[K562 cell line resistance to nilotinib induced in vitro and preliminary investigation of its mechanisms].
OBJECTIVE
To establish a bcr-abl(+) cell line resistance to nilotinib, and to investigate the possible mechanisms of resistance.


METHODS
K562 cells were treated with gradually increasing concentrations of nilotinib to generate resistance cell line K562-RN. The folder of drug-resistance was evaluated by MTT assay. Cells apoptosis rate was detected by flow cytometry, the mRNA level of bcr-abl fusion gene by FISH, and the expression of apoptosis relative gene mRNA and protein (such as bcr-abl, HO-1, mdr1, Bcl-2 and caspase-3) by RQ-PCR and western blot.


RESULTS
The resistant cell line K562-RN was successfully established, with 2.01 fold resistant to nilotinib compared with K562 cell line \[the IC(50) value of nilotinib to K562 and K562-RN were (12.320 ± 1.720) µmol/L and (24.742 ± 2.310) µmol/L, respectively\]. It also had the cross resistance to adriamycin, homoharringtonine, etoposide and imatinib. Treated with different concentrations of nilotinib, cell apoptosis rate of K562-RN was significantly lower than that of K562 cells. The rate of bcr-abl gene positive cells was 92% in K562-RN by FISH assay. The mRNA and protein levels of bcr-abl, HO-1 and mdr1 expression up-regulated in K562-RN cells, while those of caspase-3 expression down-regulated, being significantly statistical difference when compared with K562 cells (P < 0.05).


CONCLUSIONS
Human leukemic cell line resistance to nilotinib, K562-RN is established successfully by gradually increasing concentrations of drug. The mechanisms of resistance in K562-RN is probably associated with increasing expression of bcr-abl, HO-1, mdr1 and decreasing expression of caspase-3 mRNA and protein levels.
Department of Hematology, Affiliated Hospital of Guiyang Medical College, Guiyang, China. jswang_yg@yahoo.com
Ji-shi
JS
Wang
Chang
C
Yang
Qin
Q
Fang
Si-xi
SX
Wei
Cheng
C
Chen
Yuan
Y
Yang
Ya-ting
YT
Wang
Xiu-ying
XY
Hu
Dan
D
Ma
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D015973:Gene Expression Regulation, Leukemic; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006801:Humans; D020014:K562 Cells / Q000187:drug effects*; D011743:Pyrimidines / Q000494:pharmacology*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011743:Pyrimidines; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D016044:Fusion Proteins, bcr-abl; C505407:CASP3 protein, human; D053148:Caspase 3; C498826:nilotinib