Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and MDR1 shRNA expression vector in leukemia cells.
In many instances, multidrug resistance (MDR) is mediated by increasing the expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic Fe(3)O(4) nanoparticle [MNP (Fe(3)O(4))] and MDR1 shRNA expression vector in K562/A02 cells. For stable reversal of "classical" MDR by short hairpin RNA (shRNA) aiming directly at the target sequence (3491-3509, 1539-1557, and 3103-3121 nucleotide) of MDR1 mRNA. PGC silencer-U6-neo-GFP-shRNA/MDR1 called PGY1-1, PGY1-2, and PGY1-3 were constructed and transfected into K562/A02 cells by lipofectamine 2000. After transfected and incubated with or without MNP (Fe(3)O(4)) for 48 hours, the transcription of MDR1 mRNA and the expression of P-gp were detected by quantitative real-time PCR and Western-blot assay respectively. Meanwhile intracellular concentration of DNR in K562/A02 cells was detected by flow cytometry (FCM). PGC silencer-U6-neo-GFP-shRNA/MDR1 was successfully constructed, which was confirmed by sequencing and PGY1-2 had the greatest MDR1 gene inhibitory ratio. Analysis of the reversal ratio of MDR, the concentration of daunorubicin (DNR) and the transcription of MDR1 gene and expression of P-gp in K562/A02 showed that combination of DNR with either MNP (Fe(3)O(4)) or PGY1-2 exerted a potent cytotoxic effect on K562/A02 cells, while combination of MNP (Fe(3)O(4)) and PGY1-2 could synergistically reverse multidrug resistance. Thus our in vitro data strongly suggested that a combination of MNP (Fe(3)O(4)) and shRNA expression vector might be a more sufficient and less toxic anti-MDR method on leukemia.
Chen
Bao-an
BA
Department of Hematology, The Affiliated Zhongda Hospital, Clinical Medical School, Southeast University, Nanjing, People's Republic of China.
Mao
Pei-pei
PP
Cheng
Jian
J
Gao
Feng
F
Xia
Guo-hua
GH
Xu
Wen-lin
WL
Shen
Hui-lin
HL
Ding
Jia-hua
JH
Gao
Chong
C
Sun
Qian
Q
Chen
Wen-ji
WJ
Chen
Ning-na
NN
Liu
Li-jie
LJ
Li
Xiao-mao
XM
Wang
Xue-mei
XM
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics; D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D003630:Daunorubicin / Q000008:administration & dosage*; D004317:Doxorubicin / Q000008:administration & dosage; D018432:Drug Resistance, Multiple / Q000187:drug effects* / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics*; D019450:Genes, MDR; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D058185:Magnetite Nanoparticles / Q000008:administration & dosage*; D008969:Molecular Sequence Data; D050997:Nanomedicine; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics*; D014162:Transfection
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D017931:DNA Primers; D058185:Magnetite Nanoparticles; D034741:RNA, Small Interfering; D004317:Doxorubicin; D003630:Daunorubicin