Melatonin prevents chemical-induced haemopoietic cell death

Abstract

Melatonin (MEL), a methoxyindole synthesized by the pineal gland, is a powerful antioxidant in tissues as well as within cells, with a fundamental role in ameliorating homeostasis in a number of specific pathologies. It acts both as a direct radical scavenger and by stimulating production/activity of intracellular antioxidant enzymes. In this work, some chemical triggers, with different mechanisms of action, have been chosen to induce cell death in U937 hematopoietic cell line. Cells were pre-treated with 100 µM MEL and then exposed to hydrogen peroxide or staurosporine. Morphological analyses, TUNEL reaction and Orange/PI double staining have been used to recognize ultrastructural apoptotic patterns and to evaluate DNA behavior. Chemical damage and potential MEL anti-apoptotic effects were quantified by means of Tali® Image-Based Cytometer, able to monitor cell viability and apoptotic events. After trigger exposure, chromatin condensation, micronuclei formation and DNA fragmentation have been observed, all suggesting apoptotic cell death. These events underwent a statistically significant decrease in samples pre-treated with MEL. After caspase inhibition and subsequent assessment of cell viability, we demonstrated that apoptosis occurs, at least in part, through the mitochondrial pathway and that MEL interacts at this level to rescue U937 cells from death.

Figure 1.

Control (A,C–E) and MEL alone (B,F–H) treated cells analysed by means of supravital PI (A,B), SEM (C,F), TEM (D,G) and CLSM (E,H) reveal a negligible cell death and normal morphological features. Histograms (A,B) show in white and grey area, living and dead cells, respectively. Scale bars: (C) 5 μm; (D,F,G) 2 μm; (E,H) 10 μm.

Figure 2.

Cells exposed to H₂O₂ (A,C–F) or MEL+ H₂O₂ (B,G–I), analysed by means of supravital PI, SEM, TEM and CLSM, prove a cell viability decrease (A) in the presence of morphological apoptotic patterns: membrane blebbing (C), micronuclei (D,E) and necrotic cell death (D). In addition, several positive nuclei appear after TUNEL reaction (F). MEL administration is able to ameliorate cell viability (B) and to reduce apoptotic events (G–I). Scale bars: (C,D,F,G) 5 μm; (E,H) 2 μm; (I) 10 μm.

Figure 3.

Cells exposed to staurosporine (A,C–E) or MEL + staurosporine (B,F–H), analysed by means of supravital PI (A,B), SEM (C,F), TEM (D,G) and CLSM (E,H). The trigger induces an evident cell death increase (A); apoptotic features can be observed after ultrastructural analyses (C–E). MEL added before staurosporine treatment restores cell viability (B) and morphology (F,G) and prevents DNA fragmentation in situ (H). Scale bars: (C,F) 5 μm; (D) 1 μm; (E,H) 10 μm; (G) 2 μm.

Figure 4.

Fluorescent micrographs of AO and PI double-stained U937 cells. Intact green nuclei appear in control (A) and MEL alone (B) conditions suggesting a good cell viability. After H₂O₂ (C) or staurosporine (E and inset) exposure apoptotic cells appear and their decrease can be observed in MEL pre-treated samples (D,F). Scale bars: (A–F) 25 μm; (E and inset) 10 μm.

Figure 5.

Annexin V/PI dot plots in control condition (A), MEL alone (F), H₂O₂ (B) and staurosporine (G)-treated cells. In (C) and (H), dot plots are relative to MEL administration before H₂O₂ and staurosporine respectively. Dot plots of caspase-9, −3 inhibitor pre-treatment before apoptosis induction appear in (D–E) and (I–L).

Figure 6.

Percentage study of viable, apoptotic and necrotic cells based on Annexin V analyses. All data are expressed as mean values of percentages for each group ± DS. MEL significantly (yellow in the table) restore cell viability (A,D) and prevents apoptotic (B,E) and necrotic cell death (C,F), induced by H₂O₂ (A,B,C) or staurosporine (D,E,F) with caspase-9 involvement.