The Effect of HO-1 on AMN107 In Promoting K562/A02 Apoptosis.
Authors: Wang Jishi , Chai Baisheng , Chen Cheng , Fang Qin 
Published: October 2019

Abstract
Abstract 1231

Aim:
Heme oxygenase-1(HO-1), also known as heat shock protein 32(Hsp32), has recently been identified as a stress-related survival molecule that acts anti-apoptotic and cytoprotective in inflammatory reactions. In the present study, we provide evidence that HO-1 is effective on AMN107(Nilotinib) in promoting K562/A02 apoptosis which is induced by STI571(Imatinib),and investigate AMN107 on the mechanism of resistance to STI571.

Method:
HO-1 gene was cloned from rat liver by RT-PCR. And the retrovirus vector pQCXIP-EGFP-C1 was constructed. K562/A02 cell which was expressed HO-1 highly was seemed as gene-transfected group. At the same time, we set the empty vector transfected group and untransfected group. K562/A02 cell was cultivated with AMN107 in gene-transfected, empty vector transfected and untransfected groups. Expression of HO-1 mRNA was demonstrable by RT-PCR, Real-time PCR, and the HO-1 protein by Western blotting. Constant MTT assay and cell number count were used to detect the proliferation of leukemia cells after treatment with AMN107. Apoptosis was determined by morphological observation and flow cytomertry analysis after AnnexinV/PI double labeling. Also we detected the intracellular drug concentration by HPLC after treatment with the same concentration of AMN107.

Result:
HO-1 gene was cloned from rat liver successfully. The sequences were confirmed by restriction enzyme digestion analysis and sequencing. The virus was packaged in 293T cells and titer of virus was tested by Real-Time PCR, 8.09×1010v.p./mL. Following transfer the retrovirus vector into K562/A02, 72 hours after transfection, it showed that HO-1 was expressed highest by fluorescence micrope. High expression of HO-1 was detected by RT-PCR, Real-Time PCR and Western blotting. After 10umol/L AMN107 treated, the expression of HO-1 was clearly lower in empty vector transfected group and untransfected group (p <0.05), but in transfected group there was no obvious change. MTT assay showed that after AMN107 treated, IC50 value in transfected group was 5.62174 mmol/L, significantly higher than empty vector transfected and untransfected group(P<0.05). Cell number count showed that after AMN107 treated the activity of cells were decreased sharply in empty vector transfected and untransfected group, but no significant change in transfected group. At 48 hours after treatment with 10umol/L AMN107 by flow cytometry, the survival rate in transfected group was lowest which is 8.3±1.2%(P>0.05). And in empty vector transfected and untransfected group the survival rate was 35.3±1.6% and 37.5±1.3%(P<0.01). HPLC analysis showed that intracellular drug concentration was lowest in transfected group which treatment with the same concentration of AMN107.

Conclusion:
STI571 resistance is a major cause of STI571 treatment failure in chronic myeloid leukemia(CML) patients. Our data showed that HO-1/Hsp32 is a novel survival factor and interesting target in K562/A02. In addition, it could predict the risk of resistance to STI571 therapy. Down-regulating of HO-1/Hsp32 in chronic myeloid leukemia cell is associated with reduced tumor cell growth and induction of apoptosis. Based on these data, it seems desirable to explore the value of the Hsp32-targeting drug in clinical trials in patients with leukemia and solid tumors.

Disclosures:
No relevant conflicts of interest to declare.