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Reactive oxygen species: Biological stimuli of neuroblastoma cell response
Cancer Letters 228 (2005) 111–116 www.elsevier.com/locate/canlet
Reactive oxygen species: Biological stimuli of neuroblastoma cell response Barbara Marengoa, Lizzia Raffaghellob, Vito Pistoiab, Damiano Cottalassoa, Maria Adelaide Pronzatoa, Umberto Maria Marinaria, Cinzia Domenicottia,* a
Department of Experimental Medicine, Section of General Pathology, University of Genova, Via L.B. Alberti, 2, 16132, Genoa, Italy b Laboratory of Oncology, G. Gaslini Institute, Genoa, Italy Received 24 November 2004; accepted 12 January 2005
Abstract Reactive oxygen species play a critical role in differentiation, proliferation and apoptosis acting as ‘second messengers’ able to regulate sulphydryl groups in signaling molecules as protein kinase C, a family of isoenzymes involved in many cellular responses and implicated in cell transformation. Neuroblastoma is characterised by the production of oxygen intermediates and L-buthionine-S,R-sulfoximine, a glutathione-depleting agent that has been tested in the clinics, exploits this biological peculiarity to induce cell death. The latter process is mediated by the oxidative activation of PKC delta which might be involved also in the production of reactive oxygen species, thus amplifying the apoptotic cascade. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neuroblastoma; Reactive oxygen species; Protein kinase C; Apoptosis; MYCN amplification
1. Introduction Neuroblastoma is a tumor of infancy [1] that presents a high rate of spontaneous regression, a phenomenon reflecting the activation of an apoptotic and/or differentiation program [2,3]. Cell proliferation and cell suicide are thought to be important mechanism for prevention of oncogenesis and it is believed that mutations responsible for deregulated growth can lead to neoplasia only when apoptosis has been suppressed. * Corresponding author. Tel.: C39 010 353 88 35; fax: C39 010 353 88 36. E-mail address: [email protected] (C. Domenicotti).
Studies on biological and genetic features of neuroblastoma are important to define the molecular pathways that contribute to malignant transformation or progression in order to discover new possible therapeutic strategies. In this Mini-Review, we have focused on oxygen and reactive oxygen species as biological stimuli able to drive neuroblastoma cell growth and apoptosis.
2. Biology of neuroblastoma and role of oxygen in tumour cell response Neuroblastoma is the most common solid tumour in childhood. It derives from primitive cells of
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.01.046
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the sympathetic nervous system and shows remarkable biological and clinical heterogeneity. The prognosis of neuroblastoma is grim in approximately 50% of patients presenting with poorly differentiated metastatic tumours (stroma poor neuroblastoma) at diagnosis. The remaining cases show localised presentation with more differentiated tumors (ganglioneuroblastoma or ganglioneuroma), usually associated with favourable prognosis. Neuroblastoma can also spontaneously regress [4]. In order to shed new light on molecular mechanisms responsible for tumour progression or spontaneous regression, genetic alterations that characterize neuroblastoma are studied. In this context, MYCN amplification confers to neuroblastoma a worse prognosis with a more aggressive development [1,5] and represents so far the only genetic parameter used for treatment stratification [6] and mentioned in the International Neuroblastoma Staging Systems (INSS) [7]. MYCN amplification has been demonstrated to determine the inability of neuroblastoma cells to start apoptotic death induced by tumour necrosis factorrelated apoptosis inducing ligand (TRAIL) system [8]. Dysregulation of the apoptotic process and increased cell proliferation contribute to malignant transformation and it has been found that changes in cell redox state may be associated with the transformed phenotype. Neuroblastoma has been reported to produce oxygen intermediates during synthesis of catecholamines and to store high amounts of reduced iron that can interact with hydrogen peroxide to produce superoxide radicals via Fenton reaction [9]. Cell survival to oxidative stress depends on the presence of antioxidants such as glutathione (GSH), crucial to counter endogenous production of reactive oxygen species (ROS). The discovery of L-buthionine-S,R-sulfoximine (BSO) has provided a tool to test the effects of GSH depletion on cell responses. This substance is a selective inhibitor of g-glutamilcysteine synthetase, a rate-limiting enzyme in GSH biosynthesis [10,11]. Clinical trials in adults and children have documented that steady–state plasma concentrations during continuous intravenous infusion of BSO range from 300 to 500 mM concentration. Yang and collaborators [12] have demonstrated that BSO-mediated GSH depletion in neuroblastoma
cell lines cultured in normoxia (20% oxygen) caused apoptosis as a consequence of basal ROS level increase. Exposure of neuroblastoma cell lines to hypoxic conditions (2% oxygen) close to those commonly found in bone and bone marrow showed that cell lines were insensitive to BSO since hypoxia antagonized ROS generation and apoptosis. In fact, it has been shown that hypoxia decreases tumours sensitivity to chemotherapy and higher capability of invasion and metastasis [13]. Moreover, hypoxia altered the expression of differentiation marker genes in neuroblastoma cells by down-regulating genes responsible for neuronal phenotype and upregulating genes associated with a neural crest-like phenotype, with an adverse clinical impact [14]. High-risk neuroblastoma recurring after intensive therapy metastasises to high oxygen tension sites such as the lungs and central nervous system [15] and hypoxic tissues may serve as sanctuaries for neuroblastoma cells, consistent with their decreased sensitivity to chemotherapy and radiotherapy.
