Ribavirin Inhibits the Activity of mTOR/eIF4E, ERK/Mnk1/eIF4E Signaling Pathway and Synergizes with Tyrosine Kinase Inhibitor Imatinib to Impair Bcr-Abl Mediated Proliferation and Apoptosis in Ph+ Leukemia

Abstract

The eukaryotic translation initiation factor 4E (eIF4E), which is the main composition factor of eIF4F translation initiation complex, influences the growth of tumor through modulating cap-dependent protein translation. Previous studies reported that ribavirin could suppress eIF4E-controlled translation and reduce the synthesis of onco-proteins. Here, we investigated the anti-leukemic effects of ribavirin alone or in combination with tyrosine kinase inhibitor imatinib in Philadelphia chromosome positive (Ph+) leukemia cell lines SUP-B15 (Ph+ acute lymphoblastic leukemia cell line, Ph+ ALL) and K562 (chronic myelogenous leukemia cell line, CML). Our results showed that ribavirin had anti-proliferation effect; it down-regulated the phosphorylation levels of Akt, mTOR, 4EBP1, and eIF4E proteins in the mTOR/eIF4E signaling pathway, and MEK, ERK, Mnk1 and eIF4E proteins in ERK/Mnk1/eIF4E signaling pathway; reduced the expression of Mcl-1 (a translation substrates of eIF4F translation initiation complex) at protein synthesis level not mRNA transcriptional level; and induced cell apoptosis in both SUP-B15 and K562. 7-Methyl-guanosine cap affinity assay further demonstrated that ribavirin remarkably increased the eIF4E binding to 4EBP1 and decreased the combination of eIF4E with eIF4G, consequently resulting in a major inhibition of eIF4F complex assembly. The combination of ribavirin with imatinib enhanced antileukemic effects mentioned above, indicating that two drugs have synergistic anti-leukemic effect. Consistent with the cell lines, similar results were observed in Ph+ acute lymphoblastic primary leukemic blasts; however, the anti-proliferative role of ribavirin in other types of acute primary leukemic blasts was not obvious, which indicated that the anti-leukemic effect of ribavirin was different in cell lineages.

Fig 1. Anti-leukemic effect of ribavirin alone or combined with imatinib in Ph+ leukemia cell lines.

A. SUP-B15 cells were treated with 30, 60, 90, and 120μM ribavirin for 24, 48, 72, and 96h. Cell proliferation was assessed by the MTT assay and cell survival rates were presented. B. SUP-B15 cells were treated with a series of concentrations of imatinib (0.001–20μM) alone or combined with 10μM or 20μM ribavirin for 72h, the IC₅₀ values of imatinib were shown. C. K562 cells were treated with 1μM imatinib or combined with 10μM ribavirin for 72h, the IC₅₀ values of imatinib were shown. The data represent means±SD of three experiments. * represents p<0.05, ** represents p<0.01.

Fig 2. Ribavirin inhibited the signaling pathway of mTOR/eIF4E in SUP-B15.

A. The SUP-B15 cells were treated with different concentrations of ribavirin for 48h, and the proteins in mTOR/eIF4E signaling pathway and Mcl-1 were detected by western bolt analysis. B. The SUP-B15 cells were incubated in 60μM ribavirin for 24, 48, 72h, and the mTOR/eIF4E pathway expression was detected by western bolt analysis. C. The expression of mTOR/eIF4E pathway and Mcl-1 in SUP-B15 cells after treated with ribavirin (30μM), imatinib (10μM) alone, and 30μM ribavirin plus 10μM imatinib for 48h.

Fig 3. Ribavirin inhibited the MEK/ERK/Mnk1/eIF4E signaling pathway in SUP-B15.

