Reactive oxygen species and PI3K/Akt signaling play key roles in the induction of Nrf2-driven heme oxygenase-1 expression in sulforaphane-treated human mesothelioma MSTO-211H cells

The nuclear factor erythroid-derived 2 related factor 2 (Nrf2)/heme oxygenase (HO)-1 induction plays cytoprotective roles against oxidative injury, apoptosis, and anticancer therapy; however, little is known about its regulation in human mesothelioma MSTO-211H cells. In this study, we investigated Nrf2/HO-1 induction in response to sulforaphane and determined the signaling pathways involved in this process. Sulforaphane treatment decreased cell viability and triggered a rapid and transient increase in the intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) prevented sulforaphane-induced cytotoxicity. Erk1/2 was activated within 1h of sulforaphane addition, whereas Akt phosphorylation was suppressed until the first 8h, and was then maintained at an elevated level until 72h, displaying a biphasic regulatory feature. Nrf2 protein levels in both nuclear and whole cell lysates were increased after sulforaphane treatment and were decreased by pretreatment with NAC, actinomycin D and cycloheximide. Activation of the Nrf2/HO-1 system after sulforaphane treatment was suppressed by pretreatment with NAC or Ly294002, a PI3K inhibitor. Knockdown of Nrf2 with siRNA decreased cell viability and attenuated sulforaphane-induced HO-1 up-regulation. Overall, our results indicate that ROS generation and/or activation of PI3K/Akt signaling regulate cell survival and Nrf2-driven HO-1 expression in sulforaphane-treated MSTO-211H cells.     

Cadmium-induced apoptosis in murine macrophages is antagonized by antioxidants and caspase inhibitors

Cadmium is a toxic heavy metal that accumulates in the environment and is commonly found in cigarette smoke and industrial effluents. This study was designed to determine the role of reactive oxygen species (ROS) generation, and its antagonism by antioxidants, in cadmium-mediated cell signaling and apoptosis in murine macrophage cultures. Cadmium-generated ROS production was observed in J774A.1 cells at 6 h, reverting to control levels at 16 and 24 h. The ROS production was concentration related between 20 and 500 microM cadmium. Activation of caspase-3 was observed at 8 h and DNA fragmentation at 16 h in the presence of 20 microM cadmium, suggesting that caspase-3 activation is a prior step to DNA fragmentation in cadmium-induced apoptosis. Inhibitors of caspase-3, -8, -9, and a general caspase inhibitor suppressed cadmium-induced caspase-3 activation and apoptosis indicating the importance of caspase-3 in cadmium-induced toxicity in these cells. Protection against the oxidative stress with N-acetylcysteine (NAC) and silymarin (an antioxidant flavonoid) blocked cadmium-induced apoptosis. Pretreatment of cells with NAC and silymarin prevented cadmium-induced cell injury, including growth arrest, mitochondrial impairment, and necrosis, and reduced the cadmium-elevated intracellular calcium ([Ca2+]i), suggesting that the oxidative stress is a source of increased [Ca2+]i. NAC inhibited cadmium-induced activation of mitogen-activated protein kinases, the c-Jun NH2-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK). However, silymarin provided only a partial protection for JNK activation, and only at the low concentration did it inhibit cadmium-induced ERK activation. Inhibition of caspase-3 protected oxidative stress produced by cadmium, suggesting that the activation of caspase-3 also contributes to generation of reactive oxygen species (ROS). Results emphasized the role of ROS, Ca2+ and mitogen-activated protein kinases in cadmium-induced cytotoxicity in murine macrophages.     

