Mitochondrial dysfunction and effect of antiglycolytic bromopyruvic acid in GL15 glioblastoma cells

Abstract

Most cancer cells, including GL15 glioblastoma cells, rely on glycolysis for energy supply. The effect of antiglycolytic bromopyruvate on respiratory parameters and viability of GL15 cells was investigated. Bromopyruvate caused Δψ(m) and MTT collapse, ATP decrease, and cell viability loss without involving apoptotic or necrotic pathways. The autophagy marker LC3-II was increased. Δψ(m) decrease was accompanied by reactive oxygen species (ROS) increase and cytochrome c (cyt c) disappearance, suggesting a link between free radical generation and intramitochondrial cyt c degradation. Indeed, the free radical inducer menadione caused a decrease in cyt c that was reversed by N-acetylcysteine. Cyt c is tightly bound to the inner mitochondrial membrane in GL15 cells, which may confer protein peroxidase activity, resulting in auto-oxidation and protein targeting to degradation in the presence of ROS. This process is directed towards impairment of the apoptotic cyt c cascade, although cells are committed to die.

Regulation of Death Induction and Chemosensitizing Action of 3-Bromopyruvate in Myeloid Leukemia Cells: Energy Depletion, Oxidative Stress, and Protein Kinase Activity Modulation

“…Cell Death Induction by 3-Bromopyruvate in Leukemia apoptosis or necrosis of glioblastoma cells, but rather it activates autophagy in association with ATP depletion and a decrease in the DCm (Macchioni et al, 2011). In our experiments, 20-30 mM 3-BrP decreased ATP and induced mIMP, and preliminary observations also point to a decrease in p62/SQSTM1 levels (data not shown) that is a characteristic of autophagy.…”

Bromopyruvate mediates autophagy and cardiolipin degradation to monolyso-cardiolipin in GL15 glioblastoma cells

“…Previously, we observed a collapse of both MTT and mitochondrial membrane potential in the 70-80 μM range, accompanied by massive degradation of cyt c and Bax within mitochondria. ATP levels did not change up to 60 μM bromopyruvate and reached about 7% of the control in 80 μM bromopyruvate-treated cells (Macchioni et al 2011a). Cyt c degradation following the treatment of GL15 cells with 80-100 μM bromopyruvate was confirmed in the experiments reported in Fig.…”

“…Moreover, since the inhibition of glycogen synthase kinase-3β (GSK3β) by Akt is involved in the association of hexokinase II to VDAC in the mitochondria (Pastorino et al 2005;Pastorino and Hoek 2008;Pedersen 2007Pedersen , 2008Mathupala et al 2006), we also evaluated the possibility that bromopyruvate activates GSK3β, constitutively inhibited in GL15 cells (Macchioni et al 2011a). After 30 min of treatment with 80 μM bromopyruvate, the activity of Akt was significantly inhibited, as demonstrated by the decrease of pAkt.…”

“…Bromopyruvate is a potent antiglycolytic, able to induce severe ATP reduction and viability loss in many cancer cell lines, including GL15 glioblastoma cells (Macchioni et al 2011a). A variety of biochemical mechanisms by which this antiglycolytic agent leads cells to die have been described in different cell types (Xu et al 2005;Kim et al 2008;Yun et al 2009;Pereira da Silva et al 2009;Qin et al 2010).…”

“…A peculiar feature of GL15 cells is the tight binding of cyt c to the inner mitochondrial membrane that enables cells to escape the cyt c-triggered apoptotic pathway. In a recent paper we have shown that GL15 cell starvation produced by bromopyruvate forces these cells to switch to respiration for their energy supply (Macchioni et al 2011a). Thus, cyt c is targeted for degradation, possibly due to acquired peroxidase activity (Macchioni et al 2011b).…”