5-aminolevulinic acid enhances cell death under thermal stress in certain cancer cell lines

5-aminolevulinic acid (5-ALA) is contained in all organisms and a starting substrate for heme biosynthesis. Since administration of 5-ALA specifically leads cancer cells to accumulate protoporphyrin IX (PpIX), a potent photosensitizer, we tested if 5-ALA also serves as a thermosensitizer. 5-ALA enhanced heat-induced cell death of cancer cell lines such as HepG2, Caco-2, and Kato III, but not other cancer cell lines including U2-OS and normal cell lines including WI-38. Those 5-ALA-sensitive cancer cells, but neither U2-OS nor WI-38, accumulated intracellular PpIX and exhibited an increased reactive oxygen species (ROS) generation under thermal stress with 5-ALA treatment. In addition, blocking the PpIX-exporting transporter ABCG2 in U2-OS and WI-38 cells enhanced their cell death under thermal stress with 5-ALA. Finally, a ROS scavenger compromised the cell death enhancement by 5-ALA. These suggest that 5-ALA can sensitize certain cancer cells, but not normal cells, to thermal stress via accumulation of PpIX and increase of ROS generation.     

The coffee diterpene kahweol induces heme oxygenase-1 via the PI3K and p38/Nrf2 pathway to protect human dopaminergic neurons from 6-hydroxydopamine-derived oxidative stress

In this study, we investigated the mechanisms of kahweol protection of neuronal cells from cell death induced by the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA). Pretreatment of SH-SY5Y cells with kahweol significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. Kahweol also up-regulated heme oxygenase-1 (HO-1) expression, which conferred neuroprotection against 6-OHDA-induced oxidative injury. Moreover, kahweol induced PI3K and p38 activation, which are involved in the induction of Nrf2, HO-1 expression, and neuroprotection. These results suggest that regulation of the anti-oxidant enzyme HO-1 via the PI3K and p38/Nrf2 signaling pathways controls the intracellular levels of ROS.     

Up-regulation of heme biosynthesis during differentiation of Neuro2a cells

Heme is an iron-containing tetrapyrrole molecule that functions as a prosthetic group for proteins such as mitochondrial respiratory enzymes. Several studies have suggested that heme has essential functions in the construction and maintenance of the nervous system. In this study, the contents of three biologically important forms of heme (types a, b, and c) and the expression of heme biosynthetic enzymes were examined in differentiating Neuro2a cells. During neuronal differentiation, there were increases in the cellular heme levels and increases in the mRNA levels for the rate-limiting enzymes of heme biosynthesis, such as aminolevulinic acid synthase (ALAS; EC 2.3.1.37) and coproporphyrinogen oxidase (EC 1.3.3.3). With respect to heme contents, heme b increased in the late phase of differentiation, but no apparent increase in heme a or b was observed in the early phase. In contrast, heme c (cytochrome c) markedly increased during the early phase of differentiation. This change preceded the increase in heme b and the up-regulation of the mRNA levels for heme biosynthetic enzymes. This study suggests the up-regulation of heme biosynthesis and differential regulation of the heme a, b, and c levels during neuronal differentiation.     

Non-catalytic roles for TET1 protein negatively regulating neuronal differentiation through srGAP3 in neuroblastoma cells

The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.     

Tat-indoleamine 2,3-dioxygenase 1 elicits neuroprotective effects on ischemic injury

It is well known that oxidative stress participates in neuronal cell death caused production of reactive oxygen species (ROS). The increased ROS is a major contributor to the development of ischemic injury. Indoleamine 2,3-dioxygenase 1 (IDO-1) is involved in the kynurenine pathway in tryptophan metabolism and plays a role as an anti-oxidant. However, whether IDO-1 would inhibit hippocampal cell death is poorly known. Therefore, we explored the effects of cell permeable Tat-IDO-1 protein against oxidative stress-induced HT-22 cells and in a cerebral ischemia/reperfusion injury model. Transduced Tat-IDO-1 reduced cell death, ROS production, and DNA fragmentation and inhibited mitogen-activated protein kinases (MAPKs) activation in H₂O₂ exposed HT-22 cells. In the cerebral ischemia/reperfusion injury model, Tat-IDO-1 transduced into the brain and passing by means of the blood-brain barrier (BBB) significantly prevented hippocampal neuronal cell death. These results suggest that Tat-IDO-1 may present an alternative strategy to improve from the ischemic injury.     