3. ROS: biological messengers in signal transduction All aerobic cells require oxygen for life, but paradoxically, the by-product of biological processes is represented by ROS which include free radicals such as the superoxide anion, hydroxyl radicals and hydrogen peroxide [16]. They have high chemical reactivity and can damage a variety of biomolecules, such as lipids, proteins, carbohydrates and nucleic acids. Therefore, cells have developed several defence and repair mechanisms to deal with oxidative stress: antioxidants represent the first line of defence [17] whose failure promotes proteolytic degradation of oxidatively modified proteins [18] and culminates into apoptosis of irreversibly damaged cells. Recent studies imply that ROS, although toxic, can play essential role in cell biology and physiology [19]. Therefore, as result of dynamic interplay between ROS generation and antioxidant action, high concentrations of ROS are pathogenic, while moderate amounts of oxidant species take part in cell regulation, acting as mediators and signal transduction molecules [20]. The dosage of the redox signal is important in the modulation of cell function: indeed, the cell cycle
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is arrested when cells are exposed to moderate levels of oxidative stress, whereas very low doses of ROS stimulate cell proliferation. The mitogenic signals mediated through the generation of ROS activate redox sensitive transcription factors influencing molecular and biochemical processes responsible for changes observed during cell differentiation, senescence and transformation [21]. The primary target of this redox regulation may be a sulphydryl group (RSH) on cysteine residues of critical molecules. Oxygen free radicals derived from cell exposure to radiations, hydrogen peroxide, chemotherapeutic agents and from cell stimulation with inflammatory cytokines have been shown to modulate the activity of many signaling proteins [22] including MAP kinases [23], phospholipase C g1 [24] and protein kinase C [25]. MAPKs are a well studied signal transduction system which transduce signals for cell proliferation/differentiation and cell survival/ death. Activated phospholipase C (PLC) g1 catalyzes the hydrolysis of phosphatidylinositol 4,5-diphosphate to inositol 1,4,5-triphosphate and diacylglycerol, which act as second messengers inducing the mobilization of Ca2C and activation of protein kinase C (PKC), respectively [26].
