Mitochondrial-dependent Mechanisms Research Articles

Effect of Lipocalin‐2 expression on mitochondrial function in renal tubular cells in chronic kidney disease

Kidney cells metabolic status requires high abundance of mitochondria. It has been reported in chronic kidney disease (CKD) a loss of mitochondrial mass and function leading to reactive oxygen species (ROS) production and subsequent tubular cell death. Hence, mitochondrial dysfunction participates to CKD onset. Nevertheless, the cellular mechanisms inducing mitochondrial dysfunction in kidney cells are poorly understood. We have recently shown that in CKD, Lipocalin‐2 (Lcn2) expression is strongly induced in kidney cells leading to ROS production and apoptosis. However, the mechanisms by which Lcn2 triggers these phenomena are unknown. Considering that mitochondria can induce oxidative stress and apoptosis, we hypothesized that Lcn2 participates to CKD onset via an effect on mitochondrial dependent mechanisms. Using renal tubular cell lines overexpressing (OE) or knock‐down (KD) for Lcn2, we aimed at studying the effect of Lcn2 protein expression on mitochondrial function. First, we demonstrated that Lcn2 regulates mitochondrial dynamics, mass and function. Indeed, we observed by immunofluorescence and electron microscopy that kidney cells expressing Lcn2 present smaller and rounded mitochondria while Lcn2 KD induces mitochondrial elongation. Moreover, Lcn2 expression increases mitochondrial fission marker abundance and decreases fusion marker expression. Mitotracker staining and mtDNA content measurement experiments demonstrated that Lcn2 expression decreased mitochondrial mass while Lcn2 KD increased it. Furthermore, we observed that Lcn2 expression decrease mitochondrial membrane potential and accordingly increased Lactate production compared to cell KD for Lcn2. Using iron chelator or Lcn2 mutant unable to transport iron, we demonstrated that Lcn2 expression induces mitochondrial dysfunction independently of its iron transport effect. In addition, we observed that extracellular Lcn2 did not rescue Lcn2 effects on mitochondria while intracellular Lcn2 did. Finally, using a subtotal nephrectomy mice model, that recapitulates different features of human CKD, we demonstrated that Lcn2 invalidation by knock‐out in mice decreased the mitochondrial fission and the subsequent mitochondrial fragmentation and mass loss in kidney tubular cells normally observed during CKD onset in wild‐type mice.Altogether, these results demonstrated that Lcn2 expression regulates mitochondrial dynamics, mass and function in kidney tubular cells. This suggests that Lcn2 driven mitochondrial dysfunction might by responsible at least in part of CKD onset.

Hexachloronaphthalene Induces Mitochondrial-Dependent Neurotoxicity via a Mechanism of Enhanced Production of Reactive Oxygen Species.

Hexachloronaphthalene (PCN67) is one of the most toxic among polychlorinated naphthalenes. Despite the known high bioaccumulation and persistence of PCN67 in the environment, it is still unclear to what extent exposure to these substances may interfere with normal neuronal physiology and lead to neurotoxicity. Therefore, the primary goal of this study was to assess the effect of PCN67 in neuronal in vitro models. Neuronal death was assessed upon PCN67 treatment using differentiated PC12 cells and primary hippocampal neurons. At 72 h postexposure, cell viability assays showed an IC50 value of 0.35 μg/ml and dose-dependent damage of neurites and concomitant downregulation of neurofilaments L and M. Moreover, we found that younger primary neurons (DIV4) were much more sensitive to PCN67 toxicity than mature cultures (DIV14). Our comprehensive analysis indicated that the application of PCN67 at the IC50 concentration caused necrosis, which was reflected by an increase in LDH release, HMGB1 protein export to the cytosol, nuclear swelling, and loss of homeostatic control of energy balance. The blockage of mitochondrial calcium uniporter partially rescued the cell viability, loss of mitochondrial membrane potential (ΔΨm), and the overproduction of reactive oxygen species, suggesting that the underlying mechanism of neurotoxicity involved mitochondrial calcium accumulation. Increased lipid peroxidation as a consequence of oxidative stress was additionally seen for 0.1 μg/ml of PCN67, while this concentration did not affect ΔΨm and plasma membrane permeability. Our results show for the first time that neuronal mitochondria act as a target for PCN67 and indicate that exposure to this drug may result in neuron loss via mitochondrial-dependent mechanisms.

