Gallic acid sensitizes paclitaxel-resistant human ovarian carcinoma cells through an increase in reactive oxygen species and subsequent downregulation of ERK activation.

Paclitaxel (PTX) is currently used as a front-line chemotherapeutic agent for several types of cancer, including ovarian carcinoma; however, PTX-resistance frequently arises through multiple mechanisms. The development of new strategies using natural compounds and PTX in combination has been the aim of several prior studies, in order to enhance the efficacy of chemotherapy. In this study, we found the following: (i) gallic acid (GA), a phenolic compound, potentiated the capacity of PTX to decrease proliferation and to cause G2/M cycle arrest in the PTX-resistant A2780AD ovarian cancer cell line; (ii) GA exerted a pro-oxidant action by increasing the production of reactive oxygen species (ROS), and co-treatment with the antioxidant agent N­acetyl-L­cysteine (NAC) prevented GA+PTX-induced cell proliferation inhibition and G2/M phase arrest; (iii) PTX stimulated ERK phosphorylation/activation, and co-treatment with the MEK/ERK inhibitor PD98049 potentiated the proliferation inhibition and G2/M phase arrest; (iv) and finally, GA abrogated the PTX-induced stimulation of ERK phosphorylation, a response that was prevented by co-treatment with NAC. Taken together, these results indicate that GA sensitizes PTX-resistant ovarian carcinoma cells via the ROS­mediated inactivation of ERK, and suggest that GA could represent a useful co-adjuvant to PTX in ovarian carcinoma treatment.

Selected polyphenols potentiate the apoptotic efficacy of glycolytic inhibitors in human acute myeloid leukemia cell lines. Regulation by protein kinase activities.

BACKGROUND: The glycolysis inhibitor 2-deoxy-d-glucose (2-DG) is a safe, potentially useful anti-tumour drug, but its efficacy is normally low when used alone. Recent studies indicated that 2-DG stimulates the PI3K/Akt and MEK/ERK defensive pathways, which limits the apoptotic efficacy in tumour cell lines. We hypothesized that co-treatment with selected polyphenols could improve 2-DG-provoked apoptosis by preventing defensive kinase activation. METHODS: Cell proliferation was measured by cell counting or the MTT assay. Cell cycle, apoptosis and necrosis were determined by propidium iodide staining and/or annexin V labeling followed by flow cytometry. Mitochondria pore transition and depolarization were determined by calcein-ATM or rhodamine 123 labeling followed flow cytometry. Intracellular reactive oxygen species and GSH were determined by dichlorodihydrofluorescein diacetate or monochlorobimane labeling followed by flow cytometry or fluorimetry. Expression and phosphorylation of protein kinases were analyzed by the Western blot. RESULTS: (i) 2-DG-provoked apoptosis was greatly potentiated by co-treatment with the sub-lethal concentrations of the flavonoid quercetin in human HL60 acute myeloblastic leukemia cells. Allowing for quantitative differences, apoptosis potentiation was also obtained using NB4 promyelocytic and THP-1 promonocytic cells, using curcumin or genistein instead of quercetin, and using lonidamine instead of 2-DG, but not when 2-DG was substituted by incubation in glucose-free medium. (ii) Quercetin and 2-DG rapidly elicited the opening of mitochondria pore transition, which preceded the trigger of apoptosis. (iii) Treatments did not affect GSH levels, and caused disparate effects on reactive oxygen species generation, which did not match the changes in lethality. (iv) 2-DG and lonidamine stimulated defensive Akt and ERK phosphorylation/activation, while glucose starvation was ineffective. Polyphenols prevented the stimulation of Akt phosphorylation, and in some cases also ERK phosphorylation. In addition, quercetin and 2-DG stimulated GSK-3α,ß phosphorylation/inactivation, although with different isoform specificity. The use of pharmacologic inhibitors confirmed the importance of these kinase modifications for apoptosis. CONCLUSIONS: The present in vitro observations suggest that co-treatment with low concentrations of selected polyphenols might represent a manner of improving the poor anti-tumour efficacy of some glycolytic inhibitors, and that apoptosis potentiation may be at least in part explained by the regulation of defensive protein kinase activities.