4. Oxidative stress and PKC PKC is a family of structurally correlated enzymatic isoforms that are differentially involved in the transduction of signals for cell proliferation, differentiation and apoptosis [27]. PKC isoforms are classified into three subfamilies on the basis of sequence similarities and modes of activation: conventional or classic (a, bI, bII, g) PKCs, which are calciumdependent and stimulated by second-messenger diacylglycerol (DAG); novel (d, 3, h, q) PKCs, which are calcium-independent but stimulated by DAG; and atypical (i, l, z) PKCs, which require neither calcium nor DAG for optimal activity [28]. PKC is subjected to a complicated cellular redox regulation; this enzyme is activated by phorbol 12-myristate 13 acetate (PMA) and is regulated by phosphorylation, lipids and calcium [29]. Treatment of cells with H2O2 [30] and redox-cycling quinones [31] leads to a stimulation of PKC activity. Moreover, selective oxidative modification at the amino-terminal
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regulatory domain led to PKC activation [32] while alteration at the carboxy-terminal catalytic domain resulted in complete inactivation of the kinase [33]. Our previous in vivo and in vitro studies [34–37] have confirmed the biphasic behaviour of PKC in response to different oxidative injuries; in fact, high doses of pro-oxidant compounds (carbon tetrachloride and ethanol) cause hepatic PKC inactivation and proteolytic degradation, while low doses induce stimulation of kinase activity. Ward et al. [38] have suggested that depletion of GSH during oxidative stress removes a mechanism for negative regulation of PKC and, consequentely, provides a permissive environment for PKC activity and tumor promotion. In a previous work, we have demonstrated that a decrease in liver GSH, induced by BSO or diethylmaleate (DEM) treatment, was accompanied by inactivation of classic PKCs and increased activity of PKC-d, a pro-apoptotic isoenzyme [39]. In agreement with these findings, Chu and coll. [40] have reported that PKC-d was stimulated by GSSG and cystine, while the oncogenic isoenzyme PKC-3 was fully inactivated. Demonstration of oxidative regulation of cellular PKC-d and PKC-3 might offer novel targets for development of cancer preventive or therapeutic agents that selectively inactivate 3 or stimulate d isoform [41]. To further investigate the molecular mechanisms of cell response to GSH loss, we exposed SK-N-BE-2C human neuroblastoma cells to BSO which has been demonstrated to be efficacious in neuroblastoma treatment [9]. Longer times (24 h) of BSO treatment (1 mM) were characterized by marked (44%) ROS generation, and by a shift in the GSH/GSSG balance; these oxidative events were accompanied by selective stimulation (5-fold) of PKC-d activity and inactivation (60%) of PKC-a [42,43]. Previous studies of Tonini et al. [44] have reported that a decrease in PKC-a expression was associated with down regulation of MYCN mRNA accompanied by the reversion of the malignant phenotype. Recently, we have observed that two MYCN amplified neuroblastoma cell lines [45] (LAN-5 and SK-N-BE-2-C) showed basal ROS production three times higher than that detected in three MYCN nonamplified cell lines (SHSY5Y, GIMEN and ACN). Moreover, we have found that LAN5 and SK-NBE-2C cells presented 2.5 and 4 times higher ratio of
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PKC-d activity/level, respectively, than ACN, SHSY5Y and GIMEN cells. Taken together these findings confirm a correlation between intracellular ROS levels and the enhanced activity of the proapoptotic kinase.
5. Apoptosis triggered by ROS in neuroblastoma therapy Apoptosis is a cell death program that plays a crucial role in the physiological regulation of tissue development and homeostasis. Imbalance between cell death and proliferation is involved in the pathogenesis of many disorders: thus, for example, excessive apoptosis is associated with neurodegenerative diseases, while defective apoptosis contribute to cancer development [46,47]. MYCN amplified neuroblastoma is characterized by an apoptotic defect due to a down-regulation of caspase-8, which strongly correlates with TRAIL unresponsiveness. This lack of expression is associated with hypermethylation of caspase-8 promoter and results into more aggressive neuroblastoma development [8,48]. Apoptosis can also develop through the activation of caspase 9 involving the release of mitochondrial cytochrome c into the cytosol. Tumour cell apoptosis induced by chemotherapeutic drugs is considered to involve mainly the mitochondrial pathway [49]. Many MYCN amplified tumours manifest ab initio or acquire chemoresistance and consequently, novel therapeutic strategies must be addressed to interfere with these pathways in order to restore sensitivity of cells to MYCN and to traditional anti-neoplastic drugs [50]. Drugs that generate ROS can be used in cancer treatment since they may enhance the antitumour effect of radiation therapy. Redox metabolism, the homeostasis of ROS and their detoxification, are critical in cell signalling and in the regulation of programmed cell death. An increase in the generation of ROS, or a reduction in antioxidant defences, such as depletion of GSH, results in the induction of mitochondrial pathway of apoptosis [51,52]. Our recent reports have shown that BSO treatment of SK-N-BE-2-C (MYCN amplified) cells associated with ROS production and PKC-d activation, induced
morphological changes characteristic of early apoptosis (20% annexin-V-positive cells). Pre-treatment with Rottlerin, a selective PKC-delta inhibitor, and with Vitamin C, a known antioxidant, were able to suppress ROS generation and apoptosis of neuroblastoma cells [42,43]. These results suggest that the increase in ROS production and in pro-apoptotic PKC isoform activity are two necessary steps that cells activate in order to trigger the apoptotic process. A large body of evidence implicates PKC-d in cell death even if this isoenzyme has been also defined a ‘co-oncogene’ for its role in cell proliferation [53]. The pro-apoptotic effects of PKC-d in response to cytotoxic drugs have been attributed to its ability to translocate and cleave caspases. Activated and/or cleaved PKC-d could participate in phosphorylation and inactivation of nuclear enzymes responsible for DNA repair [54] or in the phosphorylation of laminin, which has been shown to contribute to apoptosis induced by chemotherapeutic agents [55]. Alternatively, PKC-d could participate in chemotherapy-induced apoptosis through activation of mitochondrial damage [56] and in support of this hypothesis, we have recently demonstrated that BSO exposure of neuroblastoma cells induces translocation of PKC-d from cytosol to the mitochondria [42].
