Treatment with 0.2 mM hydrogen peroxide (H₂O₂) or with 0.5 mM cisplatin caused caspase-9 and caspase-3 activation and death by apoptosis in U-937 human promonocytic cells. However, treatment with 2 mM H₂O₂, or incubation with the glutathione suppressor DL-buthionine-(S,R)-sulfoximine (BSO) prior to treatment with cisplatin, suppressed caspase activation and changed the mode of death to necrosis. Treatment with 2 mM H₂O₂ caused a great decrease in the intracellular ATP level, which was partially prevented by 3-aminobenzamide (3-ABA). Correspondingly, 3-ABA restored the activation of caspases and the execution of apoptosis. By contrast, BSO plus cisplatin did not decrease the ATP levels, and the generation of necrosis by this treatment was not affected by 3-ABA. On the other hand, while all apoptosis-inducing treatments and treatment with 2 mM H₂O₂ caused Bax translocation from the cytosol to mitochondria as well as cytochrome c release from mitochondria to the cytosol, treatment with BSO plus cisplatin did not. Treatment with cisplatin alone caused Bid cleavage, while BSO plus cisplatin as well as 0.2 and 2 mM H₂O₂ did not. Bcl-2 overexpression reduced the generation of necrosis by H₂O₂, but not by BSO plus cisplatin. These results indicate the existence of different apoptosis/necrosis regulatory mechanisms in promonocytic cells subjected to different forms of oxidative stress.

Arsenic trioxide (ATO, Trisenox) is an important antileukemic drug, but its efficacy is frequently low when used as a single agent. Here, we demonstrate that the apoptotic action of ATO is greatly increased when combined with subcytotoxic curcumin concentrations in U937 and HL60 human acute myeloid leukemia cells, and with lower efficacy in K562 chronic myelogenous leukemia cells. Curcumin exerts similar cooperative effect with the mitochondria-targeting drug lonidamine, whereas the response is negligible in combination with the DNA-targeting drug cisplatin. Curcumin plus ATO or lonidamine stimulates typical events of the mitochondrial executioner pathway (Bax and Bid activation, cytochrome c release, X-linked inhibitor of apoptosis down-regulation, and caspase-9/-3 activation) and causes mitochondrial transmembrane potential dissipation, which nevertheless represents a late event in the apoptotic response. Curcumin increases anion superoxide production, and its proapoptotic action in combination with ATO and lonidamine is mimicked by pro-oxidant agents (2-methoxyestradiol and H₂O₂) and prevented by antioxidant agents [Mn(III)tetrakis(4-benzoic acid)porphyrin chloride and N-acetyl-l-cysteine]. Within the assayed time period (16–24 h), curcumin does not significantly modify p38-mitogen-activated protein kinase and c-Jun NH₂-terminal kinase phosphorylation/activation or nuclear factor-κB activity, but it greatly stimulates extracellular signal-regulated kinase (ERK) phosphorylation, and decreases Akt phosphorylation. Experiments using mitogen-activated protein kinase kinase/ERK inhibitors [2′-amino-3′-methoxyflavone (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126)] and phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) indicate that ERK activation does not mediate and even restrains apoptosis potentiation, whereas Akt down-regulation facilitates apoptosis generation. In summary, cotreatment with curcumin may represent a useful manner of increasing the efficacy of ATO and lonidamine as antitumor drugs in myeloid leukemia cells.

The observation that genistein may behave as a pro‐oxidant agent lead us to examine the capacity of this isoflavone to modulate the toxicity of the oxidation‐sensitive anti‐leukemic agent arsenic trioxide (ATO), and for comparison other anti‐tumor drugs. Co‐treatment with genistein increased ATO‐provoked apoptosis and activated apoptosis regulatory events (Bcl‐X_L down‐regulation, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP decrease and caspase‐8/Bid and caspase‐3 activation) in U937 promonocytes and other human leukemia cell lines (HL60, THP‐1, Jurkat, RPMI‐8866), but not in phytohemagglutinin‐stimulated non‐tumor peripheral blood lymphocytes (PBLs). Genistein, alone and with ATO, stimulated reactive oxygen species generation, and apoptosis was attenuated by N‐acetyl‐L‐cysteine and butylated hydroxyanisole. Addition of low H₂O₂ concentrations mimicked the capacity of genistein to increase ATO‐provoked apoptosis in leukemia cells, but not in PBLs. By contrast, co‐treatment with genistein or H₂O₂ failed to potentiate the toxicity of DNA‐targeting agent cisplatin, the proteasome inhibitor MG‐132 and the histone deacetylase inhibitor MS‐275. Within the here used time‐period (14 hr) genistein, alone or with ATO, did not significantly affect Akt phosphorylation and NF‐κB binding activity, nor decreased intracellular GSH content. However, it elicited N‐acetyl‐L‐cysteine‐inhibitable phosphorylation of p38‐MAPK and AMPK, and apoptosis was attenuated by pharmacologic inhibitors against these kinases. The pro‐oxidant capacity of genistein might be exploited to improve the efficacy of ATO as anti‐leukemic agent, and perhaps the efficacy of other oxidation‐based therapeutic approaches.

