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Expression profile of Notch-related genes in multidrug resistant K562/A02 cells compared with parental K562 cells
1Department of Hematology, Qilu Hospital, Shandong University, Jinan, Shandong, China.
Abstract:
Drug resistance is the major setback of acute myeloid leukemia (AML) therapy. Notch proteins have demonstrated functional regulation in cell proliferation, differentiation, and apoptosis and thus may affect drug resistance. Our study aimed to identify the Notch-related gene profile in drug-resistant AML cells and provide potential strategies for resistant AML therapy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to detect the cytotoxicity of adriamycin toward K562 and drug-resistant K562/A02. Intracellular mean fluorescence intensity was monitored to reflect the intake of adriamycin by confocal microscopy. cDNA microarray was used to test the expression of 113 Notch signaling pathway-related genes in K562/A02 and K562. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot were used to validate the results from microarray. K562/A02 cells showed a 65-fold higher IC(50) to adriamycin and less intracellular accumulation of adriamycin than K562. cDNA microarray showed marked increases in binding of collagen and cell proliferation-related genes (CD44, DLL3, IL17B, NUMB, and NUMBL) and decreases in signal transduction and transcription factor activity related genes (FZD9, GBP2, GLI1, GLI2, IFNG, KRT5, Notch2, and Notch3). The change of gene expression was further validated by real-time RT-PCR and western blot. Notch signaling pathway-related genes may contribute to the drug resistance of AML.
Insights
Drug resistance in acute myeloid leukemia (AML) is a major challenge. This study identified Notch-related gene profiles in resistant AML cells, revealing potential therapeutic targets for overcoming drug resistance.
Area of Science:
- Hematology
- Molecular Biology
- Cancer Research
Background:
- Drug resistance significantly hinders effective treatment for acute myeloid leukemia (AML).
- Notch signaling pathway proteins play crucial roles in regulating cell functions relevant to cancer, including proliferation, differentiation, and apoptosis, suggesting a potential link to drug resistance.
Purpose of the Study:
- To investigate the Notch-related gene expression profile in adriamycin-resistant AML cells.
- To identify potential molecular targets within the Notch pathway for developing novel therapeutic strategies against resistant AML.
Main Methods:
- Cytotoxicity assays (MTT) and confocal microscopy were used to assess adriamycin resistance and intracellular drug accumulation in K562 and K562/A02 cells.
- cDNA microarray analysis was performed to screen 113 Notch signaling pathway-related genes.
- Real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot were employed for validation of microarray findings.
Main Results:
- Drug-resistant K562/A02 cells exhibited a 65-fold higher IC50 to adriamycin and reduced intracellular drug accumulation compared to sensitive K562 cells.
- Microarray analysis revealed significant upregulation of genes associated with collagen binding and cell proliferation (e.g., CD44, DLL3, IL17B, NUMB, NUMBL) and downregulation of genes involved in signal transduction and transcription factor activity (e.g., FZD9, GBP2, GLI1, GLI2, IFNG, KRT5, Notch2, Notch3).
- RT-PCR and western blot confirmed the differential expression patterns observed in the microarray analysis.
Conclusions:
- The Notch signaling pathway and its related genes are implicated in the development of adriamycin resistance in acute myeloid leukemia.
- Identifying specific Notch pathway gene alterations in resistant AML could pave the way for targeted therapeutic interventions to improve patient outcomes.