303 - SQSTM1/p62 deficiency induces oxidative stress and senescence in vivo and in vitro in VSMCs

Aging is a major contributor to age-related diseases including cardiovascular disease (CVD). Vascular senescence, a hallmark of mammalian aging, has been linked to vascular disease like atherosclerosis. We previously showed that the NADPH oxidase Nox1 mediates senescence induced by angiotensin II, a major inducer of vascular aging and CVD. Disruption of other processes, like autophagy, also promotes senescence due to the accumulation of dysfunctional mitochondria leading to upregulation of ROS levels. SQSTM1, known as p62, is an autophagy adaptor involved in the targeting of protein aggregates and dysfunctional organelles, including mitochondria, into the autophagy degradation pathway. The interaction between NADPH oxidases and autophagy in the senescence pathway is incompletely understood. Using vascular smooth muscle cells (VSMCs) isolated from aortas of p62+/+ and p62-/- mice, we found that lack of p62 causes senescence by a ROS-dependent and Nox1-independent mechanism. Downregulation of p62 decreased cell proliferation, increased the expression of the senescence markers (senescence associated-β-galactosidase (SA-β-gal) and p21) and increased superoxide, hydrogen peroxide and mitochondrial ROS levels. Treatment with the antioxidant N-acetyl cysteine (NAC) and the mitochondrial ROS scavenger MitoTEMPO reduced SA-β-gal activity in p62-/- VSMCs, suggesting that cytosolic ROS, as well as mitochondrial ROS contribute to the development of senescence. These effects were not associated with upregulation of Nox1. Further, senescence was also upregulated in vivo in aortas of male and female p62-/-, compared with p62+/+ mice. Altogether, these data suggest lack of p62 induces oxidative stress by a Nox1-independent and mitochondrial-dependent mechanisms.

Ex Vivo Cardiotoxicity of Antineoplastic Casiopeinas Is Mediated through Energetic Dysfunction and Triggered Mitochondrial-Dependent Apoptosis.

Casiopeinas are a group of copper-based antineoplastic molecules designed as a less toxic and more therapeutic alternative to cisplatin or Doxorubicin; however, there is scarce evidence about their toxic effects on the whole heart and cardiomyocytes. Given this, rat hearts were perfused with Casiopeinas or Doxorubicin and the effects on mechanical performance, energetics, and mitochondrial function were measured. As well, the effects of Casiopeinas-triggered cell death were explored in isolated cardiomyocytes. Casiopeinas III-Ea, II-gly, and III-ia induced a progressive and sustained inhibition of heart contractile function that was dose- and time-dependent with an IC50 of 1.3 ± 0.2, 5.5 ± 0.5, and 10 ± 0.7 μM, correspondingly. Myocardial oxygen consumption was not modified at their respective IC50, although ATP levels were significantly reduced, indicating energy impairment. Isolated mitochondria from Casiopeinas-treated hearts showed a significant loss of membrane potential and reduction of mitochondrial Ca2+ retention capacity. Interestingly, Cyclosporine A inhibited Casiopeinas-induced mitochondrial Ca2+ release, which suggests the involvement of the mitochondrial permeability transition pore opening. In addition, Casiopeinas reduced the viability of cardiomyocytes and stimulated the activation of caspases 3, 7, and 9, demonstrating a cell death mitochondrial-dependent mechanism. Finally, the early perfusion of Cyclosporine A in isolated hearts decreased Casiopeinas-induced dysfunction with reduction of their toxic effect. Our results suggest that heart cardiotoxicity of Casiopeinas is similar to that of Doxorubicin, involving heart mitochondrial dysfunction, loss of membrane potential, changes in energetic metabolites, and apoptosis triggered by mitochondrial permeability.