Arsenic trioxide (As₂O₃) caused apoptosis in U-937 human promonocytic cells. This effect was potentiated by the simultaneous addition of the glutathione (GSH) synthesis inhibitor dl-buthionine-(R,S)-sulfoximine or the protein kinase C activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin 1. In addition TPA decreased the intracellular GSH content, caused ERK activation, and potentiated the As₂O₃-provoked activation of p38 and JNK. The addition of N-acetyl-l-cysteine, the PKC inhibitor GF109203X, and the MEK/ERK inhibitors PD98059 and U0126 attenuated both apoptosis induction and GSH decrease, whereas the p38 inhibitor SB203580 and the JNK inhibitor SP600125 were ineffective. TPA also potentiated ERK activation and GSH depletion when added simultaneously to cadmium chloride (CdCl₂) and doxorubicin. However, TPA only enhanced apoptosis in the case of CdCl₂, which is a GSH-sensitive agent, whereas it reduced the toxicity of doxorubicin and other DNA-specific drugs. Finally, preincubation for 14-24 h with TPA did not potentiate but, instead, attenuated the As₂O₃- and CdCl₂-provoked apoptosis. The same result was obtained by preincubation with bryostatin 1 and other differentiation inducers. It is concluded that TPA increases the apoptotic action of As₂O₃, an effect mediated by ERK activation and GSH depletion. However, the increase in apoptosis is only effective in non-differentiated cells.

Fatty acid synthesis and oxidation are frequently exacerbated in leukemia cells, and may therefore represent a target for therapeutic intervention. In this work we analyzed the apoptotic and chemo-sensitizing action of the fatty acid oxidation inhibitor etomoxir in human acute myeloid leukemia cells. Etomoxir caused negligible lethality at concentrations up to 100 µM, but efficaciously cooperated to cause apoptosis with the anti-leukemic agent arsenic trioxide (ATO, Trisenox), and with lower efficacy with other anti-tumour drugs (etoposide, cisplatin), in HL60 cells. Etomoxir-ATO cooperation was also observed in NB4 human acute promyelocytic cells, but not in normal (non-tumour) mitogen-stimulated human peripheral blood lymphocytes. Biochemical determinations in HL60 cells indicated that etomoxir (25–200 µM) dose-dependently inhibited mitochondrial respiration while slightly stimulating glycolysis, and only caused marginal alterations in total ATP content and adenine nucleotide pool distribution. In addition, etomoxir caused oxidative stress (increase in intracellular reactive oxygen species accumulation, decrease in reduced glutathione content), as well as pro-apoptotic LKB-1/AMPK pathway activation, all of which may in part explain the chemo-sensitizing capacity of the drug. Etomoxir also cooperated with glycolytic inhibitors (2-deoxy-D-glucose, lonidamine) to induce apoptosis in HL60 cells, but not in NB4 cells. The combined etomoxir plus 2-deoxy-D-glucose treatment did not increase oxidative stress, caused moderate decrease in net ATP content, increased the AMP/ATP ratio with concomitant drop in energy charge, and caused defensive Akt and ERK kinase activation. Apoptosis generation by etomoxir plus 2-deoxy-D-glucose was further increased by co-incubation with ATO, which is apparently explained by the capacity of ATO to attenuate Akt and ERK activation. In summary, co-treatment with etomoxir may represent an interesting strategy to increase the apoptotic efficacy of ATO and (with some limitations) 2-deoxy-D-glucose which, although clinically important anti-tumour agents, exhibit low efficacy in monotherapy.