A. The expression of MEK/ERK/Mnk1/eIF4E pathway and Mcl-1 in SUP-B15 cells was detected after treated with ribavirin (30μM), imatinib (10μM) alone, and 30μM ribavirin plus10μM imatinib for 48h. B. Indicated concentrates of U0126 (MEK1/2 inhibitor) or CGP57380 (Mnk1 inhibitor) alone treated SUP-B15 cells and the MEK/ERK/Mnk1/ eIF4E signaling pathway expression were analyzed. C. The SUP-B15 cells were treated with 60μM ribavirin alone or plus 10μM U0126 (MEK1/2 inhibitor) for 6, 24, 48, and 72h, and the expression of MEK/ERK/Mnk1/eIF4E pathway and Mcl-1 was detected by western bolt analysis.

Fig 4. eIF4F complex formation is inhibited by ribavirin in SUP-B15 and K562 cell lines.

A. The SUP-15 cells were treated with ribavirin, imatinib alone, or ribavirin plus imatinib for 48h. 1×10⁷ cells were lysed in 500μl of RIPA lysis buffer. 7^mGTP-Sepharose beads were added into part of supernatants, incubated for 1–2h, and solubilized in 50μl of SDS-PAGE sample buffer. The buffer was boiled for 7 min and was immunoblotted with the primary antibodies against eIF4G, eIF4E, and 4EBP1. The retained supernatants were analyzed by western blot with primary antibodies against eIF4G, eIF4E, 4E-BP1, and GAPDH, as control. B. The K562 cells were conducted by 7^mGTP pull-down analysis, like SUP-B15 cells.

Fig 5. The effect of ribavirin on apoptosis rates in SUP-B15 and K562 cell lines.

A. SUP-B15 cells were cultured with ribavirin at the 60μM for 24, 48, and 72h, and apoptosis rates were assessed by flow cytometry after staining of the cells with annexin V-FITC and PI, PBS was used as a negative control. B. The apoptosis rates of SUP-B15 cells when cultured with ribavirin at 30 and 60μM for 48h. C. The apoptosis rates of SUP-B15 cells when cultured with1μM imatinib, 30μM ribavirin alone, or combined for 48h. D. K562 cells were cultured with ribavirin at the indicated doses for 24, 48 and 72h, and apoptosis rates were assessed by flow cytometry after staining of the cells with annexin V-FITC and PI. E. The apoptosis rates of K562 cells when treated with 100μM ribavirin alone or combined with 0.2μM imatinib for 24 or 48h. F. K562 cells were treated with 100μM ribavirin, 0.2μM imatinib alone or combined for 48h, and apoptosis rates were assessed by flow cytometry after staining of the cells with annexin V-FITC and PI. * represents p<0.05, ** represents p<0.01, *** represents p<0.001.

Fig 6. Ribavirin suppressed the cell growth and activation of mTOR/eIF4E, ERK/Mnk1/eIF4E signaling pathways in Ph+ ALL primary blasts.

A. The primary leukemia bone marrow and peripheral blood samples were collected and treated at a series of concentrations of imatinib alone or combined with 100μM ribavirin. The MTT assay was performed and IC₅₀ values of imatinb alone or in combination were calculated by SPSS17.0. The differences of IC₅₀ values between imatinib alone and combination with ribavirin in four groups (Ph+ ALL, Ph–ALL, AML, CML) were analyzed by two paired sample t-tests, * represents p<0.05. B. The primary leukemia blasts were treated with 10μM imatinib, 500μM ribavirin, or combination, and the whole cell lysate was analyzed by western blot with the indicated antibodies, PBS was used as a negative control. The expression of mTOR/eIF4E signaling pathway in one of Ph+ ALL primary blasts was shown. C. The expression of ERK/Mnk1/eIF4E signaling pathways in the primary blasts from one Ph+ ALL patient. D. The 7^mGTP pull-down analysis was performed in the primary blasts from one Ph+ ALL patient and the expressions of 4EBP1, eIF4E, and eIF4G in 7^m-GTP pull down or whole cell lysate were exhibited.