Study on the mechanism of luteolin reversing multidrug resistance in leukemia K562/ADR cells

doi:10.11958/20230722

Abstract

Abstract:

Objective To investigate the mechanism of luteolin′s (Lut) reversal effect on multidrug resistance of chronic myeloid leukemia K562/ADR cells. Methods CCK-8 assay was used to detect drug resistance in K562 and K562/ADR cells 24 hours after treatment with different doses of adriamycin (ADR). CCK-8 assay was used to assess the cytotoxicity and sensitizing effect of Lut on ADR after K562/ADR cells were treated with Lut alone or in combination with ADR for 24 hours. K562/ADR cells in logarithmic growth phase were separated into three group: 0 μmol/L Lut, 2 μmol/L and 4 μmol/L Lut groups. ADR accumulation in cells was measured using flow cytometry. Nuclear factor erythroid-2-related factor 2 (Nrf2), multidrug resistance associated protein 1 (MRP1), P-glycoprotein (P-gp) and glutathione-S-transferase-PI (GST-pi) mRNA and protein expressions were identified using RT-PCR and Western blot assay. Glutathione (GSH) kit was used to detect intracellular GSH content. Results Compared with K562 cells, K562/ADR cell line was significantly resistant to ADR, and the drug resistance was 53.69 times. K562/ADR cell proliferation was decreased to variable degrees by different doses of Lut when compared to the 0 μmol/L Lut group (P<0.05). The proliferation inhibition rates of K562/ADR cells treated with 2 and 4 μmol/L Lut were less than 10%, indicating that the concentration of Lut was non-toxic. Compared with the 0 μmol/L Lut group, the 2 μmol/L Lut group and the 4 μmol/L Lut group showed significantly increased ADR growth inhibition rate on K562/ADR and increased accumulation of ADR in cells, improved the reversal resistance fold, and decreased GSH content in cells. MRP1, P-gp, GST-pi and Nrf2 mRNA and protein expression were reduced in cells (P<0.05). The effect of 4 mol/L Lut was greater than that of 2 mol/L Lut. Conclusion Lut may decrease K562/ADR cell proliferation and reverse ADR medication resistance. The mechanism could be connected to the downregulation of Nrf2, MRP1, P-gp and GST-pi expression, which leads to an increase in ADR accumulation in K562/ADR cells.

Key words: leukemia, myelogenous, chronic, BCR-ABL positive, Luteolin, drug resistance, multiple, nuclear factor erythroid2-related factor 2, multidrug resistance associated protein 1, P-glycoprotein, glutathione-S-transferase-PI

CLC Number:

R733.72

Figures/Tables 5

References 18

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基因名称	引物序列（5'→3'）	产物大小/bp
MRP1	上游：CCTGGGCATTTCACAAGGGAT	139
MRP1	下游：GGTCCGCTCAAAGAAGCTCA	139
P-gp	上游：CCACAGAGGGGATGGTCAGT	142
P-gp	下游：GGTCCGCTCAAAGAAGCTCA	142
GST-pi	上游：AGCCTTTTGAGACCCTGCTGC	122
GST-pi	下游：AGGGGCTAGGACCTCATGGAT	122
Nrf2	上游：TAGTGCCCCTGGAAGTGTCAA	112
Nrf2	下游：ATCATGCACGTGAGTGCTCTG	112
GAPDH	上游：GCACCGTCAAGGCTGAGAAC	138
GAPDH	下游：TGGTGAAGACGCCAGTGGA	138

基因名称	引物序列（5'→3'）	产物大小/bp
MRP1	上游：CCTGGGCATTTCACAAGGGAT	139
MRP1	下游：GGTCCGCTCAAAGAAGCTCA	139
P-gp	上游：CCACAGAGGGGATGGTCAGT	142
P-gp	下游：GGTCCGCTCAAAGAAGCTCA	142
GST-pi	上游：AGCCTTTTGAGACCCTGCTGC	122
GST-pi	下游：AGGGGCTAGGACCTCATGGAT	122
Nrf2	上游：TAGTGCCCCTGGAAGTGTCAA	112
Nrf2	下游：ATCATGCACGTGAGTGCTCTG	112
GAPDH	上游：GCACCGTCAAGGCTGAGAAC	138
GAPDH	下游：TGGTGAAGACGCCAGTGGA	138

组别	MRP1	P-gp	Nrf2	GST-pi
0 μmol/L Lut组	1	1	1	1
2 μmol/L Lut组	0.76±0.05^a	0.53±0.07^a	0.57±0.14^a	0.88±0.04^a
4 μmol/L Lut组	0.67±0.02^ab	0.36±0.05^ab	0.35±0.20^a	0.64±0.08^ab
F	101.700^＊	130.300^＊	15.650^＊	48.690^＊

组别	MRP1	P-gp	Nrf2	GST-pi
0 μmol/L Lut组	1	1	1	1
2 μmol/L Lut组	0.76±0.05^a	0.53±0.07^a	0.57±0.14^a	0.88±0.04^a
4 μmol/L Lut组	0.67±0.02^ab	0.36±0.05^ab	0.35±0.20^a	0.64±0.08^ab
F	101.700^＊	130.300^＊	15.650^＊	48.690^＊

组别	MRP1	P-gp	Nrf2	GST-pi
0 μmol/L Lut组	1.15±0.10	1.42±0.03	1.89±0.19^a	1.20±0.11
2 μmol/L Lut组	0.78±0.13^a	1.04±0.03^a	1.18±0.17^a	0.78±0.05^a
4 μmol/L Lut组	0.51±0.06^ab	0.69±0.06^ab	0.85±0.17^a	0.47±0.07^ab
F	31.370^＊	237.800^＊	27.090^＊	63.940^＊