Etoposide induces pancreatic ß-cells cytotoxicity via the JNK/ERK/GSK-3 signaling-mediated mitochondria-dependent apoptosis pathway.

Etoposide is widely used in the treatment of the different types of tumors such as pancreatic cancer. However, etoposide also causes several unwanted side-effects in normal viable cells, including pancreatic ß-cells, which are vulnerable to chemical-induced injuries, and the molecular mechanisms underlying etoposide-induced apoptosis are still unclear. Here, the results showed that in RIN-m5F cells (a ß-cell-derived cell line), the number of viable cells was significantly decreased after 24h of etoposide treatment and underwent mitochondria-dependent apoptotic signals accompanied by mitochondrial dysfunction, and increases in the population of sub-G1 hypodiploid cells and apoptotic cells, caspase-3 activity, and the activation of caspase cascades. Etoposide also increased the phosphorylation levels of glycogen synthase kinase (GSK)-3α/ß in treated RIN-m5F cells. Pretreatment with LiCl, a GSK-3 inhibitor, prevented etoposide-induced mitochondria-dependent apoptosis and GSK-3 protein phosphorylation in RIN-m5F cells. Furthermore, exposure of the cells to etoposide induced the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK)1/2 but not p38-MAPK, which was suppressed by the specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059), respectively. Additionally, pretreatment with both SP600125 and PD98059 effectively suppressed etoposide-induced ß-cell cytotoxicity, apoptosis, and GSK-3 protein phosphorylation; however, LiCl did not reverse JNK and ERK1/2 phosphorylation. Taken together, these results suggest that etoposide is capable of causing cytotoxicity on pancreatic ß-cells by inducing apoptosis through the JNK/ERK-mediated GSK-3 downstream-triggered mitochondria-dependent signaling pathway.

Cantharidin Induces Apoptosis Through the Calcium/PKC-Regulated Endoplasmic Reticulum Stress Pathway in Human Bladder Cancer Cells.

Bladder cancer is a common malignancy worldwide. However, there is still no effective therapy for bladder cancer. In this study, we investigated the cytotoxic effects of cantharidin [a natural toxin produced (pure compound) from Chinese blister beetles (Mylabrisphalerata or Mylabriscichorii) and Spanish flies (Cantharis vesicatoria)] in human bladder cancer cell lines (including: T24 and RT4 cells). Treatment of human bladder cancer cells with cantharidin significantly decreased cell viability. The increase in the expressions of caspase-3 activity and cleaved form of caspase-9/-7/-3 were also increased in cantharidin-treated T24 cells. Furthermore, cantharidin increased the levels of phospho-eIF2α and Grp78 and decreased the protein expression of procaspase-12, which was accompanied by the increase in calpain activity in T24 cells. Cantharidin was capable of increasing the intracellular Ca (2+) and the phosphorylation of protein kinase C (PKC) in T24 cells. The addition of BAPTA/AM (a Ca (2+) chelator) and RO320432 (a selective cell-permeable PKC inhibitor) effectively reversed the increase in caspase-3 and calpain activity, the phosphorylation levels of PKC and eIF2α and Grp78 protein expression, and the decrease in procaspase-12 expression induced by cantharidin. Importantly, cantharidin significantly decreased the tumor volume (a dramatic 71% reduction after 21 days of treatment) in nude mice xenografted with T24 cells. Taken together, these results indicate cantharidin induced human bladder cancer cell apoptosis through a calcium/PKC-regulated ER stress pathway. These findings suggest that cantharidin may be a novel and potential anticancer agent targeting on bladder cancer cells.

Chloroacetic acid triggers apoptosis in neuronal cells via a reactive oxygen species-induced endoplasmic reticulum stress signaling pathway.