6. Conclusions and future directions The production of ROS consequent to GSH depletion induced by BSO can trigger mitochondrial pathway of apoptosis in neuroblastoma cells with
Fig. 1. Modulation of intracellular ROS levels influences both upstream and downstream PKC-d activity triggering mitochondrial apoptosis in MYCN amplified neuroblastoma cells characterized by unresponsiveness to extrinsic pathway.
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MYCN amplification. This process is mediated by the oxidative activation of the pro-apoptotic PKC delta which might be involved also in the production of ROS amplifying the apoptotic cascade (Fig. 1). Future studies will be addressed to alter the expression of PKC isoenzymes, utilising antisense and/or over expression techniques, in order to induce cancer cell death; these strategies would offer hope of increasing the efficacy of chemotherapy for neuroblastoma treatment. Acknowledgements This work was supported by grants from the Italian Ministry of Health (Progetti Ricerca Corrente) and Compagnia di S. Paolo, from Genoa University (ex 60%) and from the Italian Ministry of University: COFIN 2002 #2002063817_001 (National co-ordinator Prof. U.M. Marinari).
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Reactive oxygen species: Biological stimuli of neuroblastoma cell response Barbara Marengoa, Lizzia Raffaghellob, Vito Pistoiab, Damiano Cottalassoa, Maria Adelaide Pronzatoa, Umberto Maria Marinaria, Cinzia Domenicottia,* a
Department of Experimental Medicine, Section of General Pathology, University of Genova, Via L.B. Alberti, 2, 16132, Genoa, Italy b Laboratory of Oncology, G. Gaslini Institute, Genoa, Italy Received 24 November 2004; accepted 12 January 2005
Abstract Reactive oxygen species play a critical role in differentiation, proliferation and apoptosis acting as ‘second messengers’ able to regulate sulphydryl groups in signaling molecules as protein kinase C, a family of isoenzymes involved in many cellular responses and implicated in cell transformation. Neuroblastoma is characterised by the production of oxygen intermediates and L-buthionine-S,R-sulfoximine, a glutathione-depleting agent that has been tested in the clinics, exploits this biological peculiarity to induce cell death. The latter process is mediated by the oxidative activation of PKC delta which might be involved also in the production of reactive oxygen species, thus amplifying the apoptotic cascade. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neuroblastoma; Reactive oxygen species; Protein kinase C; Apoptosis; MYCN amplification
1. Introduction Neuroblastoma is a tumor of infancy [1] that presents a high rate of spontaneous regression, a phenomenon reflecting the activation of an apoptotic and/or differentiation program [2,3]. Cell proliferation and cell suicide are thought to be important mechanism for prevention of oncogenesis and it is believed that mutations responsible for deregulated growth can lead to neoplasia only when apoptosis has been suppressed. * Corresponding author. Tel.: C39 010 353 88 35; fax: C39 010 353 88 36. E-mail address: [email protected] (C. Domenicotti).
Studies on biological and genetic features of neuroblastoma are important to define the molecular pathways that contribute to malignant transformation or progression in order to discover new possible therapeutic strategies. In this Mini-Review, we have focused on oxygen and reactive oxygen species as biological stimuli able to drive neuroblastoma cell growth and apoptosis.
