The Anti-Leukemia Role and Mechanism of Ribavirin in Ph+ Leukemia

Purposes: The eukaryotic translation initiation factor 4E (eIF4E), a main composition factor of eIF4F translation initiation complex, influences the growth of tumor through modulating translation functions of eIF4F initiation complex and cap depending protein expression. Ribavirin (a broad-spectrum antiviral drug) is a physical mimic of the m⁷G cap depending protein, it plays antitumor roles by supressing eIF4E-controlled translation and interfering the synthesis of oncoproteins including a number of cell growth-related, proliferation-related and apoptosis-related proteins, such as anti-apoptotic factor Mcl-1, the cell cycle regulators cyclin D1 and D3, pro-vascular endothelial growth factor VEGF, and oncoprotein c-Myc. Here, we studied the anti-leukemia roles and mechanism of ribavirin alone and in combination with imatinib in Ph+ leukemia.

Methods: The Ph+ acute lymphoblastic leukemia cell line SUP-B15, chronic myeloid leukemia cell line K562 and primary Ph+ leukemia blasts were treated with ribavirin alone or in combination with imatinib. Cell proliferation was evaluated using the MTT assay. The phosphorylation expression of the key proteins of mTOR/eIF4E and ERK/Mnk1/eIF4E signaling pathway, and expression of eIF4F translation substrates Mcl-1 (an apoptosis-related protein) were assessed by western blot analysis. The assembly of eIF4F translation initiation complex was examined with 7-Methyl-guanosine cap affinity assay. Apoptosis was detected by flow cytometry with Annexin-V and PI double staining.

Results: 1. The MTT assay showed that ribavirin or imatinib alone had anti-proliferation effect in SUP-B15 and K562 cell lines. The IC₅₀ values of ribavirin against to SUP-B15 and K562 were 65.18 µmol/L and 78.30 µmol/L, the IC₅₀ values of imatinib against to SUP-B15 and K562 were 1.44 µmol/L and 0.18 µmol/L respectively. Combined with 10 µmol/L of ribavirin, the IC₅₀ values of imatinib decreased to 0.075umol/L and 0.077 µmol/L in SUP-B15 and K562 cell lines, the combination index (CI) was 0.206 and 0.556, which indicated the combination of imatinib and ribavirin had synergistic anti-leukemia effect. 2. Ribavirin down-regulated the phosphorylation levels of Akt, mTOR, 4EBP1, eIF4E proteins in the mTOR/eIF4E signaling pathway, and MEK, ERK, Mnk1, eIF4E proteins in ERK/Mnk1/eIF4E signaling pathway, resulted in reduction of their translation substrates Mcl-1 in both SUP-B15 and K562 cell lines. The combination of ribavirin with imatinib down-regulated the phosphorylation level of these proteins more significantly than ribavirin alone. 3. MEK inhibitor U0126 or Mnk1 inhibitor CGP57380 alone down-regulated the phosphorylation of ERK and eIF4E proteins, the combination of ribavirin with U0126 or CGP57380 further reduced the phosphorylation levels of ERK and eIF4E. 4. 7-Methyl-guanosine cap affinity assay showed that ribavirin could increase the combination of eIF4E and 4EBP1 while decrease the combination of eIF4E and eIF4G, therefore, inhibit the assembly of eIF4F translation initiation complex. When combined with imatinib, inhibition effect on assembly of eIF4F translation initiation complex was more obvious. 5. Both ribavirin and imatinib induced apoptosis analysized by flow cytometry in SUP-B15 and K562 cell lines, while combination of the two drugs exerted a more significant effect. 6. MTT assay showed that ribavirin at concentration that was lower than the clinical dosage enhanced the inhibition of imatinib to Ph + ALL primary blasts, however, the same concentrations of ribavirin did not significantly effect on the anti-proliferation of imatinib to the primary blasts of chronic myeloid leukemia. Western blot assay demonstrated that ribavirin alone significantly reduced the protein phosphorylation of mTOR/eIF4E, ERK/Mnk1/eIF4E signaling pathways and Mcl-1 in Ph+ ALL primary blasts, the combination of ribavirin and imatinib exhibited more significant effect on the signaling pathways, as described in the cell lines. However, the anti-proliferative role of ribavirin in the other types of acute leukemia primary blasts was not obvious, which indicated that the anti-leukemia effect of ribavirin exist the cell lineages differences.

Conclusions: Our studies demonstrated that ribavirin combined with imatinib exerted a strong synergistic anti-leukemia role in Ph+ ALL.