The Effect of Tyrosine Kinase Inhibitor Resistant to CML Cells Which Was Transfected with HO-1 Lentiviral Vector

Heme oxygenase-1(HO-1) is well characterized survival factor that inhibits apoptosis in tumor cells. Chronic myeloid leukemia (CML) cells constitutively express HO-1 in chronic phase, but express highly in accelerated phase (AP) and blast phase (BP). However, resistance against tyrosine kinase inhibitor (TKI) can occur during therapy with TKI, particularly in AP and BP. This study was designed to confirm the relationship between HO-1 and drug resistant in CML, and seek ways to reversal of TKI resistant.

HO-1 gene was cloned from human liver by RT-PCR. And the lentiviral vector pLenti6-GFP-HO-1 was constructed. K562 cells 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 cells was cultivated with dasatinib in gene-transfected, empty vector transfected and untransfected groups. Expression of HO-1 mRNA was demonstrable by RT-PCR and the HO-1 protein by Western blotting. Gene mutation was detected by high performance liquid chromatography (HPLC) analysis. HO-1, multi-drug resistance gene(MDR1), lung reristance-related protein (LRP), glutathione S-transferase-π (GST-π), topoisomerase-I (Topo-I) in mRNA and protein level were deteceted by RT-PCR and Western blot. Apoptosis and cell cycle were determined by flow cytomertry analysis after treated with dasatinib. The activity of HO-1 against dasatinib for CML cells in vivo was evaluated by using the nude mouse xenograft model. The virus was injected into mouse through tail vein.

HO-1 gene was cloned successfully from human liver. The sequences were confirmed by restriction enzyme digestion analysis and sequencing. The virus was packaged in 293 cells and titer of virus was tested by Real-Time PCR, 1.02×10⁹v.p./mL. Following transfer the lentiviral vector into K562, 72 hours after transfection, it showed that HO-1 was expressed highest by fluorescence micrope. The expression of HO-1 was detected by RT-PCR and Western blotting. HPLC results showed that there were not gene mutation after transfection□GRT-PCR and Western blot showed that the expression of MDR1 was significantly higher than transfection (P<0.01), also LRP and Topo-I levels were higher than transfection (P<0.05), there was no obvious changes of GST-π after transfection. At 48 hours after treatment with 10umol/L dasatinib by flow cytometry, the survival rate in transfected group was lowest which is 7.2±0.9% (P<0.05). And in empty vector transfected and untransfected group the survival rate was 38.2±1.6% and 39.3±1.7%. Cell cycle results showed that the cell population in G₀/G₁ and S phases decreased significantly in empty vector transfected and untransfected group, cell cycle arrest at G₂/M checkpoint when treated by dasatinib. Nude mouse xenegraft models bearing carcinoma were established successfully. HO-1 in nude mouse xenegraft models was associated with protection of tumor cell against dasatinib.

Up-regulating of HO-1 in chronic myeloid leukemia cell is associated with cell growth and anti-apoptotic effect. At the same time, there was a close relationship between HO-1 and resistance gene. HO-1 expression was accompanied by the expression of resistance genes. Based on these data, it seems desirable to explore the value of the HO-1-targeting drug in clinical trials in patients with leukemia and solid tumors.

No relevant conflicts of interest to declare.