2. Biology of neuroblastoma and role of oxygen in tumour cell response Neuroblastoma is the most common solid tumour in childhood. It derives from primitive cells of
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.01.046
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the sympathetic nervous system and shows remarkable biological and clinical heterogeneity. The prognosis of neuroblastoma is grim in approximately 50% of patients presenting with poorly differentiated metastatic tumours (stroma poor neuroblastoma) at diagnosis. The remaining cases show localised presentation with more differentiated tumors (ganglioneuroblastoma or ganglioneuroma), usually associated with favourable prognosis. Neuroblastoma can also spontaneously regress [4]. In order to shed new light on molecular mechanisms responsible for tumour progression or spontaneous regression, genetic alterations that characterize neuroblastoma are studied. In this context, MYCN amplification confers to neuroblastoma a worse prognosis with a more aggressive development [1,5] and represents so far the only genetic parameter used for treatment stratification [6] and mentioned in the International Neuroblastoma Staging Systems (INSS) [7]. MYCN amplification has been demonstrated to determine the inability of neuroblastoma cells to start apoptotic death induced by tumour necrosis factorrelated apoptosis inducing ligand (TRAIL) system [8]. Dysregulation of the apoptotic process and increased cell proliferation contribute to malignant transformation and it has been found that changes in cell redox state may be associated with the transformed phenotype. Neuroblastoma has been reported to produce oxygen intermediates during synthesis of catecholamines and to store high amounts of reduced iron that can interact with hydrogen peroxide to produce superoxide radicals via Fenton reaction [9]. Cell survival to oxidative stress depends on the presence of antioxidants such as glutathione (GSH), crucial to counter endogenous production of reactive oxygen species (ROS). The discovery of L-buthionine-S,R-sulfoximine (BSO) has provided a tool to test the effects of GSH depletion on cell responses. This substance is a selective inhibitor of g-glutamilcysteine synthetase, a rate-limiting enzyme in GSH biosynthesis [10,11]. Clinical trials in adults and children have documented that steady–state plasma concentrations during continuous intravenous infusion of BSO range from 300 to 500 mM concentration. Yang and collaborators [12] have demonstrated that BSO-mediated GSH depletion in neuroblastoma
cell lines cultured in normoxia (20% oxygen) caused apoptosis as a consequence of basal ROS level increase. Exposure of neuroblastoma cell lines to hypoxic conditions (2% oxygen) close to those commonly found in bone and bone marrow showed that cell lines were insensitive to BSO since hypoxia antagonized ROS generation and apoptosis. In fact, it has been shown that hypoxia decreases tumours sensitivity to chemotherapy and higher capability of invasion and metastasis [13]. Moreover, hypoxia altered the expression of differentiation marker genes in neuroblastoma cells by down-regulating genes responsible for neuronal phenotype and upregulating genes associated with a neural crest-like phenotype, with an adverse clinical impact [14]. High-risk neuroblastoma recurring after intensive therapy metastasises to high oxygen tension sites such as the lungs and central nervous system [15] and hypoxic tissues may serve as sanctuaries for neuroblastoma cells, consistent with their decreased sensitivity to chemotherapy and radiotherapy.
