Intracellular Acidification Triggered by Mitochondrial-derived Hydrogen Peroxide Is an Effector Mechanism for Drug-induced Apoptosis in Tumor Cells

Abstract

We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.

Alterations of intracellular pH homeostasis in apoptosis: origins and roles

“…In this scheme, the activation of the poly(ADP-ribose) polymerase (PARP) by H 2 O 2 would play an essential role; indeed, its activation could rapidly decrease NAD þ and subsequently ATP levels, thereby affecting NHE1, and this has been supported by experiments showing that prior inhibition of PARP by 3-aminobenzamide or phenanthridinone completely blocked acidification induced by H 2 O 2 . 25 It should nevertheless be emphasized that the way pH i varies following mitochondrial production of H 2 O 2 might depend upon intracellular H 2 O 2 concentration; indeed, as observed by Clé ment et al, 27 an acidification was induced by apoptotic doses of exogenous H 2 O 2 (0.25-0.5 mM) whereas necrotic concentrations (41 mM) promoted intracellular alkalinization. Besides H 2 O 2 , other factors, such as nitric oxide, which has been shown to be proapoptotic under certain circumstances 28 and to induce cytosolic acidification, for example, in neurons, 29 or more simply a collapse of the sodium gradient, 30 might also be involved in the downregulation of NHE activity ( Figure 4); this remains as yet to be tested.…”

“…NO: nitric oxide; H 2 O 2 : hydrogen peroxide; PARP: poly(ADP-ribose) polymerase. Inspired from Matsuyama et al, 21 Hirpara et al, 25 Marches et al, 26 Vincent et al 29 and Koike et al 30 Intracellular pH in apoptosis D Lagadic-Gossmann et al cyclosporin A, used in that study as a specific inhibitor of calcineurin phosphatase, inhibited this decrease, the authors then postulated that NHE1 dephosphorylation was dependent on the action of this phosphatase. However, downregulation of NHE-controlling kinases might also be involved.…”

“…Among these mechanisms, NHE has been the most commonly studied. Thus, following, for example, chemical insult 25 or anti-IgM treatment, 26 a decrease in NHE activity has been detected. The main factors suggested to underlie this altered activity are: (1) NHE1 dephosphorylation or (2) downregulation of NHE1 via a decrease in intracellular ATP concentration (Figure 4).…”

“…Other mechanisms might also be involved such as trapping of organic bases in the mitochondrial matrix or increased exchange of OH À for P i via the mitochondrial OH À /P i as a result of ATP hydrolysis. 5,21 Such an ATP hydrolysis, possibly resulting from H 2 O 2 -triggered PARP activation, 25 ? ?…”

Reactive oxygen species-mediated regulation of the Na+–H+ exchanger 1 gene expression connects intracellular redox status with cells' sensitivity to death triggers

“…[1][2][3][4] Indeed, an increase in intracellular O 2 KÀ concentration, achieved by either its direct overproduction, 5 drug induction, 6,7 activation of the small GTPase Rac1 8 or as a result of an inhibition of the O 2 KÀ scavenger Cu/Zn superoxide dismutase (Cu/Zn SOD), 6 inhibits tumor cell apoptosis triggered by either the ligation of the CD95 receptor or anticancer drugs. In contrast, H 2 O 2 is a widely accepted trigger of cell death 9 and nontoxic levels of H 2 O 2 sensitize cells to death stimuli. 10 Earlier reports have highlighted the regulatory role of intracellular redox status on death signaling by demonstrating an effect on caspase family protease, the central executioners of apoptotic signals.…”