4-Nerolidylcatechol: apoptosis by mitochondrial mechanisms with reduction in cyclin D1 at G0/G1 stage of the chronic myelogenous K562 cell line

Context: 4-Nerolidylcatechol (4-NRC) has showed antitumor potential through apoptosis. However, its apoptotic mechanisms are still unclear, especially in leukemic cells. Objectives: To evaluate the cytotoxic potential of 4-NRC and its cell death pathways in p53-null K562 leukemic cells. Materials and methods: Cytotoxicity of 4-NRC (4.17–534.5 μM) over 24 h of exposure was evaluated by MTT assay. 4-NRC-induced apoptosis in K562 cells was investigated by phosphatidylserine (PS) externalization, cell cycle, sub-G1, mitochondrial evaluation, cytochrome c, cyclin D1 and intracellular reactive oxygen species (ROS) levels, and caspase activity analysis. Results: IC50 values obtained were 11.40, 27.31, 15.93 and 15.70 μM for lymphocytes, K562, HL-60 and Jurkat cells, respectively. In K562 cells, 4-NRC (27 μM) promoted apoptosis as verified by cellular morphological changes, a significant increase in PS externalization and sub-G1 cells. Moreover, it significantly arrested the cells at the G0/G1 phase due to a reduction in cyclin D1 expression. These effects of 4-NRC also significantly promoted a reduction in mitochondrial activity and membrane depolarization, accumulation of cytosolic cytochrome c and ROS overproduction. Additionally, it triggered an increase in caspases -3/7, -8 and -9 activities. When the cells were pretreated with N-acetyl-l-cysteine ROS scavenger, 4-NRC-induced apoptosis was partially blocked, which suggests that it exerts cytotoxicity though not exclusively through ROS-mediated mechanisms. Discussion and conclusion: 4-NRC has antileukemic properties, inducing apoptosis mediated by mitochondrial-dependent mechanisms with cyclin D1 inhibition. Given that emerging treatment concepts include novel combinations of well-known agents, 4-NRC could offer a promising alternative for chemotherapeutic combinations to maximize tumour suppression.

Mitochondrial-dependent mechanisms are involved in angiotensin II-induced apoptosis in dopaminergic neurons.

Introduction:We recently demonstrated that angiotensin II (Ang II) was involved in the etiology of Parkinson’s disease (PD) via induction of apoptosis of dopaminergic neurons, but the mechanisms are not completely elucidated. Here, we asked whether mitochondrial-dependent mechanisms contributed to the Ang II-induced dopaminergic neuronal apoptosis.Materials and methods:CATH.a cells were incubated with Ang II in combination with mitochondrial permeability transition pore (mPTP) inhibitors or angiotensin receptor antagonists, and apoptosis rate, caspase-3 activity, cytochrome c levels, and mPTP opening were assessed.Results:We showed that Ang II triggered apoptosis via a mitochondrial-dependent pathway, as elevated cytochrome c levels were observed in the cytosol. By employing cyclosporin A and sanglifehrin A, two specific mPTP inhibitors, we revealed that cytochrome c release from mitochondria into cytoplasm was ascribed to mPTP opening. Meanwhile, the aforementioned effects could be abrogated by an AT1 receptor antagonist losartan rather than an AT2 receptor antagonist PD123319.Conclusion:This study demonstrates that Ang II triggers mitochondrial-dependent apoptosis via facilitating mPTP opening through an AT1 receptor-mediated fashion in dopaminergic neurons. These findings give insight into the effect of Ang II in the etiology of PD, and reinforce the application of AT1 receptor antagonists for PD treatment.

Apoptotic effects of 1,5-bis-(5-nitro-2-furanyl)-1, K]4-pentadien-3-one on Drosophila SL2 cells

In recent years, concerns over the potential impact of synthetic pesticides on the environment have promoted the research and development of environmentally friendly “green” pesticides. In the current study, we utilized a green biomimetic insecticide, 1,5-bis-(5-nitro-2-furanyl)-1,4-pentadien-3-one (compound C), to examine its cytotoxicity on an insect cell line, Drosophila melanogaster (SL2). Results from MTT assay showed cells treated with 100 µM of compound C for 48 h significantly inhibited the growth of SL2 cells by 87.71±0.96%. We subsequently attempted to illustrate the act on mechanism of compound C at the cellular level and found that it initiated apoptosis through a mitochondrial-dependent mechanism that increased the activity of caspase-3 and altered the cell cycle. These results suggest that the green biomimetic insecticide, compound C, is a novel and potent inducer of insect cell apoptosis.