3. ROS: biological messengers in signal transduction All aerobic cells require oxygen for life, but paradoxically, the by-product of biological processes is represented by ROS which include free radicals such as the superoxide anion, hydroxyl radicals and hydrogen peroxide [16]. They have high chemical reactivity and can damage a variety of biomolecules, such as lipids, proteins, carbohydrates and nucleic acids. Therefore, cells have developed several defence and repair mechanisms to deal with oxidative stress: antioxidants represent the first line of defence [17] whose failure promotes proteolytic degradation of oxidatively modified proteins [18] and culminates into apoptosis of irreversibly damaged cells. Recent studies imply that ROS, although toxic, can play essential role in cell biology and physiology [19]. Therefore, as result of dynamic interplay between ROS generation and antioxidant action, high concentrations of ROS are pathogenic, while moderate amounts of oxidant species take part in cell regulation, acting as mediators and signal transduction molecules [20]. The dosage of the redox signal is important in the modulation of cell function: indeed, the cell cycle
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is arrested when cells are exposed to moderate levels of oxidative stress, whereas very low doses of ROS stimulate cell proliferation. The mitogenic signals mediated through the generation of ROS activate redox sensitive transcription factors influencing molecular and biochemical processes responsible for changes observed during cell differentiation, senescence and transformation [21]. The primary target of this redox regulation may be a sulphydryl group (RSH) on cysteine residues of critical molecules. Oxygen free radicals derived from cell exposure to radiations, hydrogen peroxide, chemotherapeutic agents and from cell stimulation with inflammatory cytokines have been shown to modulate the activity of many signaling proteins [22] including MAP kinases [23], phospholipase C g1 [24] and protein kinase C [25]. MAPKs are a well studied signal transduction system which transduce signals for cell proliferation/differentiation and cell survival/ death. Activated phospholipase C (PLC) g1 catalyzes the hydrolysis of phosphatidylinositol 4,5-diphosphate to inositol 1,4,5-triphosphate and diacylglycerol, which act as second messengers inducing the mobilization of Ca2C and activation of protein kinase C (PKC), respectively [26].
4. Oxidative stress and PKC PKC is a family of structurally correlated enzymatic isoforms that are differentially involved in the transduction of signals for cell proliferation, differentiation and apoptosis [27]. PKC isoforms are classified into three subfamilies on the basis of sequence similarities and modes of activation: conventional or classic (a, bI, bII, g) PKCs, which are calciumdependent and stimulated by second-messenger diacylglycerol (DAG); novel (d, 3, h, q) PKCs, which are calcium-independent but stimulated by DAG; and atypical (i, l, z) PKCs, which require neither calcium nor DAG for optimal activity [28]. PKC is subjected to a complicated cellular redox regulation; this enzyme is activated by phorbol 12-myristate 13 acetate (PMA) and is regulated by phosphorylation, lipids and calcium [29]. Treatment of cells with H2O2 [30] and redox-cycling quinones [31] leads to a stimulation of PKC activity. Moreover, selective oxidative modification at the amino-terminal
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regulatory domain led to PKC activation [32] while alteration at the carboxy-terminal catalytic domain resulted in complete inactivation of the kinase [33]. Our previous in vivo and in vitro studies [34–37] have confirmed the biphasic behaviour of PKC in response to different oxidative injuries; in fact, high doses of pro-oxidant compounds (carbon tetrachloride and ethanol) cause hepatic PKC inactivation and proteolytic degradation, while low doses induce stimulation of kinase activity. Ward et al. [38] have suggested that depletion of GSH during oxidative stress removes a mechanism for negative regulation of PKC and, consequentely, provides a permissive environment for PKC activity and tumor promotion. In a previous work, we have demonstrated that a decrease in liver GSH, induced by BSO or diethylmaleate (DEM) treatment, was accompanied by inactivation of classic PKCs and increased activity of PKC-d, a pro-apoptotic isoenzyme [39]. In agreement with these findings, Chu and coll. [40] have reported that PKC-d was stimulated by GSSG and cystine, while the oncogenic isoenzyme PKC-3 was fully inactivated. Demonstration of oxidative regulation of cellular PKC-d and PKC-3 might offer novel targets for development of cancer preventive or therapeutic agents that selectively inactivate 3 or stimulate d isoform [41]. To further investigate the molecular mechanisms of cell response to GSH loss, we exposed SK-N-BE-2C human neuroblastoma cells to BSO which has been demonstrated to be efficacious in neuroblastoma treatment [9]. Longer times (24 h) of BSO treatment (1 mM) were characterized by marked (44%) ROS generation, and by a shift in the GSH/GSSG balance; these oxidative events were accompanied by selective stimulation (5-fold) of PKC-d activity and inactivation (60%) of PKC-a [42,43]. Previous studies of Tonini et al. [44] have reported that a decrease in PKC-a expression was associated with down regulation of MYCN mRNA accompanied by the reversion of the malignant phenotype. Recently, we have observed that two MYCN amplified neuroblastoma cell lines [45] (LAN-5 and SK-N-BE-2-C) showed basal ROS production three times higher than that detected in three MYCN nonamplified cell lines (SHSY5Y, GIMEN and ACN). Moreover, we have found that LAN5 and SK-NBE-2C cells presented 2.5 and 4 times higher ratio of
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PKC-d activity/level, respectively, than ACN, SHSY5Y and GIMEN cells. Taken together these findings confirm a correlation between intracellular ROS levels and the enhanced activity of the proapoptotic kinase.
