4-Methylumbelliferone induces anti-tumor effects independently of its role as a hyaluronan synthesis inhibitor on human aml cells

Abstract

Despite continuous improvement in the treatment for acute myeloid leukemia (AML), newtherapies are still needed to overcome resistance and reduce adverse effects. We previouslyproposed that 4-methylumbelliferone (4MU), known as an inhibitor of hyaluronic acid (HA)synthesis, would be an interesting drug for acute leukemia treatment. Previous results in our lab,showed that 4MU inhibited cell proliferation in a dose-dependent manner in human AML cell lines.Moreover, this drug was able to modulate CD44 expression levels, mitochondrial status and ROSproduction. However, it remains to assess whether the observed effects are explained only by theinhibition of HA synthesis. The aim of this work was to analyze if the anti-tumor activity of 4MUon U937 and THP-1 AML cell lines could be explained by HA synthesis inhibition. Resultsshowed, that both AML cell lines were able to produce significant quantities of AH as assessed byELISA. Surprisingly, at the doses tested in this work 4MU was able to partially inhibit HAsynthesis in THP-1 cells but not in U937 cells. Furthermore, the addition of exogenous HA failedto prevent the effects of 4MU on metabolic activity and cell proliferation in both cell lines,evaluated by XTT and 3H-T uptake, respectively. Moreover, in both cell lines 4MU+HA cotreatment failed to prevent the increase in mitochondrial mass and ROS production such as H2O2and O2.-, which were studied by FC using nonyl acridine orange (NAO), DCF-DA and MitoSoxstaining respectively. These results suggested that there would be 4MU mechanisms that areindependent of HA synthesis inhibition. To delve further into these mechanisms, we conducted aproteomic study in U937 cells after 4MU treatment by nano LC-MS/MS. Data analysis performedwith the free software RStudio resulted in fifteen proteins modulated by 4MU. These proteins weremainly related with cell metabolism signatures, but not directly linked to HA synthesismechanisms. In line with this, we performed a Single Cell Metabolism assay (SCENITH)a whichshowed that 4MU would modulate U937 cellular metabolism by significantly reducing itsglycolytic phenotype and increasing mitochondrial energetics dependency compared to untreatedcondition. Taken together, these results show that independently of its role as a HA synthesisinhibitor, 4MU is able to produce a metabolic switch inhibiting tumor progression in AML cells.We consider that these findings expand the knowledge of 4MU mechanisms of action and openthe possibility for its potential use in AML treatment.