Chloroacetic acid (CA), a chlorinated analog of acetic acid and an environmental toxin that is more toxic than acetic, dichloroacetic, or trichloroacetic acids, is widely used in chemical industries. Furthermore, CA has been found to be the major disinfection by-products (DBPs) of drinking water. CA has been reported to be highly corrosive and to induce severe tissue injuries (including nervous system) that lead to death in mammals. However, the effects and underlying mechanisms of CA-induced neurotoxicity remain unknown. In the present study, we found that CA (0.5-2.0 mM) significantly increased LDH release, decreased the number of viable cells (cytotoxicity) and induced apoptotic events (including: increases in the numbers of apoptotic cells, the membrane externalization of phosphatidylserine (PS), and caspase-3/-7 activity) in Neuro-2a cells. CA (1.5 mM; the approximate to LD50) also triggered ER stress, which was identified by monitoring several key molecules that are involved in the unfolded protein responses (including the increase in the expressions of p-PERK, p-IRE-1, p-eIF2α, ATF-4, ATF-6, CHOP, XBP-1, GRP 78, GRP 94, and caspase-12) and calpain activity. Transfection of GRP 78- and GRP 94-specific si-RNA effectively abrogated CA-induced cytotoxicity, caspase-3/-7 and caspase-12 activity, and GRP 78 and GRP 94 expression in Neuro-2a cells. Additionally, pretreatment with 2.5 mM N-acetylcysteine (NAC; a glutathione (GSH) precursor) dramatically suppressed the increase in lipid peroxidation, cytotoxicity, apoptotic events, calpain and caspase-12 activity, and ER stress-related molecules in CA-exposed cells. Taken together, these results suggest that the higher concentration of CA exerts its cytotoxic effects in neuronal cells by triggering apoptosis via a ROS-induced ER stress signaling pathway.

Lotus leaf (Nelumbo nucifera) and its active constituents prevent inflammatory responses in macrophages via JNK/NF-κB signaling pathway.

Inflammation is a serious health issue worldwide that induces many diseases, such as inflammatory bowel disease (IBD), sepsis, acute pancreatitis and lung injury. Thus, there is a great deal of interest in new methods of limiting inflammation. In this study, we investigated the leaves of Nelumbo nucifera Gaertn, an aquatic perennial plant cultivated in eastern Asia and India, in anti-inflammatory pharmacological effects in the murine macrophage cell line RAW264.7. Results showed that lipopolysaccharide (LPS) increased the protein expression of inducible nitric oxide synthase (iNOS) and COX-2, as well as the mRNA expression and level of IL-6 and TNF-α, while NNE significantly reduced these effects of LPS. LPS also induced phospho-JNK protein expression. The JNK-specific inhibitor SP600125 decreased the proteins expression of phospho-JNK, iNOS, COX-2, and the mRNAs expression and levels of IL-6 and TNF-α. Further, NNE reduced the protein expression of phospho-JNK. LPS was also found to promote the translocation of NF-κB from the cytosol to the nucleus and to decrease the expression of cytosolic IκB. NNE and SP600125 treatment recovered the LPS-induced expression of NF-κB and IκB. While phospho-ERK and phospho-p38 induced by LPS, could not be reversed by NNE. To further investigate the major components of NNE in anti-inflammatory effects, we determined the quercetin and catechin in inflammatory signals. Results showed that quercetin and catechin significantly decreased the proteins expression of iNOS, COX-2 and phospho-JNK. Besides, the mRNAs and levels of IL-6 and TNF-α also decreased by quercetin and catechin treatment in LPS-induced RAW264.7 cells. These results showed that NNE and its major components quercetin and catechin exhibit anti-inflammatory activities by inhibiting the JNK- and NF-κB-regulated pathways and could therefore be an useful anti-inflammatory agent.

Arsenic induces reactive oxygen species-caused neuronal cell apoptosis through JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-regulated pathways.