5. Apoptosis triggered by ROS in neuroblastoma therapy Apoptosis is a cell death program that plays a crucial role in the physiological regulation of tissue development and homeostasis. Imbalance between cell death and proliferation is involved in the pathogenesis of many disorders: thus, for example, excessive apoptosis is associated with neurodegenerative diseases, while defective apoptosis contribute to cancer development [46,47]. MYCN amplified neuroblastoma is characterized by an apoptotic defect due to a down-regulation of caspase-8, which strongly correlates with TRAIL unresponsiveness. This lack of expression is associated with hypermethylation of caspase-8 promoter and results into more aggressive neuroblastoma development [8,48]. Apoptosis can also develop through the activation of caspase 9 involving the release of mitochondrial cytochrome c into the cytosol. Tumour cell apoptosis induced by chemotherapeutic drugs is considered to involve mainly the mitochondrial pathway [49]. Many MYCN amplified tumours manifest ab initio or acquire chemoresistance and consequently, novel therapeutic strategies must be addressed to interfere with these pathways in order to restore sensitivity of cells to MYCN and to traditional anti-neoplastic drugs [50]. Drugs that generate ROS can be used in cancer treatment since they may enhance the antitumour effect of radiation therapy. Redox metabolism, the homeostasis of ROS and their detoxification, are critical in cell signalling and in the regulation of programmed cell death. An increase in the generation of ROS, or a reduction in antioxidant defences, such as depletion of GSH, results in the induction of mitochondrial pathway of apoptosis [51,52]. Our recent reports have shown that BSO treatment of SK-N-BE-2-C (MYCN amplified) cells associated with ROS production and PKC-d activation, induced
morphological changes characteristic of early apoptosis (20% annexin-V-positive cells). Pre-treatment with Rottlerin, a selective PKC-delta inhibitor, and with Vitamin C, a known antioxidant, were able to suppress ROS generation and apoptosis of neuroblastoma cells [42,43]. These results suggest that the increase in ROS production and in pro-apoptotic PKC isoform activity are two necessary steps that cells activate in order to trigger the apoptotic process. A large body of evidence implicates PKC-d in cell death even if this isoenzyme has been also defined a ‘co-oncogene’ for its role in cell proliferation [53]. The pro-apoptotic effects of PKC-d in response to cytotoxic drugs have been attributed to its ability to translocate and cleave caspases. Activated and/or cleaved PKC-d could participate in phosphorylation and inactivation of nuclear enzymes responsible for DNA repair [54] or in the phosphorylation of laminin, which has been shown to contribute to apoptosis induced by chemotherapeutic agents [55]. Alternatively, PKC-d could participate in chemotherapy-induced apoptosis through activation of mitochondrial damage [56] and in support of this hypothesis, we have recently demonstrated that BSO exposure of neuroblastoma cells induces translocation of PKC-d from cytosol to the mitochondria [42].
6. Conclusions and future directions The production of ROS consequent to GSH depletion induced by BSO can trigger mitochondrial pathway of apoptosis in neuroblastoma cells with
Fig. 1. Modulation of intracellular ROS levels influences both upstream and downstream PKC-d activity triggering mitochondrial apoptosis in MYCN amplified neuroblastoma cells characterized by unresponsiveness to extrinsic pathway.
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MYCN amplification. This process is mediated by the oxidative activation of the pro-apoptotic PKC delta which might be involved also in the production of ROS amplifying the apoptotic cascade (Fig. 1). Future studies will be addressed to alter the expression of PKC isoenzymes, utilising antisense and/or over expression techniques, in order to induce cancer cell death; these strategies would offer hope of increasing the efficacy of chemotherapy for neuroblastoma treatment. Acknowledgements This work was supported by grants from the Italian Ministry of Health (Progetti Ricerca Corrente) and Compagnia di S. Paolo, from Genoa University (ex 60%) and from the Italian Ministry of University: COFIN 2002 #2002063817_001 (National co-ordinator Prof. U.M. Marinari).
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