Apoptosis and necrosis: two distinct events induced by cadmium in cortical neurons in culture

(1) Cadmium is an extremely toxic metal commonly found in industrial workplaces, a food contaminant and a major component of cigarette smoke. Cadmium can severely damage several organs, including the brain. In this work, we have studied both the cadmium toxicity on rat cortical neurons in culture and the possible protective effect of serum. (2) Our results indicate that: (1) cadmium is taken up by the neurons in a dose and serum dependent way; (2) cadmium, at concentrations from 1 micro M or 10 micro M (depending on the absence or the presence of serum) up to 100 micro M, decreases the metabolic capacity, which was evaluated by the XTT (tetrazolium salt) test; (3) cadmium induces apoptosis and LDH (lactate dehydrogenase) release in a dose dependent way; (4) in a serum-free medium, the cadmium-induced apoptosis is accompanied by caspase-3 activation; (5) both the caspase-3 activation and the cadmium-induced apoptosis are reversed by N-acethyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), a selective caspase-3 inhibitor, indicating that the caspase-3 pathway is involved in cadmium-induced apoptosis in cortical neurons; and (6) the cadmium concentrations which produce caspase-3 activation do not modify the intracellular ATP levels; however, higher cadmium concentrations lead to both intracellular ATP depletion and ATP release, but do not increase the caspase-3 activity, indicating that cadmium also produces cellular death by necrosis. (3) These results suggest that cadmium induces either apoptosis or necrosis in rat cortical neurons, depending on the cadmium concentration.     

Suppression of human prostate cancer PC-3 cell growth by N-acetylcysteine involves over-expression of Cyr61

N-acetylcysteine (NAC), sulfidryl-containing thiol antioxidant, has been heralded as chemopreventive agent, generally because of its ability to scavenge free radicals. It also suppresses the proliferation of many cancer cells; however, the antiproliferative mechanism(s) remain to be fully elucidated. In this study, we investigated a growth-suppressive mechanism of NAC action in androgen-independent prostate carcinoma PC-3 cells. NAC (?1 mM) inhibited the proliferation of PC-3 cells in a dose- and time-dependent manner. Moreover, NAC treatment suppressed the activation of NF-κB induced by IKK-β as detected by the NF-κB reporter gene assay. NAC exerted a biphasic effect on the intracellular ROS levels depending on incubation time; the antioxidant effect was seen within 2 h after NAC treatment, however, a pro-oxidant effect was evident after 48 h treatment. In addition to these effects, NAC treatment elicited a dose- and time-dependent increase in the Cyr61 expression that was accompanied by an increase in its mRNA and blocked by cycloheximide pretreatment. Importantly, NAC treatment caused an early but transient activation of Akt and Erk1/2. The NAC-induced increase in Cyr61 protein levels was suppressed by the PI3K inhibitor (Ly294002) and, to a lesser extent, MEK/Erk1/2 inhibitor (PD98059). Taken together, our data suggest that the antiproliferative effect of NAC is partially mediated by intracellular ROS production, the inhibition of NF-κB activity, and the activation of PI3K- and/or MEK/Erk-related intracellular signaling pathways, which lead to up-regulation of Cyr61 expression.     

Cadmium induces apoptotic program imbalance and cell cycle inhibitor expression in cultured human astrocytes

Cadmium is a highly neurotoxic heavy metal impairing neurogenesis and induces neurodegenerative disorders. Toxic concentrations of cadmium induce astrocytic apoptosis by depleting intracellular glutathione levels, elevating intracellular calcium levels, altering mitochondria membrane potentials, and activating JNK and PI3K/Akt signaling pathways. Cadmium suppresses cell proliferation in kidney epithelial cells, lung fibroblasts, and primary myelocytes; however, cadmium’s effects on proteins regulating oxidative stress, apoptosis, and cell proliferation in astrocytes are less known. The present study hypothesized that cadmium alters levels of antioxidant enzymes, apoptotic regulator proteins, and cell cycle inhibitor proteins, resulting in apoptosis and cell cycle arrest. Concentrations ≥20 μM cadmium induced apoptosis and led to intracellular changes including DNA fragmentation, reduced mRNA expression of antioxidant enzymes (i.e., catalase and glutathione S transferase-A4), downregulation of B-cell lymphoma 2 (Bcl-2), and upregulation of Bcl-2-associated X protein (Bax). Moreover, cadmium suppressed astrocytic proliferation by inducing S and G2/M phase cell cycle arrest and promoting p53, p21, and p27 expression. In conclusion, this study provides mechanistic insight into cadmium-induced cytotoxicity of astrocytes and highlights potential targets for prevention of cadmium-induced apoptosis and cell cycle arrest.     