Involvement of membrane protein GDE2 in retinoic acid-induced neurite formation in Neuro2A cells

We show that a glycerophosphodiester phosphodiesterase homolog, GDE2, is widely expressed in brain tissues including primary neurons, and that the expression of GDE2 in neuroblastoma Neuro2A cells is significantly upregulated during neuronal differentiation by retinoic acid (RA) treatment. Stable expression of GDE2 resulted in neurite formation in the absence of RA, and GDE2 accumulated at the regions of perinuclear and growth cones in Neuro2A cells. Furthermore, a loss-of-function of GDE2 in Neuro2A cells by RNAi blocked RA-induced neurite formation. These results demonstrate that GDE2 expression during neuronal differentiation plays an important role for growing neurites.     

Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: Possible involvement in neuronal differentiation

The aim of the present study is to clarify the functional expression and physiological role in brain neurons of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring antioxidant ergothioneine (ERGO) as a substrate in vivo. After intracerebroventricular administration, the distribution of [³H]ERGO in several brain regions of octn1^−/− mice was much lower than that in wild-type mice, whereas extracellular marker [¹⁴C]mannitol exhibited similar distribution in the two strains. The [³H]ERGO distribution in wild-type mice was well correlated with the amount of ERGO derived from food intake and the OCTN1 mRNA level in each brain region. Immunohistochemical analysis revealed colocalization of OCTN1 with neuronal cell markers microtubule-associated protein 2 (MAP2) and βIII-tubulin in mouse brain and primary cultured cortical neurons, respectively. Moreover, cultured cortical neurons exhibited time-dependent and saturable uptake of [³H]ERGO. These results demonstrate that OCTN1 is functionally expressed in brain neurons. The addition of ERGO simultaneously with serum to culture medium of cortical neurons attenuated mRNA and protein expressions of MAP2, βIII-tubulin and synapse formation marker synapsin I, and induced those of sex determining region Y-box 2 (Sox2), which is required to maintain the properties of undifferentiated neural stem cells. In neuronal model Neuro2a cells, knockdown of OCTN1 by siRNA reduced the uptake of [³H]ERGO with concomitant up-regulation of oxidative stress marker HO-1 and Sox2, and down-regulation of neurite outgrowth marker GAP43. Interestingly, the siRNA knockdown decreased the number of differentiated Neuro2a cells showing long neurites, but increased the total number of cells. Thus, OCTN1 is involved in cellular differentiation, but inhibits their proliferation, possibly via the regulation of cellular oxidative stress. This is the first evidence that OCTN1 plays a role in neuronal differentiation and proliferation, which are required for brain development.     

Transferrin receptor-1 suppresses neurite outgrowth in neuroblastoma Neuro2A cells

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain.     

UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis

UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and reactive oxygen species (ROS) modulation. High levels of UCP2 mRNA were recently found in erythroid cells where UCP2 is hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. We examined UCP2 protein expression and role in mice erythropoiesis in vivo. UCP2 was mainly expressed at early stages of erythroid maturation when cells are not fully committed in heme synthesis. Iron incorporation into heme was unaltered in reticulocytes from UCP2-deficient mice. Although heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of progenitor cells from bone marrow and fetal liver both in vitro and in vivo revealed that UCP2 deficiency results in a significant decrease in cell proliferation at the erythropoietin-dependent phase of erythropoiesis. This was accompanied by reduction in the phosphorylated form of ERK, a ROS-dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid cells revealed altered distribution of ROS, resulting in decreased cytosolic and increased mitochondrial ROS. Restoration of the cytosol oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.     

Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.     

Guanosine protects human neuroblastoma SH-SY5Y cells against mitochondrial oxidative stress by inducing heme oxigenase-1 via PI3K/Akt/GSK-3β pathway

Mitochondrial perturbation and oxidative stress are key factors in neuronal vulnerability in several neurodegenerative diseases or during brain ischemia. Here we have investigated the protective mechanism of action of guanosine, the guanine nucleoside, in a human neuroblastoma cell line, SH-SY5Y, subjected to mitochondrial oxidative stress. Blockade of mitochondrial complexes I and V with rotenone plus oligomycin (Rot/oligo) caused a significant decrease in cell viability and an increase in ROS production. Guanosine that the protective effect of guanosine incubated concomitantly with Rot/oligo abolished Rot/oligo-induced cell death and ROS production in a concentration dependent manner; maximum protection was achieved at the concentration of 1mM. The cytoprotective effect afforded by guanosine was abolished by adenosine A(1) or A(2A) receptor antagonists (DPCPX or ZM241385, respectively), or by a large (big) conductance Ca(2+)-activated K(+) channel (BK) blocker (charybdotoxin). Evaluation of signaling pathways showed that the protective effect of guanosine was not abolished by a MEK inhibitor (PD98059), by a p38(MAPK) inhibitor (SB203580), or by a PKC inhibitor (cheleritrine). However, when blocking the PI3K/Akt pathway with LY294002, the neuroprotective effect of guanosine was abolished. Guanosine increased Akt and p-Ser-9-GSK-3β phosphorylation confirming this pathway plays a key role in guanosine's neuroprotective effect. Guanosine induced the antioxidant enzyme heme oxygenase-1 (HO-1) expression. The protective effects of guanosine were prevented by heme oxygenase-1 inhibitor, SnPP. Moreover, bilirubin, an antioxidant and physiologic product of HO-1, is protective against mitochondrial oxidative stress. In conclusion, our results show that guanosine can afford protection against mitochondrial oxidative stress by a signaling pathway that implicates PI3K/Akt/GSK-3β proteins and induction of the antioxidant enzyme HO-1.     

Anti-oxidizing effect of the dichloromethane and hexane fractions from Orostachys japonicus in LPS-stimulated RAW 264.7 cells via upregulation of Nrf2 expression and activation of MAPK signaling pathway

Orostachys japonicus shows various biological activities. However, the molecular mechanisms remain unknown in LPS-stimulated macrophages. Here, we investigated the anti-oxidizing effect of the dichloromethane (DCM) and hexane fractions from O. japonicus (OJD and OJH) against oxidative stress in RAW 264.7 cells stimulated by LPS. OJD and OJH significantly increased the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Additionally, it was found that the expression of HO-1 was stimulated by Nrf2 activated via degradation of Keap1. ERK and p38 inhibitors repressed HO-1 induced by OJD and OJH in LPS-stimulated cells, respectively. In conclusion, these results suggest that OJD and OJH may block oxidative damage stimulated by LPS, via increasing the expression of HO-1 and Nrf2, and MAPK signaling pathway. [BMB Reports 2014; 47(2): 98-103]     

Capsaicin Ameliorates Cisplatin-Induced Renal Injury through Induction of Heme Oxygenase-1