Pro‐oxidant effects of Ecstasy and its metabolites in mouse brain synaptosomes

3,4-Methylenedioxymethamphetamine (MDMA or 'Ecstasy') is a worldwide major drug of abuse known to elicit neurotoxic effects. The mechanisms underlying the neurotoxic effects of MDMA are not clear at present, but the metabolism of dopamine and 5-HT by monoamine oxidase (MAO), as well as the hepatic biotransformation of MDMA into pro-oxidant reactive metabolites is thought to contribute to its adverse effects. Using mouse brain synaptosomes, we evaluated the pro-oxidant effects of MDMA and its metabolites, α-methyldopamine (α-MeDA), N-methyl-α-methyldopamine (N-Me-α-MeDA) and 5-(glutathion-S-yl)-α-methyldopamine [5-(GSH)-α-MeDA], as well as those of 5-HT, dopamine, l-DOPA and 3,4-dihydroxyphenylacetic acid (DOPAC). 5-HT, dopamine, l-DOPA, DOPAC and MDMA metabolites α-MeDA, N-Me-α-MeDA and 5-(GSH)-α-MeDA, concentration- and time-dependently increased H(2) O(2 ) production, which was significantly reduced by the antioxidants N-acetyl-l-cysteine (NAC), ascorbic acid and melatonin. From experiments with MAO inhibitors, it was observed that H(2) O(2) generation induced by 5-HT was totally dependent on MAO-related metabolism, while for dopamine, it was a minor pathway. The MDMA metabolites, dopamine, l-DOPA and DOPAC concentration-dependently increased quinoproteins formation and, like 5-HT, altered the synaptosomal glutathione status. Finally, none of the compounds modified the number of polarized mitochondria in the synaptosomal preparations, and the compounds' pro-oxidant effects were unaffected by prior mitochondrial depolarization, excluding a significant role for mitochondrial-dependent mechanisms of toxicity in this experimental model. MDMA metabolites along with high levels of monoamine neurotransmitters can be major effectors of neurotoxicity induced by Ecstasy.

Homology Modeling and Docking Studies of Human Bcl-2L10 Protein

Cancer, an unrestrained proliferation of cells, is one of the lead cause of death. Nearly 12.5 million people are diagnosed with cancer worldwide, 7.5 million people die of which 2.5 million cases are from India. Major cause for cancer is restriction of programmed cell death (apoptosis). Multiple signaling pathways regulate apoptosis. Bcl-2 (B—Cell Lymphomas-2) family proteins play a vital role as central regulators of apoptosis. Bcl-2L10, a novel anti- apoptotic protein, blocks apoptosis by mitochondrial dependent mechanism. The present study evaluates the 3D structure of Bcl-2L10 protein using homology modeling and aims to understand plausible functional and binding interactions between Bcl-2L10 with BH3 domain of BAX using protein—protein docking. The docking studies show binding of BH3 domain at Lys 110, Trp-111, Pro-115, Glu-119 and Asp-127 in the groove of BH 1, 2 and 3 domains of Bcl-2L10. Heterodimerization of anti-apoptotic Bcl-2 and BH3 domain of pro- apoptotic Bcl-2 proteins instigates apoptosis. Profound understanding of Bcl-2 pathway may prove useful in identification of future therapeutic targets for cancer.

The role of mitochondrial complex III in melatonin‐induced ROS production in cultured mesangial cells

Melatonin is a potent scavenger of reactive oxygen (ROS) and reactive nitrogen species (RNS). At pharmacological concentrations, however, melatonin is documented to cause ROS/RNS production, especially in cultured cancerous cells. Currently, the mechanism responsible for melatonin-induced ROS generation remains elusive. In this study, we provided evidence that melatonin, at micromolar concentrations, induced rapid ROS generation by a mitochondrial-dependent mechanism in primary human mesangial (HM) cells. The melatonin-induced ROS production occurred independent of changes in Ca(2+) concentrations in the cytosol and/or in mitochondria. In mitochondria isolated from HM cells and mice kidney tissues, melatonin caused ROS production; this melatonin response was completely blocked by the complex III inhibitor antimycin A. In contrast, both the mitochondrial complex I inhibitor, rotenone, and another complex III inhibitor, myxothiazol, which interacts with complex III at a distinct site, had no significant inhibitory effect on melatonin-induced ROS generation. These results demonstrate that melatonin induced rapid ROS generation via the antimycin A-sensitive site of mitochondrial complex III.

