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. 7mGTP-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 7mGTP 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 7mGTP pull-down analysis was performed in the primary blasts from one Ph+ ALL patient and the expressions of 4EBP1, eIF4E, and eIF4G in 7m-GTP pull down or whole cell lysate were exhibited.