Cadmium-induced induction of cell death in human lens epithelial cells: Implications to smoking associated cataractogenesis

Cadmium is reported to accumulate in human eye tissues suggesting its implication in diverse ocular pathology. Using an in vitro cell culture model we investigated the effects of cadmium on human lens epithelial cells (HLECs) (HLE-B3). We observed cadmium-induced dose- as well as time-dependent decline in HLECs viability which was exacerbated significantly upon reduction of intracellular glutathione levels by buthionine sulfoximine (BSO). There was a dose-dependent significant increase in lactate dehydrogenase (LDH) release from HLECs suggesting cadmium-induced alteration of membrane integrity as well as necrotic cell death. The decline in cell viability was also due to apoptosis of the HLECs as determined by quantifying % apoptotic cells as well as PARP cleavage. Moreover, release of apoptosis inducing factor (AIF) into the cytosol was also detected. Cadmium was also observed to increase oxidative stress, lipid peroxidation and activation of MAPK pathway in HLECs. Antioxidants like N-acetylcysteine (NAC) and α-Tocopherol significantly prevented cadmium-induced toxicity in HLECs. Our findings suggest that cadmium-induced elevated oxidative stress as well as activation of MAPK signaling cascade eventually led to cell death of HLECs through apoptosis as well as necrosis. The loss of HLECs by cadmium could possibly explain its implication in cataract development particularly associated with smoking.     

Reduced Nrf2 activation in PI3K phosphorylation‐impaired vitiliginous keratinocytes increases susceptibility to ROS‐generating chemical‐induced apoptosis

Keratinocytes in affected epidermis of vitiligo patients are known to have impaired activation of the PI3K/AKT pathway. Based on critical roles of keratinocytes and oxidative stress in vitiligo development, this study examined whether keratinocytes with impaired PI3K activation were more vulnerable to apoptosis caused by oxidative stress from phenolic compounds, p‐tert‐butylphenol (4‐TBP) and hydroquinone (HQ). Cell viability assay, FACS analysis, ELISA for TNF‐α or IL‐1a, ROS assay, Western blot analysis for Nrf2 expression, and confocal microscopy with anti‐Nrf2 and phospho‐PI3K antibodies were done on primary cultured normal human keratinocytes with or without PI3K knockdown in the presence or absence of chemical treatment or antioxidant. Immunofluorescence staining using anti‐Nrf2, phospho‐PI3K, TNF‐ɑ, and IL‐1ɑ antibodies, ROS assay using dihydroethidium, and TUNEL assay were performed on sets of depigmented and normally pigmented skin from vitiligo patients. Results showed that 4‐TBP or HQ treatment increased apoptosis and the expression levels of TNF‐ɑ, IL‐1ɑ, and ROS in PI3K‐knockdown keratinocytes which reduced Nrf2 nuclear translocation compared to control keratinocytes. These changes were significantly recovered by an antioxidant treatment. Depigmented epidermis of vitiligo patients also showed lower levels of Nrf2 and phospho‐PI3K but higher levels of ROS, TNF‐ɑ, IL‐1ɑ, and ROS with more TUNEL‐positive cells. Therefore, impaired PI3K activation in keratinocytes in depigmented epidermis of vitiligo patients are vulnerable to apoptosis caused by ROS‐generating chemicals due to reduced Nrf2 activation.     