Apoptotic efficacy of etomoxir in human acute myeloid leukemia cells. Cooperation with arsenic trioxide and glycolytic inhibitors, and regulation by oxidative stress and protein kinase activities.

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.

Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 µM and a pure necrotic response at 60 µM. Low concentrations of 3-BrP (10-20 µM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 µM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.

2-Deoxy-D-glucose cooperates with arsenic trioxide to induce apoptosis in leukemia cells: involvement of IGF-1R-regulated Akt/mTOR, MEK/ERK and LKB-1/AMPK signaling pathways.

While the anti-tumor efficacy of 2-deoxy-D-glucose (2-DG) is normally low in monotherapy, it may represent a valuable radio- and chemo-sensitizing agent. We here demonstrate that 2-10 mM 2-DG cooperates with arsenic trioxide (ATO) and other antitumor drugs to induce apoptosis in human myeloid leukemia cell lines. Using ATO and HL60 as drug and cell models, respectively, we observed that 2-DG/ATO combination activates the mitochondrial apoptotic pathway, as indicated by Bid-, and Bax-regulated cytochrome c and Omi/HtrA2 release, XIAP down-regulation, and caspase-9/-3 pathway activation. 2-DG neither causes oxidative stress nor increases ATO uptake, but causes inner mitochondria membrane permeabilization as well as moderate ATP depletion, which nevertheless do not satisfactorily explain the pro-apoptotic response. Surprisingly 2-DG causes cell line-specific decrease in LKB-1/AMPK phosphorylation/activation, and also causes Akt/mTOR/p70S6K and MEK/ERK activation, which is prevented by co-treatment with ATO. The use of kinase-specific pharmacologic inhibitors and/or siRNAs reveals that apoptosis is facilitated by AMPK inactivation and restrained by Akt and ERK activation, and that Akt and ERK activation mediates AMPK inhibition. Finally, 2-DG stimulates IGF-1R phosphorylation/activation, and co-treatment with IGF-1R inhibitor prevents 2-DG effects on Akt, ERK and AMPK, and facilitates 2-DG-provoked apoptosis. In summary 2-DG elicits IGF-1R-mediated AMPK inactivation and Akt and ERK activation, which facilitates or restrain apoptosis, respectively. 2-DG-provoked AMPK inactivation increases the apoptotic efficacy of ATO, while in turn ATO-provoked Akt and ERK inactivation may increase the efficacy of 2-DG as anti-tumor drug.

Expression of NF-κB-related proteins and their modulation during TNF-α-provoked apoptosis in prostate cancer cells.

BACKGROUND: The involvement of TNF-α in cancer development is controversial, since this cytokine was reported to act either as tumor promoter or suppressor. TNF-α may activate signaling pathways critical for life/death decisions, such as mitogen-activated protein kinases (MAPKs) and the anti-apoptotic NF-κB pathway. In this work, we investigate the activation status of NF-κB-related proteins in human prostate cancerous versus normal epithelium, and the alterations in the NF-κB pathway in relation to cell death in TNF-α-treated LNCaP (androgen-independent cells) and PC3 (androgen-independent) prostate cancer cell lines. METHODS: The expression of phospho-p38-MAPK, phospho-IKK-α/ß and phospho-IκB-α, total IκB-α, and p65- and p50-NF-κB, were analyzed by immunohistochemistry in cancerous and normal prostate samples. The toxicity of TNF-α in LNCaP and PC3 cells, with or without kinase and NF-κB inhibitors, was assessed by changes on viability (MTT assay) and apoptosis (loss of DNA, annexin-V binding, and caspase cleavage/activation). Expression of NF-κB-related proteins in these cell lines was measured by Western blot. RESULTS: Phospho-IκB-α, phospho-IKK-α/ß and phospho-p38 levels, cytoplasmic p50 to IκB-α ratio, and nuclear p50 and p65, levels, were increased in cancerous epithelium, suggesting activation of the NF-κB pathway in prostatic malignance. TNF-α caused apoptosis with higher efficacy in LNCaP cells, and this response was potentiated by p38-MAPK inhibitor (LNCaP cells) and IKK-ß inhibitor (both cell lines). However, the protective action of IKK-ß was mediated by NF-κB only in LNCaP cells. CONCLUSIONS: IKK-ß mediates both NF-κB-dependent and -independent anti-apoptotic functions in prostate cancerous epithelium. IKK-ß and p38-MAPK may represent useful therapeutic targets against prostate cancer.

Identification of virtual signal transducers and activators of transcription response elements in the human insulin receptor gene promoter.

In this study, we look for the existence of signal transducers and activators of transcription response elements (STATREs) in the human insulin receptor (hIR) gene promoter and their possible relation with the estradiol-provoked transcriptional repression of the hIR gene and cellular insulin resistance in U-937 human promonocytic cells. Potential STATREs in the region from -1819 to -271 bp of the hIR gene promoter were identified by their homology with the consensus STATRE (5'TTCnnnGAA3') using the SEQFIND programme developed in our laboratory. We located five virtual STATRE-like sites: [(I): -1472/-1464], [(II): -1548/-1540], [(III): -1552/-1544], [(IV): -1587/-1579] and [(V): -1678/-1670] showing a difference of only one base from this consensus. These STATREs-like sites were situated between 33 bp upstream the 5' half-element of the estrogen response element 1 (ERE1)-like (-1430/-1418) and 102 bp upstream the 5' half-element of the ERE2-like (-1567/-1555) complexed with AP-1-like sites. A principal complex constituted by STATREs (II-IV) the ERE2 and AP-1 sites (IV and V) was located between -1587/-1540 bp of the hIR gene promoter. In conclusion, these results represent the first identification of virtual STATREs in the hIR gene promoter. These STATREs appear to be specifically located in the surroundings of the two EREs overlapped by various AP-1 sites. These complexes could mediate crosstalk among STATs, estrogen receptor ß (ERß), and AP-1 regulating the ERß-mediated transcriptional repression of the hIR gene and insulin resistance in U-937 cells.

Increased apoptotic efficacy of lonidamine plus arsenic trioxide combination in human leukemia cells. Reactive oxygen species generation and defensive protein kinase (MEK/ERK, Akt/mTOR) modulation.

Lonidamine is a safe, clinically useful anti-tumor drug, but its efficacy is generally low when used in monotherapy. We here demonstrate that lonidamine efficaciously cooperates with the anti-leukemic agent arsenic trioxide (ATO, Trisenox) to induce apoptosis in HL-60 and other human leukemia cell lines, with low toxicity in non-tumor peripheral blood lymphocytes. Apoptosis induction by lonidamine/ATO involves mitochondrial dysfunction, as indicated by early mitochondrial permeability transition pore opening and late mitochondrial transmembrane potential dissipation, as well as activation of the intrinsic apoptotic pathway, as indicated by Bcl-X(L) and Mcl-1 down-regulation, Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release to the cytosol, XIAP down-regulation, and caspase-9 and -3 cleavage/activation, with secondary (Bcl-2-inhibitable) activation of the caspase-8/Bid axis. Lonidamine stimulates reactive oxygen species production, and lonidamine/ATO toxicity is attenuated by antioxidants. Lonidamine/ATO stimulates JNK phosphorylation/activation, and apoptosis is attenuated by the JNK inhibitor SP600125. In addition, lonidamine elicits ERK and Akt/mTOR pathway activation, as indicated by increased ERK, Akt, p70S6K and rpS6 phosphorylation, and these effects are reduced by co-treatment with ATO. Importantly, co-treatment with MEK/ERK inhibitor (U0126) and PI3K/Akt (LY294002) or mTOR (rapamycin) inhibitors, instead of ATO, also potentiates lonidamine-provoked apoptosis. These results indicate that: (i) lonidamine efficacy is restrained by drug-provoked activation of MEK/ERK and Akt/mTOR defensive pathways, which therefore represent potential therapeutic targets. (ii) Co-treatment with ATO efficaciously potentiates lonidamine toxicity via defensive pathway inhibition and JNK activation. And (iii) conversely, the pro-oxidant action of lonidamine potentiates the apoptotic efficacy of ATO as an anti-leukemic agent.

Development of chromanes as novel inhibitors of the uncoupling proteins.

The uncoupling proteins (UCPs) are mitochondrial carriers that modulate the energetic efficiency and, as a result, can lower superoxide levels. Here, we describe the discovery of a small-molecule inhibitor of the UCPs. Screening of potential UCP1 regulators led to the identification of chromane derivatives that inhibit its proton conductance. Members of the UCP family can act as a defense against oxidative stress and, thus, UCP2 plays a protective role in tumor cells. High UCP2 levels have been associated with chemoresistance. We demonstrate that chromanes also inhibit UCP2 and, in HT-29 human carcinoma cells, cause oxidative stress. The chromane derivatives can act synergistically with chemotherapeutic agents; for instance, they increase the toxicity of arsenic trioxide in HT-29 cells. These findings open a promising line in the development of novel anticancer agents.

Curcumin stimulates reactive oxygen species production and potentiates apoptosis induction by the antitumor drugs arsenic trioxide and lonidamine in human myeloid leukemia cell lines.

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 H(2)O(2)) 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 NH(2)-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.

Modulation of arsenic trioxide-induced apoptosis by genistein and functionally related agents in U937 human leukaemia cells. Regulation by ROS and mitogen-activated protein kinases.

The proved radio- and chemo-sensitizing capacity of genistein supports the potential use of this isoflavone in antitumour therapies. In this regard, we recently reported that genistein potentiates apoptosis induction by the anti-leukaemic agent arsenic trioxide (ATO) via reactive oxygen species (ROS) generation and p38-MAPK activation. In the present study we analyze the action of agents sharing functional similarities with the isoflavone, namely 17-beta-estradiol, the DNA topoisomerase II poison etoposide, and the tyrosine kinase (PTK) inhibitors herbimycin A, epigallocatechin-3-gallate (EGCG) and adaphostin, in U937 and other human acute myeloid leukaemia cell lines. Co-treatment with 17-beta-estradiol or etoposide failed to stimulate ROS production and potentiate ATO-provoked apoptosis, although etoposide caused G(2)/M cycle arrest, in the same manner as genistein. By contrast, all PTK inhibitors increased ATO-provoked apoptosis, with similar efficacy as genistein. Daidzein, a genistein analogue without PTK-inhibiting activity, failed to potentiate apoptosis, and co-treatment with orthovanadate attenuated the sensitizing capacity of genistein. Apoptosis potentiation by PTK inhibitors was associated to ROS over-accumulation and stimulation of p38-MAPK phosphorylation, was mimicked by conventional pro-oxidant agents (exogenous H(2)O(2) and the glutathione-depleting agent dl-buthionine-(S,R)-sulfoximine), and was attenuated by the antioxidant agent N-acetyl-l-cysteine, and by the p38-MAPK inhibitor SB203580 or p38-MAPK-directed siRNAs. On the other hand, the PTK inhibitors caused disparate effects on ERK phosphorylation, and co-treatment with the MEK/ERK inhibitor PD98059 enhanced the pro-apoptotic capacity of the PTK inhibitors. These results suggest that PTK inhibition, together with ROS generation and p38-MAPK activation, are responsible for the chemo-sensitizing action of genistein and functionally related agents in leukaemia cells.

Inhibition of p38-MAPK potentiates cisplatin-induced apoptosis via GSH depletion and increases intracellular drug accumulation in growth-arrested kidney tubular epithelial cells.

We were interested in analyzing the regulation by mitogen-activated protein kinases (MAPKs) of cisplatin-provoked toxicity in epithelial renal tubule cell lines, when assayed under culture conditions (cell confluence plus serum deprivation), which mimic the characteristics of a nonproliferating epithelium. Under these restrictive growth conditions, cisplatin induced apoptosis with lower efficacy than in exponentially growing cells, and decreased p38-MAPK phosphorylation in NRK-52E and other (LLC-PK1, MDCK, HK2) cell lines. Moreover, cisplatin-provoked apoptosis was potentiated by cotreatment with p38-MAPK-specific inhibitors (SB203580, SB220025) or transfection with a kinase-negative mutant of MKK6, whereas c-Jun NH2-terminal kinase or extracellular signal-regulated kinase/MAPK and ERK Kinase inhibitors were ineffective. By contrast, when applied to exponentially growing cells, cisplatin stimulated p38-MAPK phosphorylation and apoptosis, was attenuated by kinase inhibitors. Treatment of confluent/serum-deprived cells with cisplatin caused mitochondrial transmembrane potential disruption and activated the mitochondrial apoptotic pathway, as indicated by the decrease in Bcl-X(L) expression, increase in Bax expression and cytochrome c release, and these effects were potentiated by cotreatment with SB203580. Treatment of confluent/serum-deprived cells with cisplatin plus SB203580 decreased the intracellular reduced glutathione (GSH) content, and increased intracellular cisplatin accumulation as well as cisplatin binding to DNA. Cotreatment with the GSH-depleting agent D,L-buthionine-R,S-sulfoximine also potentiated cisplatin-provoked apoptosis. In summary, p38-MAPK inhibition potentiates cisplatin-provoked apoptosis in growth-arrested epithelial renal tubule cells, a result that may be explained at least in part by GSH depletion and drug transport alteration.

Regulation of genistein-induced differentiation in human acute myeloid leukaemia cells (HL60, NB4) Protein kinase modulation and reactive oxygen species generation.

While it has been reported that genistein induces differentiation in multiple tumour cell models, the signalling and regulation of isoflavone-provoked differentiation are poorly known. We here demonstrate that genistein causes G(2)/M cycle arrest and expression of differentiation markers in human acute myeloid leukaemia cells (HL60, NB4), and cooperates with all-trans retinoic acid (ATRA) in inducing differentiation, while ATRA attenuates the isoflavone-provoked toxicity. Genistein rapidly stimulates Raf-1, MEK1/2 and ERK1/2 phosphorylation/activation, but does not stimulate and instead causes a late decrease in Akt phosphorylation/activation which is attenuated by ATRA. Both differentiation and G(2)/M arrest are attenuated by MEK/ERK inhibitors (PD98059, U0126) and ERK1-/ERK2-directed small interfering RNAs (siRNAs), and by the PI3K inhibitor LY294002, but not by the p38-MAPK inhibitor SB203580. Genistein stimulates p21(waf1/cip1) and cyclin B1 expression, phosphorylation/activation of ATM and Chk2 kinases, and Tyr15-phosphorylation/inactivation of Cdc2 (Cdk1) kinase, and these effects are attenuated by MEK/ERK inhibitors, while LY294002 also attenuates ERK and ATM phosphorylation. Caffeine abrogates the genistein-provoked G(2)/M blockade and alterations in cell cycle regulatory proteins, and also suppresses differentiation. Finally, genistein causes reactive oxygen species (ROS) over-accumulation, but the antioxidant N-acetyl-L-cysteine fails to prevent ERK activation, G(2)/M arrest, and differentiation induction. By contrast, N-acetyl-L-cysteine and p38-MAPK inhibitor attenuate the apoptosis-sensitizing (pro-apoptotic) action of genistein when combined with the antileukaemic agent arsenic trioxide. In summary, genistein-induced differentiation in acute myeloid leukaemia cells is a ROS-independent, Raf-1/MEK/ERK-mediated and PI3K-dependent response, which is coupled and co-regulated with G(2)/M arrest, but uncoupled to the pro-apoptotic action of the drug.

Genistein selectively potentiates arsenic trioxide-induced apoptosis in human leukemia cells via reactive oxygen species generation and activation of reactive oxygen species-inducible protein kinases (p38-MAPK, AMPK).

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 H(2)O(2) 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 H(2)O(2) 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-kappaB 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.

Quercetin decreases intracellular GSH content and potentiates the apoptotic action of the antileukemic drug arsenic trioxide in human leukemia cell lines.

Arsenic trioxide (ATO) is an effective therapeutic agent for the treatment of acute promyelocytic leukemia, but successful application of this agent may occasionally require the use of sensitizing strategies. The present work demonstrates that the flavonoids quercetin and chrysin cooperate with ATO to induce apoptosis in U937 promonocytes and other human leukemia cell lines (THP-1, HL-60). Co-treatment with ATO plus quercetin caused mitochondrial transmembrane potential dissipation, stimulated the mitochondrial apoptotic pathway, as indicated by cytochrome c and Omi/Htra2 release, XIAP and Bcl-X(L) down-regulation, and Bax activation, and caused caspase-8/Bid activation. Bcl-2 over-expression abrogated cytochrome c release and apoptosis, and also blocked caspase-8 activation. Quercetin and chrysin, alone or with ATO, decreased Akt phosphorylation as well as intracellular GSH content. GSH depletion was regulated at the level of L-buthionine-(S,R)-sulfoximine (BSO)-sensitive enzyme activity, and N-acetyl-L-cysteine failed both to restore GSH content and to prevent apoptosis. Treatment with BSO caused GSH depletion and potentiated ATO-provoked apoptosis, but did not affect apoptosis induction by ara-C and cisplatin. As an exception, ATO plus quercetin failed to elicit Akt de-phosphorylation and GSH depletion in NB4 acute promyelocytic leukemia cells, and correspondingly exhibited low cooperative effect in inducing apoptosis in this cell line. It is concluded that GSH depletion explains at least in part the selective potentiation of ATO toxicity by quercetin, and that this flavonoid might be used to increase the clinical efficacy of the antileukemic drug.

Arsenic trioxide sensitizes promonocytic leukemia cells to TNFalpha-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways.

Treatment with the anti-leukemic drug arsenic trioxide (As(2)O(3), 1-4 microM) sensitizes U937 promonocytes and other human myeloid leukemia cell lines (HL60, NB4) to apoptosis induction by TNFalpha. As(2)O(3) plus TNFalpha increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIP(L) expression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. The treatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, and caspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIP(L) down-regulation and caspase-8 activation, but not TNF-R1 over-expression. As(2)O(3) does not affect Akt phosphorylation/activation or intracellular GSH content, nor prevents the TNFalpha-provoked stimulation of p65-NF-kappaB translocation to the nucleus and the increase in NF-kappaB binding activity. Treatments with TNFalpha alone or with As(2)O(3) plus TNFalpha cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuate the As(2)O(3) plus TNFalpha-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, the sensitization by As(2)O(3) to TNFalpha-induced apoptosis in promonocytic leukemia cells is an Akt/NF-kappaB-independent, p38-MAPK-regulated process, which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways.

Trans platinum complexes design: one novel water soluble oxime derivative that contains aliphatic amines in trans configuration.

In an attempt to design new antitumoral drugs based on transplatin complexes, we determined the experimental conditions for the preparation of trans-[Pt((CH(3))(2)CNOH)((CH(3))(2)CHNH(2))Cl(2)], and solved the crystal structure. The cytotoxicity of the novel complex, the cis counterpart, cisplatin, and a trans complex with aliphatic amines, as well as the capacity of some of these complexes to cause either apoptotic or necrotic cell death, was comparatively examined in NRK-52E rat renal tubular cells and HepG2 human hepatoma cells. The results indicate that the oxime complex with trans geometry, but not the one with cis geometry, causes death by apoptosis, making the complex potentially suitable for therapeutic purposes. However cytotoxicity values are higher in the case of cis geometry than in trans geometry in both tumoral and non-tumoral cell lines.

Pharmacologic inhibitors of extracellular signal-regulated kinase (ERKs) and c-Jun NH(2)-terminal kinase (JNK) decrease glutathione content and sensitize human promonocytic leukemia cells to arsenic trioxide-induced apoptosis.