More than regulation of skewed Th1/Th2 cytokines in allergic asthma: A case treated with omalizumab

presence or absence of anaerobic glycolysis (i.e. glucose or galactose in the culture medium). Glucose allows ATP to be produced by both aerobic and anaerobic glycolysis, whereas galactose only allows cells to produce ATP by mitochondrial oxidative phophorylations. Data was analysed by the Student’s t-test or the Mann–Whitney U-test as appropriate. Results: In the presence of Glucose/10% FCS, the maximal cell count was (mean±SEM, ×106 cellules) 2.79±0.31 vs. 0.33±0.04 in immature & adult ASMC respectively (p< 0.05). DNA synthesis (thymidine incorporation) in immature AMSC (fold increase vs. ITS control) was 6.55±1.02 vs. 2.70±0.43 in adult ASMC (p< 0.05). A similar trend was observed with PDGF-AA (2.83±0.62 vs. 1.47±0.12, p < 0.05). This was not observed in the presence of the other agents. C/EBPa & TRPC1 expression were similar in immature & adult ASMC. DNA synthesis in Glucose-free (Galactose)/10% FCS medium were much greater in immature vs. adult ASMC (4.02±0.80 vs. 0.34±0.07 fold increase, p < 0.05). Conclusion: Enhanced proliferation of immature ASMC in the presence of 10% FCS or PDGF-AA appears to be related to a mitochondrial-dependent mechanism, similar to what has been reported in adult asthmatic ASMC. This may contribute to early airway remodelling.

Overactivation of the MEK/ERK Pathway in Liver Tumor Cells Confers Resistance to TGF-β–Induced Cell Death through Impairing Up-regulation of the NADPH Oxidase NOX4

Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, being considered a liver tumor suppressor. However, many human hepatocellular carcinoma (HCC) cells escape from its proapoptotic effects, gaining response to this cytokine in terms of malignancy. We have recently reported that the apoptosis induced by TGF-beta in hepatocytes requires up-regulation of the NADPH oxidase NOX4, which mediates reactive oxygen species (ROS) production. TGF-beta-induced NOX4 expression is inhibited by antiapoptotic signals, such as the phosphatydilinositol-3-phosphate kinase or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. The aim of the present work was to analyze whether resistance to TGF-beta-induced apoptosis in HCC cells is related to the impairment of NOX4 up-regulation due to overactivation of survival signals. Results indicate that inhibition of the MAPK/ERK kinase (MEK)/ERK pathway in HepG2 cells, which are refractory to the proapoptotic effects of TGF-beta, sensitizes them to cell death through a mitochondrial-dependent mechanism, coincident with increased levels of BIM and BMF, decreased levels of BCL-XL and MCL1, and BAX/BAK activation. Regulation of BMF, BCL-XL, and MCL1 occurs at the mRNA level, whereas BIM regulation occurs post-transcriptionally. ROS production and glutathione depletion are only observed in cells treated with TGF-beta and PD98059, which correlates with NOX4 up-regulation. Targeting knockdown of NOX4 impairs ROS increase and all the mitochondrial-dependent apoptotic features by a mechanism that is upstream from the regulation of BIM, BMF, BCL-XL, and MCL1 levels. In conclusion, overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-beta-induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis.

