In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Abstract

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

Figure 1. Effects of imatinib mesylate on the growth of colony-forming unit-granulocyte/macrophage (CFU-GM) obtained from mouse bone marrow. Quantification of colonies is reported as percent growth in relation to the control group (100% growth). Data are reported as the mean ± SEM obtained from at least 3 independent experiments performed in triplicate under the same conditions. *P < 0.05 compared to control (one-way ANOVA followed by the Tukey test). The same letters indicate no statistically significant difference between groups (P > 0.05).

Figure 2. Morphology of colony-forming unit-granulocyte/macrophage colonies in cultures treated with imatinib mesylate (IM). Numerous and large colonies were initially present (A, control) and their size decreased between (B) 1.25 and (C) 20 µM doses of IM. At 25 µM (D), colonies were rare and only small clusters were present. Cells were stained with May-Grünwald and Giemsa.

Figure 3. Morphology of the cells in the bone marrow of mice after cultivation with imatinib mesylate (IM). A, Control cells stained with May-Grünwald and Giemsa; B, cells treated with 5 µM IM; C, cells treated with 10 µM IM stained with May-Grünwald and Giemsa. The black arrow indicates cells with fibroblastoid features and the white arrow indicates round cells.

Figure 4. Effects of imatinib mesylate on bone marrow stromal cells from mice. The absorbance of the control group represents 100% cell viability. Data are reported as the mean ± SEM obtained from at least 3 independent experiments performed in triplicate under the same conditions. *P < 0.05 compared to control group (one-way ANOVA followed by the Tukey test). The same letters indicate no statistically significant difference between groups (P > 0.05).

Figure 5. Cytomorphological alterations of bone marrow stromal cells from mice treated with imatinib mesylate (IM). A, Control; B, 10 µM IM; C, 20 µM IM. Viable cells showed green fluorescent staining (acrydine orange) and apoptotic cells exhibited orange staining (ethidium bromide; white arrow).

Figure 6. Identification of adipocytes by staining with oil red O. Bone marrow stromal cells of mice were cultured in the presence of imatinib mesylate for 14 days. Observation of the cultures by light microscopy shows lipid refractile vacuoles (black arrow) (A) in the control group with no staining. Cells stained with oil red O reveal reddish lipid vacuoles (black arrow) of different sizes (B).

Figure 7. Assessment of cell proliferation by BrdU incorporation into bone marrow stromal cells from mice treated with imatinib mesylate. The cells were cultured for 14 days, quantified according to BrdU incorporation into the nucleus and compared to the control group (100%). Data are reported as the mean ± SEM obtained from at least 3 independent experiments performed in triplicate under the same conditions. *P < 0.05 compared to control (one-way ANOVA followed by the Tukey test). The same letters indicate no statistically significant difference between groups (P > 0.05).

Figure 8. Analysis of BrdU incorporation and immunofluorescence for alpha-smooth muscle actin (α-SMA) in cells cultured with imatinib mesylate for 14 days. In red, nuclei of cells that incorporated BrdU; in blue, nuclei stained with DAPI; in green, α-SMA-positive cells. Control group cells (A, B, C), cells treated with 5 µM (D, E, F), and 15 µM (G, H, I) imatinib mesylate. No cells treated with 10 µM (J) or 20 µM (L) incorporated BrdU. K, α-SMA-positive cells treated with 10 µM. C, F, I, Representation of the overlapping BrdU, DAPI, and α-SMA images.

Figure 9. Quantification of alpha-smooth muscle actin (α-SMA)-positive cells. Bone marrow stromal cells of mice were cultured for 14 days and subjected to immunocytochemical reaction to verify α-SMA expression. Data are reported as percentage in relation to the control group (100%).