Cisplatin is one of the most potent chemotherapy agents. However, its use is limited due to its toxicity in normal tissues, including the kidney and ear. In particular, nephrotoxicity induced by cisplatin is closely associated with oxidative stress and inflammation. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the heme metabolism, has been implicated in a various cellular processes, such as inflammatory injury and anti-oxidant/oxidant homeostasis. Capsaicin is reported to have therapeutic potential in cisplatin-induced renal failures. However, the mechanisms underlying its protective effects on cisplatin-induced nephrotoxicity remain largely unknown. Herein, we demonstrated that administration of capsaicin ameliorates cisplatin-induced renal dysfunction by assessing the levels of serum creatinine and blood urea nitrogen (BUN) as well as tissue histology. In addition, capsaicin treatment attenuates the expression of inflammatory mediators and oxidative stress markers for renal damage. We also found that capsaicin induces HO-1 expression in kidney tissues and HK-2 cells. Notably, the protective effects of capsaicin were completely abrogated by treatment with either the HO inhibitor ZnPP IX or HO-1 knockdown in HK-2 cells. These results suggest that capsaicin has protective effects against cisplatin-induced renal dysfunction through induction of HO-1 as well as inhibition oxidative stress and inflammation.     

Activation of catalase by apelin prevents oxidative stress-linked cardiac hypertrophy

Adipose tissue secretes a variety of bioactive factors, which can regulate cardiomyocyte hypertrophy via reactive oxygen species (ROS). In the present study we investigated whether apelin affects ROS-dependent cardiac hypertrophy. In cardiomyocytes apelin inhibited the hypertrophic response to 5-HT and oxidative stress induced by 5-HT- or H₂O₂ in a dose-dependent manner. These effects were concomitant to the increase in mRNA expression and activity of catalase. Chronic treatment of mice with apelin attenuated pressure-overload-induced left ventricular hypertrophy. The prevention of hypertrophy by apelin was associated with increased myocardial catalase activity and decreased plasma lipid hydroperoxide, as an index of oxidative stress. These results show that apelin behaves as a catalase activator and prevents cardiac ROS-dependent hypertrophy.     

Role of reactive oxygen species and NADPH-oxidase in the development of rat cerebellum

Experimental evidence suggests that reactive oxygen species (ROS) could participate in the regulation of some physiological conditions. In the nervous system, ROS have been suggested to act as signaling molecules involved in several developmental processes including cell differentiation, proliferation and programmed of cell death. Although ROS can be generated by several sources, it has been suggested that NADPH oxidase (NOX) could be critical in the production of ROS acting as a signal in some of these events. It has been reported that ROS production by NOX enzymes participate in neuronal maturation and differentiation during brain development. In the present study, we found that during rat cerebellar development there was a differential ROS generation at different ages and areas of the cerebellum. We also found a differential expression of NOX homologues during rat cerebellar development. When we treated developing rats with an antioxidant or with apocynin, an inhibitor of NOX, we found a marked decrease of the ROS levels in all the cerebellar layers at all the ages tested. Both treatments also induced a significant change in the cerebellar foliation as well as an alteration in motor behavior. These results suggest that both ROS and NOX have a critical role during cerebellar development.     

Chebulinic acid attenuates glutamate-induced HT22 cell death by inhibiting oxidative stress,calcium influx and MAPKs phosphorylation

Glutamate-induced excitotoxicity and oxidative stress is a major causative factor in neuronal cell death in acute brain injuries and chronic neurodegenerative diseases. The prevention of oxidative stress is a potential therapeutic strategy. Therefore, in the present study, we aimed to examine a potential therapeutic agent and its protective mechanism against glutamate-mediated cell death. We first found that chebulinic acid isolated from extracts of the fruit of Terminalia chebula prevented glutamate-induced HT22 cell death. Chebulinic acid significantly reduced intracellular reactive oxygen species (ROS) production and Ca²⁺ influx induced by glutamate. We further demonstrated that chebulinic acid significantly decreased the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38, as well as inhibiting pro-apoptotic Bax and increasing anti-apoptotic Bcl-2 protein expression. Moreover, we demonstrated that chebulinic acid significantly reduced the apoptosis induced by glutamate in HT22 cells. In conclusion, our results in this study suggest that chebulinic acid is a potent protectant against glutamate-induced neuronal cell death via inhibiting ROS production, Ca²⁺ influx, and phosphorylation of MAPKs, as well as reducing the ratio of Bax to Bcl-2, which contribute to oxidative stress-mediated neuronal cell death.     

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease.