Apomorphine attenuates 6-hydroxydopamine-induced apoptotic cell death in SH-SY5Y cells

Abstract

Enhanced oxidative stress is implicated in the pathogenesis of Parkinson's disease. The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS), leading to neuronal cell death. On the other hand, apomorphine, a dopamine D1/D2 receptor agonist and known as a potent antioxidant, has been reported to have a neuroprotective effect. In the present study, we investigated the effect of apomorphine on 6-OHDA-induced apoptotic cell death using the human dopaminergic neuroblastoma cell line, SH-SY5Y. The co-treatment of cells with apomorphine significantly attenuated 6-OHDA-induced ROS generation, the phosphorylation of c-Jun N-terminal kinase (JNK), DNA fragmentation and subsequent apoptotic cell death. In addition, pretreatment with apomorphine for 24 h and the following concomitant treatment enhanced the protective effects against 6-OHDA-induced toxicity except for the attenuation of JNK phosphorylation. We also demonstrated that pretreatment alone with apomorphine for 24 h prior to the exposure confers resistance against 6-OHDA-induced cell toxicity. These findings suggested that apomorphine acts principally as a radical scavenger to suppress the level of ROS and ROS-stimulated apoptotic signaling pathway, whereas the other mechanisms might be involved in the protective effects.     

Baicalein attenuates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells

It has been suggested that baicalein, a flavonoid obtained from the Scutellaria root, exerts a protective role on neurons against several neuronal insults. However, the protective mechanisms underlying this protective effect remain largely unknown. Our results indicate that baicalein protects SH-SY5Y cells, a dopaminergic neuronal cell line, from 6-hydroxydopamine (6-OHDA)-induced damage by the attenuation of reactive oxygen species (ROS). In order to determine the effects of baicalein on mitochondrial events, mitochondrial membrane potentials (deltapsim) and caspase cascades downstream of mitochondria were assessed. Baicalein inhibited the collapse of deltapsim, suggesting that baicalein reduces the mitochondrial dysfunction associated with 6-OHDA treatment. Baicalein also inhibited caspase-9 and caspase-3 activation, which can be triggered by mitochondrial malfunctions. Furthermore, baicalein induced a significant reduction in the level of phospho-JNK, which is known as an apoptotic mediator in 6-OHDA-induced neuronal cell death. Our results indicate that baicalein protects neurons from the deleterious effects of 6-OHDA via the attenuation of oxidative stress, mitochondrial dysfunction, caspase activity, and JNK activation.     

Mitochondrial-targeted active Akt protects SH-SY5Y neuroblastoma cells from staurosporine-induced apoptotic cell death

Akt is a serine/threonine protein kinase that plays a vital role in promoting cellular survival. Predominantly cytosolic, upon stimulation with growth-factors or stress, active Akt translocates into mitochondria, but the functions of Akt in mitochondria are not yet fully understood. Mitochondria play a central role in apoptotic pathways and given Akt's functions in the cytoplasm, Akt in mitochondria may help preserve mitochondrial integrity during cellular stress. To test if the translocation of Akt into mitochondria is neuroprotective, adenoviral vectors expressing a constitutively active Akt, Ad-HA-Akt (DD), and a constitutively active Akt with a mitochondrial targeting signal, Ad-Mito-HA-Akt (DD), were generated. Human SH-SY5Y neuroblastoma cells expressing the adenoviral constructs were treated with staurosporine to initiate intrinsic apoptotic cell death and several aspects of the mitochondrial apoptotic pathway were evaluated. Expression of active Akt targeted to mitochondria was found to be sufficient to significantly reduce staurosporine-induced activation of caspase-3 and caspase-9, the release of cytochrome c from mitochondria, and Bax oligomerization at mitochondria. These findings demonstrate that intramitochondrial active Akt results in efficient protection against apoptotic signaling.     

Hydrogen sulfide protects SH-SY5Y cells against 6-hydroxydopamine-induced endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative diseases, including Parkinson's disease. The present study attempted to investigate the effect of hydrogen sulfide (H(2)S) on 6-hydroxydopamine (6-OHDA)-induced ER stress in SH-SY5Y cells. We found in the present study that exogenous application of sodium hydrosulfide (NaHS; an H(2)S donor, 100 μM) significantly attenuated 6-OHDA (50 μM)-induced cell death. NaHS also reversed the upregulation of cleaved poly(ADP-ribose) polymerase and caspase 9 in 6-OHDA-treated cells. Consistent with its cytoprotective effects, NaHS markedly reduced 6-OHDA induced-ER stress responses, including the upregulated levels of eukaryotic initiation factor-2α phosphorylation, glucose-regulated protein 78, and C/EBP homologous protein expression. The protective effect of H(2)S on ER stress was attenuated by blockade of Akt activity with an Akt inhibitor or inhibition of heat shock protein (Hsp)90 with geldanamycin but not by suppression of ERK1/2 with PD-98059. Blockade of Akt also significantly decreased the protein abundance of Hsp90 in SH-SY5Y cells. Moreover, overexpression of cystathionine β-synthase (a main H(2)S-synthesizing enzyme in the brain) elevated the Hsp90 protein level and suppressed 6-OHDA-induced ER stress. In conclusion, the protective effect of H(2)S against 6-OHDA-induced ER stress injury in SH-SY5Y cells involves the Akt-Hsp90 pathway.     

Protective effect of orexin-A on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by progressive and selective death of midbrain dopaminergic neurons. Pharmacologic treatment of PD can be divided into symptomatic and neuroprotective therapies.     

Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.     

Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress

Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.     

Vanadate protects human neuroblastoma SH-SY5Y cells against peroxynitrite-induced cell death

We investigated the effect of vanadate, a tyrosine phosphatase inhibitor, on cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Vanadate prevented cell death induced by 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor; whereas SIN-1-induced cell death was not prevented by neither okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, nor cyclosporin A, an inhibitor of serine/threonine phosphatase 2B. Vanadate did not prevent cell death induced by N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine, a nitric oxide donor. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not block the protective effect of vanadate, suggesting that the protective effect of vanadate is independent on PI3-kinase. Vanadate increased tyrosine phosphorylation of several proteins including the focal adhesion protein p130 Crk-associated substrate (p130(cas)). By the treatment with SIN-1, the endogenous association of p130(cas) and Crk was disrupted, and the association was restored by vanadate treatment. These results suggest that disruption of tyrosine phosphorylation signaling may be critical for peroxynitrite-induced cell death, and that vanadate prevents cell death at least in part through the enhancement in tyrosine phosphorylation of the proteins including p130(cas).     

GRP94 reduces cell death in SH-SY5Y cells perturbated calcium homeostasis

The endoplasmic reticulum (ER) resident-94 kDa glucose-regulated protein (GRP94), plays a pivotal role in cell death due to ER stress. In our study expression of GRP94 was increased in human neuroblastoma SH-SY5Y cells due to exposure to calcium ionophore A23187. A23187-mediated cell death was associated with activation of the major cysteine proteases, caspase-3 and calpain. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) reduced viability of SH-SY5Y cells subjected to A23187 treatment compared with wild type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicated that suppression of GRP94 is associated with accelerated cell death. Moreover, expression of GRP94 suppressed A23187-induced cell death and stabilized calcium homeostasis.     

Protective effects of pinostilbene,a resveratrol methylated derivative,against 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells

Resveratrol (3,4′,5-trans-trihydroxystilbene) is a phytoalexin with emerging lines of evidence supporting its beneficial effects on cardiovascular systems and inhibition of carcinogenesis. It has also been reported that certain methylated resveratrol derivatives are more effective than resveratrol in the prevention/treatment of cancer. However, little is known about the impact of resveratrol and its derivatives on the development of Parkinson's disease. In this study, we compared the neuroprotective effects of resveratrol with four methylated (fully or partially) resveratrol derivatives against parkinsonian mimetic 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells. Release of lactate dehydrogenase and activity of caspase-3 triggered by 6-OHDA were significantly reduced by resveratrol and one of the methylated derivatives, pinostilbene (3,4′-dihydroxy-5-methoxystilbene), in a dose-dependent manner. In addition, pinostilbene exerted a potent neuroprotective effect with a wider effective concentration range than resveratrol. By using high-performance liquid chromatography, we found that uptake of pinostilbene into SH-SY5Y cells was significantly higher than that of resveratrol. Enhanced bioavailability may thus be a major factor contributing to the neuroprotective activity of pinostilbene. Moreover, Western blot analysis demonstrated that pinostilbene markedly attenuated the phosphorylation of JNK and c-Jun triggered by 6-OHDA. Besides, mammalian target of rapamycin kinase may be an intracellular target accounting for the neuroprotective effects of pinostilbene. Our findings demonstrate the potential of methylated stilbenes in neuroprotection and provide important information for further research in this field.     

Neuroprotective effects of SCM198 on 6-hydroxydopamine-induced behavioral deficit in rats and cytotoxicity in neuronal SH-SY5Y cells

6-Hydroxydopamine (6-OHDA), one of the most investigated Parkinson's disease neurotoxins, is widely used to study mechanisms of cell death in dopaminergic neurons. In the present study, we demonstrated that SCM198, a new compound based on the active component of Herba leonuri, significantly reduced 6-OHDA-induced cell death in dopaminergic SH-SY5Y cells and attenuated apomorphine-elicited rotational behavior in 6-OHDA-lesioned rats. Pretreatment with SCM198 (0.001, 0.01, 0.1, 1, and 10 μM) concentration-dependently increased the cell viability as measured in MTT and LDH leakage assays compared with 6-OHDA-injured cells. Tocopherol, an antioxidant used as positive control, had similar effect at 10 μM to SCM198 1 μM. Furthermore, we assessed oxidative stress and subsequent apoptosis, the critical players in dopaminergic neurodegeneration, with 0.1, 1, and 10 μM of SCM198 in SH-SY5Y cells exposed to 6-OHDA. Pretreatment with SCM198 significantly increased antioxidant enzyme superoxide dismutase activity, ameliorated intracellular reactive oxygen species generation, prevented the dissipation of mitochondrial membrane potential, decreased apoptotic cell death in Hoechst 33258 staining, as well as down-regulated Bax and up-regulated Bcl-2 in both mRNA and protein levels compared with 6-OHDA damaged cells. Moreover, intragastrical administration of SCM198 (18 or 60 mg kg?1 day?1) for 4 weeks significantly ameliorates apomorphine-induced contralateral rotations in 6-OHDA-lesioned rats. These results support the neuroprotective effects of SCM198 against 6-OHDA-induced toxicity in vivo and in vitro with the underlying mechanisms of inhibiting oxidative stress and apoptosis. Therefore we suggest that SCM198 might provide a useful therapeutic strategy for neurodegenerative diseases such as Parkinson's disease.     

Dosage effects of EGb761 on hydrogen peroxide-induced cell death in SH-SY5Y cells

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, is one of the most popular herbal supplements, taken for its multivalent properties. In this study, dosage effects of EGb761 on hydrogen peroxide (H₂O₂)-induced apoptosis of human neuroblastoma SH-SY5Y cells were investigated. It was found that H₂O₂-induced apoptotic cell death in SH-SY5Y cells, which was revealed in DNA fragmentation, mitochondrial membrane potential depolarization, and activation of Akt, c-Jun N-terminal kinases (JNK) and caspase 3. Low doses of EGb761 (50–100 μg/ml) inhibited H₂O₂-induced cell apoptosis via inactivation of Akt, JNK and caspase 3 while high doses of EGb761 (250–500 μg/ml) enhanced H₂O₂ toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments revealed that H₂O₂ decreased intracellular GSH content, which was also inhibited by low concentrations of EGb761 but enhanced after high concentrations of EGb761 treatment. This further suggests to us that dosage effects of EGb761 on apoptotic signaling proteins may be correlated with regulation of cell redox state. Therefore, treatment dosage may be one of the vital factors that determine the specific action of EGb761 on oxidative stress-induced cell apoptosis. To understand the mechanisms of dosage effects of EGb761 may have important clinical implications.     

Erythropoietin protects against 6-hydroxydopamine-induced dopaminergic cell death

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopaminergic neurons. In the present study, erythropoietin, a trophic factor that has both hematopoietic and neural protective characteristics, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. Using both the dopaminergic cell line, MN9D, and primary dopamine neurons, we show that erythropoietin (1-3 U/mL) is neuroprotective against the dopaminergic neurotoxin, 6-hydroxydopamine. Protection was mediated by the erythropoietin receptor, as neutralizing anti-erythropoietin receptor antibody abrogated the protection. Activation of Akt/protein kinase B (PKB), via the phosphoinositide 3-kinase pathway, is a critical mechanism in erythropoietin-induced protection, while activation of extracellular signal-regulated kinase (ERK)1/2 contributes only moderately. Indeed, transfection of constitutively active Akt/PKB into dopaminergic cells was sufficient to protect against cell death. Furthermore, erythropoietin diminished markers of apoptosis in MN9D cells, including caspase 9 and caspase 3 activation and internucleosomal DNA fragmentation, suggesting that erythropoietin interferes with the apoptosis-execution process. When erythropoietin was administered to mice unilaterally lesioned with 6-hydroxydopamine, it prevented the loss of nigral dopaminergic neurons and maintained striatal catecholamine levels for at least 8 weeks. Erythropoietin-treated mice also had significantly reduced behavioral asymmetries. These studies suggest that erythropoietin can be an effective neuroprotective agent for dopaminergic neurons, and may be useful in reversing behavioral deficits associated with Parkinson's disease.     

Methadone induces necrotic-like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis

Methadone is a widely used therapeutic opioid in narcotic addiction and neuropathic pain syndromes. Oncologists regularly use methadone as a long-lasting analgesic. Recently it has also been proposed as a promising agent in leukemia therapy, especially when conventional therapies are not effective. Nevertheless, numerous reports indicate a negative impact on human cognition with chronic exposure to opiates. Thus, clarification of methadone toxicity is required. In SH-SY5Y cells we found that high concentrations of methadone were required to induce cell death. Methadone-induced cell death seems to be related to necrotic processes rather than typical apoptosis. Cell cultures challenged with methadone presented alterations in mitochondrial outer membrane permeability. A mechanism that involves Bax translocation to the mitochondria was observed, accompanied with cytochrome c release. Furthermore, no participation of known protein regulators of apoptosis such as Bcl-X_L and p53 was observed. Interestingly, methadone-induced cell death took place by a caspases-independent pathway; perhaps due to its ability to induce a drastic depletion in cellular ATP levels. Therefore, we studied the effect of methadone on isolated rat liver mitochondria. We observed that methadone caused mitochondrial uncoupling, coinciding with the ionophoric properties of methadone, but did not cause swelling of the organelles. Overall, the effects observed for cells in the presence of supratherapeutic doses of methadone may result from a “bioenergetic crisis.” A decreased level of cellular energy may predispose cells to necrotic-like cell death.     

Bcl-x L blocks mitochondrial multiple conductance channel activation and inhibits 6-OHDA-induced death in SH-SY5Y cells

Apoptosis is an active process that is regulated by different signalling pathways. One of the more important organelles involved in apoptosis regulation is the mitochondrion. Electron chain transport disruption increases free radical production leading to multiple conductance channel opening, release of cytochrome c and caspase activation. This death pathway can be blocked by anti-apoptotic members of the Bcl-2 protein family that might shift redox potential to a more reduced state, preventing free radical-mediated damage. 6-Hydroxydopamine (6-OHDA) has been widely used to generate Parkinson's disease-like models. It is able to generate free radicals and to induce catecholaminergic cell death. In this paper we have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-x(L) as a model to gain insights into the mechanisms through which Bcl-x(L) blocks 6-OHDA-induced cell death and to identify the molecular targets for this action. Herein, we present evidence supporting that the Bcl-x(L)-anti-apoptotic signal pathway seems to prevent mitochondrial multiple conductance channel opening, cytochrome c release and caspase-3 like activity following 6-OHDA treatment in the human neuroblastoma cell line SH-SY5Y.     

Protective role of tissue transglutaminase in the cell death induced by TNF-alpha in SH-SY5Y neuroblastoma cells

Tissue transglutaminase (tTGase) regulates various biological processes, including extracellular matrix organization, cellular differentiation, and apoptosis. Here we report the protective role of tTGase in the cell death that is induced by the tumor necrosis factor alpha (TNF-alpha) and ceramide, a product of the TNF-alpha signaling pathway, in human neuroblastoma SH-SY5Y cells. Treatment with retinoic acid (RA) induced the differentiation of the neuroblastoma cells with the formation of extended neurites. Immunostaining and Western blot analysis showed the tTGase expression by RA treatment. TNF-alpha or C(2) ceramide, a cell permeable ceramide analog, induced cell death in normal cells, but cell death was largely inhibited by the RA treatment. The inhibition of tTGase by the tTGase inhibitors, monodansylcadaverine and cystamine, eliminated the protective role of RA-treatment in the cell death that is caused by TNF-alpha or C(2)-ceramide. In addition, the co-treatment of TNF-alpha and cycloheximide decreased the protein level of tTGase and cell viability in the RA-treated cells, supporting the role of tTGase in the protection of cell death. DNA fragmentation was also induced by the co-treatment of TNF-alpha and cycloheximide. These results suggest that tTGase expressed by RA treatment plays an important role in the protection of cell death caused by TNF-alpha and ceramide.     

Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death

Insulin-like growth factor I (IGF-I) and the type I IGF receptor are widely distributed in developing and adult mammalian nervous systems. In vitro, IGF-I is a mitogen for primary neurons and also for cells from the SH-SY5Y human neuroblastoma cell line, a well-characterized model system of neuronal growth. In the current study, we examined the effects of osmotic stress on SH-SY5Y cell viability and the mechanism by which IGF-I serves as a neuronal osmoprotectant. Within 24 hr, exposure of SH-SY5Y cells to hyperosmotic serum-free media decreased (1) the number of viable cells, (2) the rate of ³H-thymidine incorporation, and (3) cell cycle progression. The inclusion of 10 nM IGF-I with hyperosmotic media prevented the loss of cell viability. The osmoprotective effects of IGF-I were inhibited by α-IR_J, a blocking antibody of the type I IGF receptor. The observed loss of SH-SY5Y cell viability following hyperosmotic shock was due to an induction of programmed cell death as determined by flow cytometry and gel electrophoresis. Our results suggest that IGF-I can protect SH-SY5Y cells from hyperosmotic induced programmed cell death. © 1996 Wiley-Liss, Inc.     

Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway

Inhibition of proteasome activity and the resulting protein accumulation are now known to be important events in the development of many neurological disorders, including Alzheimer’s and Parkinson’s diseases. Abnormal or over expressed proteins cause endoplasmic reticulum and oxidative stress leading to cell death, thus, normal proteasome function is critical for their removal. We have shown previously, with cultured SH-SY5Y neuroblastoma cells, that proteasome inhibition by the drug epoxomicin results in accumulation of ubiquitinated proteins. This causes obligatory loading of the mitochondria with calcium (Ca²⁺), resulting in mitochondrial damage and cytochrome c release, followed by programmed cell death (PCD). In the present study, we demonstrate that all-trans-retinoic acid (RA) pretreatment of SH-SY5Y cells protects them from PCD death after subsequent epoxomicin treatment which causes proteasome inhibition. Even though ubiquitinated protein aggregates are present, there is no evidence to suggest that autophagy is involved. We conclude that protection by RA is likely by mechanisms that interfere with cell stress-PCD pathway that otherwise would result from protein accumulation after proteasome inhibition. In addition, although RA activates both the AKT and ERK phosphorylation signaling pathways, only pretreatment with LY294002, an inhibitor of PI3-kinase in the AKT pathway, removed the protective effect of RA from the cells. This finding implies that RA activation of the AKT signaling cascade takes precedence over its activation of ERK1/2 phosphorylation, and that this selective effect of RA is key to its protection of epoxomicin-treated cells. Taken together, these findings suggest that RA treatment of cultured neuroblastoma cells sets up conditions under which proteasome inhibition, and the resultant accumulation of ubiquitinated proteins, loses its ability to kill the cells and may likely play a therapeutic role in neurodegenerative diseases.     

Combined effects of curcumin and doxorubicin on cell death and cell migration of SH-SY5Y human neuroblastoma cells

Neuroblastoma is the most common cancer of the sympathetic nervous system in children. Here, the influences of curcumin on survival, apoptosis, migration, and its combined effects with doxorubicin were investigated in SH-SY5Y cells by cell survival assay, flow cytometry, migration assays, and RT-PCR. Curcumin inhibited SH-SY5Y cell growth and induced apoptosis in dose- and time-dependent manners. This apoptotic induction relied on the upregulation of p53 and p21. Moreover, the treatment of curcumin for 24 h significantly suppressed cell migration, together with the downregulation of matrix metalloproteinase-2 (MMP-2) and upregulation of tissue inhibitor of metalloproteinases-1 (TIMP-1). The combination of curcumin augmented the anticancer activity of doxorubicin and significantly induced apoptosis. Pretreatment with curcumin increased the fraction of doxorubicin-induced apoptotic cells from 21.76?±?0.50 to 57.74?±?2.68%. Co-treatment with doxorubicin plus curcumin further inhibited 3D tumor migration. Altogether, the results suggest that curcumin suppresses growth and migration of SH-SY5Y cells and enhances the anticancer activity of doxorubicin. The addition of curcumin to therapeutic regimens may be promising for the treatment of neuroblastomas if a number of problems related to its in vivo bioavailability can be resolved.

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