O-GlcNAc signaling attenuates ER stress-induced cardiomyocyte death

We previously demonstrated that the O-linked beta-N-acetylglucosamine (O-GlcNAc) posttranslational modification confers cardioprotection at least partially through mitochondrial-dependent mechanisms, but it remained unclear if O-GlcNAc signaling interfered with other mechanisms of cell death. Because ischemia/hypoxia causes endoplasmic reticulum (ER) stress, we ascertained whether O-GlcNAc signaling could attenuate ER stress-induced cell death per se. Before induction of ER stress (with tunicamycin or brefeldin A), we adenovirally overexpressed O-GlcNAc transferase (AdOGT) or pharmacologically inhibited O-GlcNAcase [via O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate] to augment O-GlcNAc levels or adenovirally overexpressed O-GlcNAcase to reduce O-GlcNAc levels. AdOGT significantly (P < 0.05) attenuated the activation of the maladaptive arm of the unfolded protein response [according to C/EBP homologous protein (CHOP) activation] and cardiomyocyte death (reflected by percent propidium iodide positivity). Moreover, pharmacological inhibition of O-GlcNAcase significantly (P < 0.05) mitigated ER stress-induced CHOP activation and cardiac myocyte death. Interestingly, overexpression of GCA did not alter ER stress markers but exacerbated brefeldin A-induced cardiomyocyte death. We conclude that enhanced O-GlcNAc signaling represents a partially proadaptive response to reduce ER stress-induced cell death. These results provide new insights into a possible interaction between O-GlcNAc signaling and ER stress and may partially explain a mechanism of O-GlcNAc-mediated cardioprotection.

A mitochondrial mechanism is involved in apoptosis of Robertsonian mouse male germ cells

The aim of this study was to determine whether the intrinsic mechanism of apoptosis is involved in the death of germ cells in Robertsonian (Rb) heterozygous adult male mice. Testes from 5-month-old Rb heterozygous CD1 x Milano II mice were obtained and compared with those from homozygous CD1 (2n=40) and Milano II (2n=24) mice. For histological evaluation of apoptosis, TUNEL labelling and immunohistochemistry were used to localise Bax and cytochrome c. Expression of calbindin D(28k) (CB), an anti-apoptotic molecule, was also analysed by immunohistochemistry and immunoblotting. Testicular ultrastructure was visualised by electron microscopy. Morphology and cell associations were abnormal in the Rb heterozygous seminiferous epithelium. An intense apoptotic process was observed in tubules at stage XII, mainly in metaphase spermatocytes. Metaphase spermatocytes also showed Bax and cytochrome c redistributions. Mitochondria relocated close to the paranuclear region of spermatocytes. CB was mainly expressed in metaphase spermatocytes, but also in pachytene spermatocytes, spermatids and Sertoli cells at stage XII. The co-localisation of CB and TUNEL labelling was very limited. Sixty per cent of metaphase spermatocytes were apoptotic and calbindin negative, while 40% were calbindin positive without signs of apoptosis. Ten per cent of the Bax- and cytochrome c-positive cells were also calbindin positive. These data suggest that apoptosis of the germ cells in heterozygous mice occurs, at least in part, through a mitochondrial-dependent mechanism. Calbindin overexpression might prevent or reduce the apoptosis of germ cells caused by Rb heterozygosity, which could partially explain the subfertility of these mice.

Regulation of ubiquitin–proteasome system, caspase enzyme activities, and extracellular proteinases in rat soleus muscle in response to unloading

In the present study, we determined the impact of 5 and 10 days of muscle deconditioning induced by hindlimb suspension (HS) on the ubiquitin-proteasome system of protein degradation and caspase enzyme activities in rat soleus muscles. A second goal was to determine whether activities of matrix metalloproteinase-2/9 (MMP-2/9) and urokinase-type/tissue-type plasminogen activator (PAs) were responsive to HS. As expected, HS led to a pronounced atrophy of soleus muscle. Level of ubiquitinated proteins, chymotrypsin-like activity of 20S proteasome, and Bcl-2-associated gene product-1 protein level were all transitory increased in response to 5 days of HS. These changes may thus potentially account for the decrease in muscle mass observed in response to 5 days of HS. Caspase-3 activity was significantly increased throughout the experimental period, whereas activities of caspase-6, another effector caspase, and caspase-9, the mitochondrial-dependent activator of both caspase-3 and -6, were only increased in response to 10 days of HS. This suggests that caspase-3 may be regulated through mitochondrial-independent and mitochondrial-dependent mechanisms in response to HS. Finally, MMP-2/9 activities remained unchanged, whereas PAs activities were increased after 5 days of HS. Overall, these data suggest that time-dependent regulation of intracellular and extracellular proteinases are important in setting the new phenotype of rat soleus muscle in response to HS.