Cadmium induces carcinogenesis in BEAS-2B cells through ROS-dependent activation of PI3K/AKT/GSK-3β/β-catenin signaling

Cadmium has been widely used in industry and is known to be carcinogenic to humans. Although it is widely accepted that chronic exposure to cadmium increases the incidence of cancer, the mechanisms underlying cadmium-induced carcinogenesis are unclear. The main aim of this study was to investigate the role of reactive oxygen species (ROS) in cadmium-induced carcinogenesis and the signal transduction pathways involved. Chronic exposure of human bronchial epithelial BEAS-2B cells to cadmium induced cell transformation, as evidenced by anchorage-independent growth in soft agar and clonogenic assays. Chronic cadmium treatment also increased the potential of these cells to invade and migrate. Injection of cadmium-stimulated cells into nude mice resulted in the formation of tumors. In contrast, the cadmium-mediated increases in colony formation, cell invasion and migration were prevented by transfection with catalase, superoxide dismutase-1 (SOD1), or SOD2. In particular, chronic cadmium exposure led to activation of signaling cascades involving PI3K, AKT, GSK-3β, and β-catenin and transfection with each of the above antioxidant enzymes markedly inhibited cadmium-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the cadmium-mediated increase in total and active β-catenin proteins and colony formation. Moreover, there was a marked induction of AKT, GSK-3β, β-catenin, and carcinogenic markers in tumor tissues formed in mice after injection with cadmium-stimulated cells. Collectively, our findings suggest a direct involvement of ROS in cadmium-induced carcinogenesis and implicate a role of AKT/GSK-3β/β-catenin signaling in this process.     

p53-Dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts

This study focused on the activation of cell cycle checkpoint responses in diploid human fibroblasts that were treated with cadmium chloride and the potential roles of ATM and p53 signaling pathways in cadmium-induced responses. The alkaline comet assay indicated that cadmium caused a dose-dependent increase in DNA damage. Cells that were rendered p53-defective by expression of a dominant-negative p53 allele or knockdown of p53 mRNA were more resistant to cadmium-induced inactivation of colony formation than normal and ataxia telangiectasia (AT) cells. Synchronized fibroblasts in S were more sensitive to cadmium toxicity than cells in G1, suggesting that cadmium may target some element of DNA replication. Cadmium produced a dose- and time-dependent inhibition of DNA synthesis. An immediate inhibition was associated with severe delay in progression through S phase and a delayed inhibition seen 24 h after treatment was associated with accumulation of cells in G2. AT and normal cells displayed similar patterns of inhibition of DNA synthesis and G2 delay after treatment with cadmium, while p53-defective cells displayed significantly less of the delayed inhibition of DNA synthesis and accumulation in G2 post-treatment. Total p53 protein and ser15-phosphorylated p53 were induced by cadmium in normal and AT cells. The p53 transactivation target Gadd45alpha was induced in both p53-effective and p53-defective cells after 4 h cadmium treatment, and this was associated with an acute inhibition of mitosis. Cadmium produced a very unusual pattern of toxicity in human fibroblasts, inhibiting DNA replication and inducing p53-dependent growth arrest but without induction of p21(Cip1/Waf1) or activation of Chk1.     

Cadmium induces autophagy through ROS-dependent activation of the LKB1-AMPK signaling in skin epidermal cells

Cadmium is a toxic heavy metal which is environmentally and occupationally relevant. The mechanisms underlying cadmium-induced autophagy are not yet completely understood. The present study shows that cadmium induces autophagy, as demonstrated by the increase of LC3-II formation and the GFP-LC3 puncta cells. The induction of autophagosomes was directly visualized by electron microscopy in cadmium-exposed skin epidermal cells. Blockage of LKB1 or AMPK by siRNA transfection suppressed cadmium-induced autophagy. Cadmium-induced autophagy was inhibited in dominant-negative AMPK-transfected cells, whereas it was accelerated in cells transfected with the constitutively active form of AMPK. mTOR signaling, a negative regulator of autophagy, was downregulated in cadmium-exposed cells. In addition, cadmium generated reactive oxygen species (ROS) at relatively low levels, and caused poly(ADP-ribose) polymerase-1 (PARP) activation and ATP depletion. Inhibition of PARP by pharmacological inhibitors or its siRNA transfection suppressed ATP reduction and autophagy in cadmium-exposed cells. Furthermore, cadmium-induced autophagy signaling was attenuated by either exogenous addition of catalase and superoxide dismutase, or by overexpression of these enzymes. Consequently, these results suggest that cadmium-mediated ROS generation causes PARP activation and energy depletion, and eventually induces autophagy through the activation of LKB1-AMPK signaling and the down-regulation of mTOR in skin epidermal cells.     

Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygene species

Cadmium poses a serious environmental threat in aquatic ecosystems but the mechanisms of its toxicity remain unclear. The purpose of this work was first to determine whether cadmium induced apoptosis in trout hepatocytes, second to determine whether or not reactive oxygen species (ROS) were involved in cadmium-induced apoptosis and genotoxicity. Hepatocytes exposed to increasing cadmium concentrations (in the range of 1-10 microM) showed a molecular hallmark of apoptosis which is the fragmentation of the nuclear DNA into oligonucleosomal-length fragments, resulting from an activation of endogenous endonucleases and recognized as a 'DNA ladder' on conventional agarose gel electrophoresis. Exposure of hepatocytes to cadmium led clearly to the DEVD-dependent protease activation, acting upstream from the endonucleases and considered as central mediators of apoptosis. DNA strand breaks in cadmium-treated trout hepatocytes was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique used to detect DNA primary damage in individual cells. Simultaneous treatment of trout hepatocytes with cadmium and the nitroxide radical TEMPO used as a ROS scavenger, reduced significantly DNA fragmentation, DEVD-related protease activity and DNA strand breaks formation. These results lead to a working hypothesis that cadmium-induced apoptosis and DNA strand breaks in trout hepatocytes are partially triggered by the generation of ROS. Additional studies are required for proposing a mechanistic model of cadmium-induced apoptosis and genotoxicity in trout liver cells, in underlying the balance between DNA damage and cellular defence systems in fish.     

Dexamethasone prevents acute cadmium-induced hepatic injury but exacerbates kidney dysfunction in rabbits

Cadmium is a potent hepatotoxicant for which neither effective preventive methods nor the mechanism of toxicity has been established. We investigated the preventive effect of dexamethasone against cadmium toxicity on cadmium-induced liver injury in rabbits. Pretreatment with dexamethasone at 1 mg/kg increased the rate of survival in rabbits administered 2.5 mg/kg iv cadmium. Cadmium induced acute severe liver injury characterized by hepatocellular necrosis, infiltration by inflammatory cells, and increases of plasma GOT, GPT, LDH, and LDH5. Dexamethasone mitigated the acute hepatotoxic effect of cadmium, but exacerbated cadmium-induced kidney dysfunction, with destruction of renal tubular cells and increases in excretion of protein, glucose, and amino acids into urine. The cadmium concentration in liver and kidney of rabbits administered cadmium was not changed by dexamethasone pretreatment. Although metallothionein mRNA expression induced by cadmium was not affected by dexamethasone in liver or kidney, cadmium-induced metallothionein protein production was augmented at the early phase in liver and decreased at the later phase in kidney. Neutrophilia observed after cadmium administration was enhanced initially by dexamethasone pretreatment. These results indicate that dexamethasone pretreatment potently prevented cadmium-induced liver injury, but exacerbated renal tubular dysfunction.     

Pro-oxidant status and Nrf2 levels in psoriasis vulgaris skin tissues and dimethyl fumarate-treated HaCaT cells

Reactive oxygen species (ROS) contribute to pathogenesis of many inflammatory skin diseases, including psoriasis. The aim of this study is to compare antioxidant protein expression in psoriasis vulgaris (PV) skin tissues with that in normal skin tissues in vivo and to evaluate the effects of dimethyl fumarate (DMF), used for the treatment of psoriasis, on ROS generation and apoptosis in a human keratinocyte cell line HaCaT. Compared with normal skin tissues, PV skin tissues showed increased protein oxidation as well as down-regulation of Nrf2 and its regulatory proteins such as HO-1 and AKR1C3. Using HaCaT cells to model DMF-induced pro-oxidant effects in the skin cells, we found that DMF treatment induced increased ROS levels and apoptotic cell death, as signified by increased proportion of cells with Annexin V-PE(+) staining and a sub-G₀/G₁ peak in the cell cycle. Preceding these changes, DMF treatment resulted in up-regulation of Nrf2, HO-1, and AKR1C3 proteins in these cells. Collectively, increased oxidative stress and impaired cellular anti-oxidant enzyme systems may participate in the pathogenesis of PV. DMF may exert an additive therapeutic efficacy in PV by attenuating the redox burden and subsequent oxidative damage to normal keratinocytes through activation of Nrf2 pathway relative to PV.     

Cytoprotective effects of selenium on cadmium-induced LLC-PK1 cells apoptosis by activating JNK pathway.

Extensive studies have indicated that the apoptosis pathway appears to be associated with intracellular reactive oxygen species (ROS) production in cadmium-induced nephrotoxicity, however, the precise cellular mechanism remains unclear. The purpose of this study was to determine the relationships between the activation of phosphorylated c-jun N-terminal kinase (JNK) and cadmium-induced apoptosis, and assess the possible cytoprotective mechanism of selenium. Our study clearly revealed cadmium treatment caused apoptosis in LLC-PK1 cells, which was partially suppressed by pretreatment with selenium, an antioxidant nutrient. Further studies found the phosphorylation of JNK kinase increased with exposure to cadmium for 3 h, even remained elevated at 9 h in the time course study, and the activation of phosphorylated JNK was detected in a dose-dependent manner. In addition, a concomitant time-dependent increase in caspase-3 activities was observed by cadmium treatment. During the process, selenium played the same role as N-acetyl-L-cysteine (NAC), a free radical scavenger. Pretreatment of cells with selenium partially suppressed of the phosphorylation of JNK, coupled with caspase-3 activation involved in cadmium-induced apoptosis. In conclusion, our studies provided a molecular linkage between the phosphorylation of JNK and cadmium-induced LLC-PK1 cells apoptosis, and demonstrated selenium also contributed a potentially protection to prevent cadmium-cytotoxicity.     

Calcium-mediated activation of c-Jun NH2-terminal kinase (JNK) and apoptosis in response to cadmium in murine macrophages.

Cadmium is a well-known carcinogenic and immunotoxic metal commonly found in cigarette smoke and industrial effluent. An altered intracellular calcium ([Ca(2+)](i)) level has been implicated in the pathophysiology of immune dysfunction. The present study was designed to determine the possible involvement of calcium (Ca(2+)) and mitogen-activated protein kinases (MAPKs) signaling pathways on cadmium-induced cell death in J774A.1 murine macrophage cells. Cadmium caused a low-amplitude [Ca(2+)](i) elevation at 20 microM and rapid and high-amplitude [Ca(2+)](i) elevation at 500 microM. Exposure to cadmium dose-dependently induced phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and deactivated p38 MAPK. Use of the selective JNK inhibitor SP600125 suggested that activation of JNK is pro-apoptotic and pro-necrotic. Buffering of the calcium response with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA-AM) and ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) completely blocked cadmium-induced apoptotic response. The pretreatment of cells with BAPTA-AM and EGTA suppressed the cadmium-induced cell injury, including growth arrest, mitochondrial activity impairment, and necrosis, and it also recovered the cadmium-altered JNK and p38 MAPK activity. Chelating [Ca(2+)](i) also reversed cadmium-induced hydrogen peroxide generation, suggesting that production of reactive oxygen species (ROS) is related to [Ca(2+)](i). The present study showed that cadmium induces a [Ca(2+)](i)-ROS-JNK-caspase-3 signaling pathway leading to apoptosis. Furthermore, cadmium-induced [Ca(2+)](i) regulates phosphorylation/dephosphorylation of JNK and p38, and it modulates signal transduction pathways to proliferation, mitochondrial activity, and necrosis.