Treatment with 1-4 microM As(2)O(3) slightly induced apoptosis in U-937 human promonocitic leukemia cells. This effect was potentiated by co-treatment with MEK/ERK (PD98059, U0126) and JNK (SP600125, AS601245) inhibitors, but not with p38 (SB203580, SB220025) inhibitors. However, no potentiation was obtained using lonidamine, doxorubicin, or cisplatin instead of As(2)O(3). Apoptosis potentiation by mitogen-activated protein kinase (MAPK) inhibitors involved both the intrinsic and extrinsic executionary pathways, as demonstrated by Bax activation and cytochrome c release from mitochondria, and by caspase-8 activation and Bid cleavage, respectively; and the activation of both pathways was prevented by Bcl-2 over-expression. Treatment with MEK/ERK and JNK inhibitors, but not with p38 inhibitors, caused intracellular glutathione (GSH) depletion, which was differentially regulated. Thus, while it was prevented by N-acetyl-L-cysteine (NAC) in the case of U0126, it behaved as a NAC-insensitive process, regulated at the level of DL-buthionine-(S,R)-sulfoximine (BSO)-sensitive enzyme activity, in the case of SP600125. The MEK/ERK inhibitor also potentiated apoptosis and decreased GSH content in As(2)O(3)-treated NB4 human acute promyelocytic leukemia (APL) cells, but none of these effects were produced by the JNK inhibitor. MEK/ERK and JNK inhibitors did not apparently affect As(2)O(3) transport activity, as measured by intracellular arsenic accumulation. SP600126 greatly induced reactive oxygen species (ROS) accumulation, while BSO and U0126 had little or null effects. These results, which indicate that glutathione is a target of MAP kinases in myeloid leukemia cells, might be exploited to improve the antitumor properties of As(2)O(3), and provide a rationale for the use of kinase inhibitors as therapeutic agents.

Regulation of cadmium-induced apoptosis by PKCdelta in U937 human promonocytic cells.

Pulse treatment with cadmium chloride followed by recovery caused apoptosis in U937 human promonocytic cells. In addition, the treatment-induced PKCdelta translocation from cytosol to membrane fraction, which was already detected at 30 min of treatment; and also caused PKCdelta cleavage to give a 41-kDa fragment, which was detected at 3-6 h of recovery, concomitantly with the execution of apoptosis. All these effects were reduced by the PKCdelta-specific inhibitor rottlerin. By contrast, rottlerin did not prevent the cadmium-provoked stimulation of the stress response (as measured by HSP70 expression), nor inhibited the generation of apoptosis by heat-shock, which failed to cause PKCdelta translocation. Cadmium chloride rapidly induced p38(MAPK) activation, which was not affected by rottlerin. By contrast, the p38(MAPK) inhibitor SB203580 reduced PKCdelta translocation and cleavage, indicating that p38(MAPK) activation precedes and regulates PKCdelta activation. It is concluded that PKCdelta mediates apoptosis induction by cadmium ions via early membrane translocation, and also possibly through late kinase proteolytic cleavage and phosphorylation on tyrosine residues.

Pharmacological inhibitors of extracellular signal-regulated protein kinases attenuate the apoptotic action of cisplatin in human myeloid leukemia cells via glutathione-independent reduction in intracellular drug accumulation.

It has been reported that inhibition of extracellular signal-regulated protein kinases (ERKs) attenuates the toxicity cisplatin (cis-platinum (II)-diammine dichloride) in some cell types. This response was here investigated using human myeloid leukemia cells. Cisplatin stimulated ERK1/2 phosphorylation and caused apoptosis in U-937 promonocytic cells, an effect which was attenuated by the MEK/ERK inhibitors PD98059 and U0126. While ERK1/2 activation was a general phenomenon, irrespective of the used cell type or antitumour drug, the MEK/ERK inhibitors only reduced cisplatin toxicity in human myeloid cells (THP-1, HL-60 and NB-4), but not in RAW 264.7 mouse macrophages and NRK-52E rat renal tubular cells; and failed to reduce the toxicity etoposide, camptothecin, melphalan and arsenic trioxide, in U-937 cells. U0126 attenuated cisplatin-DNA binding and intracellular peroxide accumulation, which are important regulators of cisplatin toxicity. Although cisplatin decreased the intracellular glutathione (GSH) content, which was restored by U0126, treatments with GSH-ethyl ester and dl-buthionine-(S,R)-sulfoximine revealed that GSH does not regulate cisplatin toxicity in the present experimental conditions. In spite of it, PD98059 and U0126 reduced the intracellular accumulation of cisplatin. These results suggest that GSH-independent modulation of drug transport is a major mechanism explaining the anti-apoptotic action of MEK/ERK inhibitors in cisplatin-treated myeloid cells.