The synergistic apoptotic effects of thiophenfurin, an inosine monophosphate dehydrogenase inhibitor, in combination with retinoids in HL60 cells

New effective cytotoxic agents and combinations are urgently needed in cancer treatment. The enzyme inosine monophosphate dehydrogenase is a potentially useful target for drug development, since its activity has been shown to be amplified in malignant cells. Thiophenfurin, an inhibitor of the enzyme synthesized by us, is endowed with a significant apoptotic activity in promyelocytic leukaemia HL60 cells. Since retinoids were successfully employed in the treatment of patients with leukaemia, demonstrating significant differentiation-inducing and apoptotic effects, we carried out this study to evaluate the effects of the combination of thiophenfurin and several retinoid molecules, acting in different phases of the cell cycle in vitro. The results show that thiophenfurin is capable of eliciting significant S phase-specific antiproliferative effects in different sensitive and resistant cell lines with the IC50s ranging from 6.7 to 26 microM. When HL60 cells were treated with thiophenfurin in combination with retinoids, the effects on cell growth were additive or synergistic, depending on the kind of retinoid used and the sequence of treatment. In particular, we observed additive effects when the cells were exposed to thiophenfurin and all-transretinoic acid either simultaneously or sequentially. Instead, when the new heterocyclic retinoid isoxazole benzoic acid was used, synergism was obtained in the cells treated sequentially. The combination of thiophenfurin and isoxazole benzoic acid determined synergistic apoptotic effects through a mitochondrion-dependent mechanism, suggesting the possible usefulness of this combination in the treatment of leukaemia.

A Critical Role for the Bcl-2 Pathway in Notch-Mediated B Cell Apoptosis, Where Growth Arrest Is Independent of the E2A, IL-3 and p21Waf1/p27Kip1 Pathways.

The Notch receptor pathway regulates critical cell fate decisions in multiple developmental systems, including hematopoiesis. We have previously demonstrated that Notch signaling induces growth arrest and apoptosis in a wide range of human B cell malignancies and has potential as a B cell-specific therapeutic approach. In order to identify the mechanisms of growth arrest and apoptosis we analyzed an immortalized murine progenitor B cell line derived from Bax/Bak double knockout mice. These cells are unable to undergo apoptosis since they lack the pro-apoptotic effectors of the Bcl-2 pathway, and have been shown to be resistant to multiple apoptotic stimuli.Here we report that induction of Notch signaling through expression of several family members (Notch1, Notch4, Hes1) leads to rapid growth arrest, but not apoptosis, within 48 hours in these Bax-/Bak- progenitor B cells. These findings provide the first evidence for a critical role of the Bcl-2 pathway in Notch-mediated B cell apoptosis, and establish a mitochondrial-dependent mechanism for this effect. Importantly, the kinetics of growth arrest are accelerated with the expression of the Notch downstream target Hes1 as compared to the Notch receptors 1 and 4. These results extend our observation that Hes1 is sufficient to reproduce Notch-mediated B cell death, by demonstrating that Hes1 is more proximal to the critical growth inhibiting events, and may therefore provide a therapeutic target.In this model system we can isolate growth arrest from the effects on the apoptotic cascade. This provides a unique opportunity to explore the mechanism of Notch-mediated growth arrest. Prior studies have suggested that Notch signaling may induce growth arrest through inhibition of the E2A pathway, or through upregulation of the cell cycle regulators p21Waf1 and p27Kip1. In this model system, inhibition of the E2A pathway is not sufficient to induce growth arrest. Similarly, Hes1 does not upregulate either p21Waf1 or p27Kip1, suggesting that this is not the mechanism of growth arrest. To explore whether Notch/Hes1 induce growth arrest through inhibition of the IL-3 pathway, we compared phenotypic and functional aspects of Hes1 expression and IL-3 withdrawal. Although the timing and phenotypic effects (cell size, cell cycle and metabolic studies) were quite similar, Hes1 growth arrested cells lose their ability to migrate in response to the pan-B chemo-attractant SDF1a compared to IL-3 withdrawn cells.In summary, these results demonstrate that Notch/Hes1-mediated B cell apoptosis relies critically on pro-apoptotic members of the Bcl-2 pathway, Bax/Bak. Furthermore, growth arrest when isolated from apoptosis does not rely on inhibition of the E2A or IL-3 pathways, nor upregulation of p21Waf1/ p27Kip1. These findings provide the first insight into the mechanisms of Notch/Hes1-mediated B cell growth arrest and apoptosis and will help guide the development of Notch/Hes1 signaling as a cell-type specific therapeutic approach for B cell malignancies.

