Study of the effects of 4-methylumbelliferone on human acute leukemia cell lines.

DÍAZ M.; PIBUEL M; COSTANTINO S.; MIHALEZ C.Y.; LOMBARDO T.; LEVERMANN M.; ALVAREZ E. M. C.; PAPADEMETRIO D. L.; HAJOS S. E.; LOMPARDÍA S. L.

Congreso; Hyaluronan Meeting 2017; 2017

ISHAS

4-methylumbelliferone (4MU) is a non-toxic coumarin derivative that has been approved in Europe and Asia to treat biliary spasms. During the last years, it has been proposed as a potential new drug for the treatment of different types of cancer, mainly solid tumors, as it has shown to inhibit cell proliferation, migration and invasion due to its capacity to inhibit hyaluronan synthesis. Despite these results, little is known about its effect on hematological malignancies. Previous work in our lab showed that 4MU inhibits cell proliferation and induces senescence in human chronic myeloid leukemia cell lines. Considering this, we hypothesize that 4MU would be a potential new drug for the treatment of leukemia. Therefore, the aim of this work was to evaluate the effects of 4MU on acute myelomonocytic leukemia cell line (U937), acute monocytic cell line (THP1) and on acute T-lymphoblastic leukemia cell line (Jurkat). Cells were exposed to 0.1, 0.5, 0.75, 1 and 2 mM 4MU during 48 h and 72 h. Cell viability and cell proliferation were evaluated by XTT and 3H-T uptake respectively. Results showed that 4MU abrogated all cells growth in a dose-dependent manner. Cell death was assessed by flow cytometry using FDA/IP stain. 4MU increased IP+ cells percentage at high doses in all cell lines, mostly at 72 h for THP1 and U937 cells. In view of this, we decided to evaluate the mechanism of cellular death at these doses. Apoptosis was evaluated by fluorescence microscopy using AO/EB and DAPI stains and by flow cytometry through the sub-G1 peak analysis. 4MU increased nuclear fragmented EB+ cells mainly at 72 h in doses higher than 0.75 mM in all cell lines which would indicate an increment in late apoptosis. These findings were in agreement with DAPI stain and sub-G1 peak analysis. Then, to explain the mechanisms involved in the inhibition of cell proliferation at lower doses and considering that SAHF+ cells were observed with DAPI stain we decided to evaluate senescence by SA-ẞ-galactosidase colorimetric assay. 4MU 0.75 mM incremented SA-B-Gal + cells percentage in U937 cell lines and this effect was reverted by hyaluronic acid. As senescence is characterized by an irreversible cell cycle arrest, we assessed this mechanism by flow cytometry with DAPI stain. 4MU seemed to arrest cell cycle in G0/G1 phase in U937 cell line. In view of these results, we conclude that 4MU inhibited cell proliferation in a dose dependent manner by the induction of apoptosis at high doses. By contrast, senescence would be the mechanism that governs cell suppression at lower doses and these effects could be explained by the inhibition of hyaluronic acid synthesis. These findings highlight the potential use of 4MU for acute leukemia treatment.