Effects and Mechanisms of FTI-277 Inhibiting Proliferation of Myeloid Leukemia Cells and Enhancing Apoptosis Induced by As₂O₃.

Objective:To investigate the effects and mechanisms of farnesyltransferase inhibitor FTI-277 inhibiting proliferation of myeloid leukemia cells and enhancing apoptosis induced by arsenic trioxide(As₂O₃).

Methods:HL-60, K562 cells and BMMNCs from 31 AML patients at diagnosis, 21 CML patients at diagnosis were examined. Cell viability and cell proliferation were detected by trypan blue exclusion assay and MTT assay, respectively. Cell apoptosis were determined by light microscope, DNA agrose gel electrophoresis and annexinV-FITC/PI double-staining. Cell cycle was analyzed by flow cytometry. The expression of phospho-ERK1/2 (phospho-P44/42MAPK) and ERK2 (P42MAPK) were detected by flow cytometry and Western blot. The expression of Bcl-2 and active caspapse-3 were measured by flow cytometry.

Results: FTI-277 inhibited proliferation of HL-60, K562 cells and BMMNCs from AML, CML patients at diagnosis in a time-dose dependent manner (P<0.05). However, this inhibitor had little effect on proliferation of normal cells. Treatment of myeloid leukemia cells with FTI-277 alone couldn’t induce apoptosis (P>0.05). FTI-277 and As₂O₃ in combination significantly induced apoptosis of myeloid leukemia cells compared with either FTI-277 or As₂O₃(P<0.05). Furthermore, the percentage of apoptosis increased with increasing concentration of FTI-277 in the combination group (P<0.05). In addition, the apoptosis ladder and the morphological features of apoptosis were readily observed in the combination group. Incubation with FTI-277 led to an increase of myeloid leukemia cells in G₂/M (P<0.05), whereas the sub-G₁ fraction remained unchanged(P>0.05). Incubation of myeloid leukemia cells with FTI-277 decreased the expression of phospho-ERK1/2(phospho-P44/42MAPK)(P<0.05), whereas the expression of ERK2 (P42MAPK) in myeloid leukemia cells and the expression of phospho-ERK1/2 in normal cells were unaffected by FTI-277(P>0.05). FTI-277 had no effect on Bcl-2 expression in HL-60 and K562 cells(P>0.05). As₂O₃ decreased Bcl-2 expression in HL-60 cells(P<0.05), but didn’t affect Bcl-2 expression in K562 cells(P>0.05). As₂O₃ alone or FTI-277 and As₂O₃ in combination significantly increased active caspapse-3 expression in HL-60 and K562 cells(P<0.05), the combination group being more significantly increased compared with As₂O₃ alone (P<0.05).

Conclusion:FTI-277 exerts proliferation inhibition on myeloid leukemia cells by inhibiting constitutive ERK/MAPK activation and resulting in a G₂/M block. FTI-277 doesn’t inhibit proliferation of normal cells with low steady-state levels of ERK/MAPK activity. Combination of FTI-277 and As₂O₃ results in the activities of caspases and the synergistic induction of apoptosis in HL-60 cells by simultaneous targeting of the ERK/MAPK and Bcl-2 pathways. As₂O₃ interferes with the BCR/ABL protein signal transduction, whereas FTI-277 lowers the apoptotic threshold, thereby increasing the activation of caspase-3 and generating the synergistic induction of apoptosis in K562 cells.