox cells were significantly upregulated. Additionally, THP1-Rdox cells showed decreased uptake and increased efflux of DOX. Notably, after treatment with verapamil (Ver), a specific inhibitor of P-gp, intracellular DOX accumulation was significantly increased in THP1-Rdox cells. Compared with parental THP1 cells, the THP1-Rdox cells exhibited a significantly decreased apoptosis rate, a reduced proportion of cells in the S phase, an increased proportion of cells in the G1 phase, and a marked upregulation in the expression level of the cell cycle regulatory protein Cyclin D1. RNA-seq analysis showed that among the differentially expressed genes between THP1-Rdox and parental THP1 cells, the top 10 most significantly upregulated genes in THP1-Rdox cells were ABCB1, HNRNPA1P9, SEMA3E, MICB, JAML, FGL2, VSIG1, CLEC1B, DLGAP1-AS3, and HOOK1; the top 10 most significantly downregulated genes were MAGEB2, NPTX2, CGREF1, CDO1, GBP5, ZNF595, ZNF630, DTX3, KLHL4 and ZBED6CL.
Conclusion:
Low-dose, concentration-gradient intermittent induction method can successfully establish a DOX-resistant AML cell line, THP1-Rdox. The drug resistance of this cell line is likely attributed to enhanced drug efflux mediated by the elevated expression of P-gp and LRP, as well as cell cycle dysregulation resulting from the upregulation of Cyclin D1 protein.
