Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells

Abstract

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.

Figure 1.. Thiamine antagonists amprolium, oxythiamine and pyrithiamine affect viability of PC-12 cells in time- and dose-dependent manner.

Viability of neuronally differentiated PC-12 cells was measured using WST-1 reagent and Multiscan RC (Thermolabsystems) upon treatment with 10, 100 or 1000 μM oxythiamine, pyrithiamine or amprolium in serum-reduced media for 48, 72 and 96 h. The viability rates represent the means ± S.E.M. for non-treated cells and cells treated with tested thiamine antagonists in which each value was determined from assays performed in quadruplicate. Further experimental details are given in Materials and Methods.

Figure 2.. Thiamine antagonists induce binding of annexin V to cell surface membrane and translocation of active caspase 3 to the nucleus.

Neuronally differentiated PC-12 cells were treated for 72 h with 100 μM of thiamine antagonists — amprolium (AM), pyrithiamine (PT) or oxythiamine (OT). Non-treated cells were examined as control (NT). Cells treated with 4 μM staurosporine (ST) for 24 h were used as positive control. (A) Annexin V binding (magnification 10×) and (B) translocation of cleaved caspase 3 to the nucleus (magnification 40×) were analyzed by confocal laser scanning immunofluorescence microscopy. SYTOX Green — nucleic acid stain; annexin V (annexin V-alexa fluor 568) — fluorescent conjugate which detects apoptotic cells; propidium iodide — fluorescent DNA stain; cleaved caspase 3 was detected by goat anti-rat antibody conjugated to fluorescein. Staining of cell nuclei allowed counting of cells. Each experiment was performed in triplicate.

Figure 3.. Differential effect of pyrithiamine, oxythiamine and amprolium on cleavage of caspase 3.

PC-12 cells differentiated with 100 μM NGF were treated for 72 h with 100 μM amprolium (AM), oxythiamine (OT) or pyrithiamine (PT). Non-treated cells were examined as control (NT). Western blots (A) of caspase 3 and cleaved caspase 3 after 72 h treatment were stained by appropriate antibodies as described in Materials and Methods. Equal protein loading (A) shown by Ponceau S staining of nitrocellulose membrane before application of antibodies. Data on the graph (in B) indicate ratios between cleaved caspase 3 and caspase 3 ± S.E.M. (n = 3) in response to treatment with antagonists.

Figure 4.. Activation of aif gene expression and DNA fragmentation by thiamine antagonists.

(A) Neuronally differentiated PC-12 cells were treated for 72 h with 100 μM amprolium (AM), oxythiamine (OT) or pyrithiamine (PT). Non-treated cells were examined as control (NT). Total RNA extracted, reverse transcribed and PCR was performed to quantitate the expression of aif mRNA, presented on the gel (a). Values (in b) are mean ± S.E.M. (n = 3) for aif gene expression were quantified using Versa Doc^® and QuantityOne^® software, normalized to gapdh mRNA levels and expressed as the percentage of increase over NT. (B) TUNEL fluorescence microscopy for evaluation of the apoptotic events was conducted on neuronally differentiated PC-12 cells (a) after treatment with amprolium (AM), oxythiamine (OT), or pyrithiamine (PT). Non-treated cells were examined as control (NT). Propidium iodide (PI) was used to stain all cells. Graph (in b) represents percentage of TUNEL-positive cells relative to total PI nuclei as evaluated by confocal laser scanning fluorescence microscopy at magnification 40×. Data on the graph indicate percentage of TUNEL-positive cells ± S.E.M. (n = 3) in response to treatment with antagonists.