Study on the Effects of Rapamycin Reverse AMN107 resistance on k562/AO2 Cells

Rapamycin(RAPA) is the inhibitor of m-TOR. The activation of m-TOR results in changes in multiple cellular processes, eg, catabolism, anabolism, proliferation, growth and apoptosis. Rapamycin, which directly inhibits the Akt downstream molecule mammalian target of rapamycin (mTOR), effectively inhibits survival and proliferation in the development of BCR- ABL–induced chronic myelogenous leukemia(CML) from PTENfl/fl. Nilotinib (AMN107), a BCR-ABL tyrosine kinase inhibitor, was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. We aim to introduce the CML cell line (k562/A02) resistant to AMN107 and investigate the effect of RAPA reverse AMN107 resistance on k562/A02 cell line.

K562/A02 be added in the culture medium final concentration 1μmol/ L of AMN107 drugs a week before the withdrawal. For all experiments, cells were on the logarithmic phase.K562/A02 cells were treated with 400nmol/L rapamycin(400nmol/L RAPA group),100nmol/L rapamycin(100nmol/L RAPA group), 20μmol/L nilotinib(20μmol/L AMN107 group), 400nmol/L rapamycin +20μmol/L nilotinib (400nmol/L RAPA+20μmol/L AMN107 group), 100nmol/L rapamycin +20μmol/L nilotinib (100nmol/L RAPA+20μmol/L AMN107 group). Tetrazolium—based colorimetric assay (MTT) was used to detect the inhibiton effect of cell growth. The expression of Survivin, m-TOR, Bcl-2, and Caspase-3 were detected by western blot; Cell apoptosis was inspected by Annexin V/PI double staining method; The expression of Survivin, m-TOR, Bcl-2 and Caspase-3 in mRNA level was determined using reverse transcriptase—polymerase chain reaction (RT-PCR). BCR-ABL gene was detected by Real Time-PCR when K562/AO2 cells in different groups were treated by drugs for 48h.

K562/A02 cells were successfully introduced to resistant toAMN107. Combination of 400 and 100 nmol/L RAPA with AMN107 could significantly enhance the sensitivity of k562/A02 to AMN107. The reverse factor was 1.97 and 2.73 fold respectively. RT-PCR and Western blot method indicated that the expression of Survivin and Bcl-2 was increased by RAPA+AMN107 group, but the MDR1 and Caspase-3 was decreased. The expression of BCR-ABL fusion gene in100 nmol/L RAPA group was higher than the blank groups, the copy number is 5.6×10⁻². But the copy number in 400nmol/L RAPA +20μmol/L AMN107 group is1.64×10⁻² that is lowest. Annexin V/PI showed that the apoptosis rates were (8.21±0.05)%, (32.07±0.03)%, (28.88±0.11)%, (16.57±0.05)%, (38.49±0.04)%, and(42.31±0.07)% respectively in control group, AMN107 group, 400nmol/L RAPA group, 100nmol/L RAPA group, 100nmol/L RAPA +20μmol/L AMN107 group, and 400nmol/L RAPA +20μmol/L AMN107 group.

We successfully introduced K562/A02 resistant toAMN107. Moreover, RAPA can enhance the sensitivity of AMN107 resistant 562/A02 cells. Combined of RAPA and AMN107 has significant synergistic growth inhibiting effect and apoptosis inducing effect on AMN107-resistant K562/A02 cells. The mechanism may be related at down-regulation of expression MDR1 and up-regulation of expression Survivin.

No relevant conflicts of interest to declare.