Pro-apoptotic effect of a nitric oxide-donating NSAID, NCX 4040, on bladder carcinoma cells

Nitric oxide-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs) are a promising class of compounds that cause cell cycle perturbations and induce apoptosis in cell lines from different tumors. We investigated the activity of a recently developed NO-NSAID (NCX 4040) in bladder cancer cell lines (HT1376 and MCR). Cells were treated with different drug concentrations for different exposure times. Cytostatic and cytocidal activity was tested by SRB assay and apoptosis was evaluated by TUNEL analysis, ANNEXIN V assay and fluorescence microscopy. To further investigate the cell death-inducing mechanisms of NCX 4040, we analyzed gp-170, caspase expression and mitochondrial membrane potential (Delta Psi) depolarization. NCX 4040 showed a striking cytocidal activity in both cell lines, reaching LC(50) at a 10-microM and 50-microM concentrations in HT1376 and in MCR cells, respectively, after an exposure of only 6 h followed by an 18-h washout. Apoptosis was triggered in up to 90% of cells and was associated with active caspase-3 expression and Delta Psi depolarization in both cell lines after a 6-h exposure. In conclusion, NCX 4040, which probably causes apoptosis via a mitochondrial-dependent mechanism, could prove to be a useful agent for improving bladder cancer treatment.

The promyelocytic leukemia zinc finger protein down-regulates apoptosis and expression of the proapoptotic BID protein in lymphocytes.

The promyelocytic leukemia zinc finger (PLZF) gene, involved in rare cases of acute promyelocytic leukemia, encodes a Krüppel-type zinc finger transcription factor. It has been reported that PLZF affects myeloid cell growth, differentiation, and apoptosis. However, the function of PLZF in the lymphoid compartment, where PLZF is also expressed, remains largely unknown. To investigate a potential relationship between PLZF expression in lymphocytes and programmed cell death, an inducible model of stable clones of the lymphoid Jurkat cell line was created by using the tet-off system. Although induction of PLZF expression by itself did not produce changes in the basal levels of apoptosis, PLZF had a significant anti-apoptotic effect in Jurkat cells cultured in conditions of serum starvation, as measured by annexin V staining and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. In addition, retarded loss of mitochondrial transmembrane potential was observed in the PLZF-expressing clones, suggesting that PLZF protects from cell death through a mitochondrial-dependent mechanism. To identify apoptosis-related targets of PLZF, a screen for differential expression identified BID, a proapoptotic member of the Bcl2 family, as significantly down-regulated by PLZF. Furthermore, a high-affinity PLZF-binding site element was identified upstream of the BID transcriptional start site, as assessed by electrophoretic mobility-shift assays. These results suggest that BID is a target of PLZF repression and a candidate gene to mediate the PLZF-induced resistance to apoptosis.

Tributyltin interacts with mitochondria and induces cytochrome c release.

Although triorganotins are potent inducers of apoptosis in various cell types, the critical targets of these compounds and the mechanisms by which they lead to cell death remain to be elucidated. There are two major pathways by which apoptotic cell death occurs: one is triggered by a cytokine mediator and the other is by a mitochondrion-dependent mechanism. To elucidate the mechanism of triorganotin-induced apoptosis, we studied the effect of tributyltin on mitochondrial function. We found that moderately low doses of tributyltin decrease mitochondrial membrane potential and induce cytochrome c release by a mechanism inhibited by cyclosporine A and bongkrekic acid. Tributyltin-induced cytochrome c release is also prevented by dithiols such as dithiothreitol and 2,3-dimercaptopropanol but not by monothiols such as GSH, N-acetyl-L-cysteine, L-cysteine and 2-mercaptoethanol. Further studies with phenylarsine oxide agarose revealed that tributyltin interacts with the adenine nucleotide translocator, a functional constituent of the mitochondrial permeability transition pore, which is selectively inhibited by dithiothreitol. These results suggest that, at low doses, tributyltin interacts selectively with critical thiol residues in the adenine nucleotide translocator and opens the permeability transition pore, thereby decreasing membrane potential and releasing cytochrome c from mitochondria, a series of events consistent with established mechanistic models of apoptosis.