Arsenic (As), a well-known high toxic metal, is an important environmental and industrial contaminant, and it induces oxidative stress, which causes many adverse health effects and diseases in humans, particularly in inorganic As (iAs) more harmful than organic As. Recently, epidemiological studies have suggested a possible relationship between iAs exposure and neurodegenerative disease development. However, the toxicological effects and underlying mechanisms of iAs-induced neuronal cell injuries are mostly unknown. The present study demonstrated that iAs significantly decreased cell viability and induced apoptosis in Neuro-2a cells. iAs also increased oxidative stress damage (production of malondialdehyde (MDA) and ROS, and reduction of Nrf2 and thioredoxin protein expression) and induced several features of mitochondria-dependent apoptotic signals, including: mitochondrial dysfunction, the activations of PARP and caspase cascades, and the increase in caspase-3 activity. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these iAs-induced responses. iAs also increased the phosphorylation of JNK and ERK1/2, but did not that p38-MAPK, in treated Neuro-2a cells. NAC and the specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) abrogated iAs-induced cell cytotoxicity, caspase-3/-7 activity, and JNK and ERK1/2 activation. Additionally, exposure of Neuro-2a cells to iAs triggered endoplasmic reticulum (ER) stress identified through several key molecules (GRP 78, CHOP, XBP-1, and caspase-12), which was prevented by NAC. Transfection with GRP 78- and CHOP-specific si-RNA dramatically suppressed GRP 78 and CHOP expression, respectively, and attenuated the activations of caspase-12, -7, and -3 in iAs-exposed cells. Therefore, these results indicate that iAs induces ROS causing neuronal cell death via both JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-triggered apoptosis pathways.

Cadmium induces apoptosis in pancreatic ß-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation.

Cadmium (Cd), one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM). However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic ß-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic ß-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP) and the increase of cytosolic cytochrome c release), the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose) polymerase (PARP) cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK)1/2, and p38-mitogen-activated protein kinase (MAPK), which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA) transfection suppressed Cd-induced ß-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580) did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic ß-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.

Mercuric compounds induce pancreatic islets dysfunction and apoptosis in vivo.

Mercury is a toxic heavy metal that is an environmental and industrial pollutant throughout the world. Mercury exposure leads to many physiopathological injuries in mammals. However, the precise toxicological effects of mercury on pancreatic islets in vivo are still unclear. Here, we investigated whether mercuric compounds can induce dysfunction and damage in the pancreatic islets of mice, as well as the possible mechanisms involved in this process. Mice were treated with methyl mercuric chloride (MeHgCl, 2 mg/kg) and mercuric chloride (HgCl(2), 5 mg/kg) for more than 2 consecutive weeks. Our results showed that the blood glucose levels increased and plasma insulin secretions decreased in the mice as a consequence of their exposure. A significant number of TUNEL-positive cells were revealed in the islets of mice that were treated with mercury for 2 consecutive weeks, which was accompanied by changes in the expression of the mRNA of anti-apoptotic (Bcl-2, Mcl-1, and Mdm-2) and apoptotic (p53, caspase-3, and caspase-7) genes. Moreover, plasma malondialdehyde (MDA) levels increased significantly in the mice after treatment with mercuric compounds for 2 consecutive weeks, and the generation of reactive oxygen species (ROS) in the pancreatic islets also markedly increased. In addition, the mRNA expression of genes related to antioxidation, including Nrf2, GPx, and NQO1, were also significantly reduced in these islets. These results indicate that oxidative stress injuries that are induced by mercuric compounds can cause pancreatic islets dysfunction and apoptosis in vivo.

Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury.

Methylmercury (MeHg) is well-known for causing irreversible damage in the central nervous system as well as a risk factor for inducing neuronal degeneration. However, the molecular mechanisms of MeHg-induced neurotoxicity remain unclear. Here, we investigated the effects and possible mechanisms of MeHg in the mouse cerebrum (in vivo) and in cultured Neuro-2a cells (in vitro). In vivo study showed that the levels of LPO in the plasma and cerebral cortex significantly increased after administration of MeHg (50µg/kg/day) for 7 consecutive weeks. MeHg could also decrease glutathione level and increase the expressions of caspase-3, -7, and -9, accompanied by Bcl-2 down-regulation and up-regulation of Bax, Bak, and p53. Moreover, treatment of Neuro-2a cells with MeHg significantly reduced cell viability, increased oxidative stress damage, and induced several features of mitochondria-dependent apoptotic signals, including increased sub-G1 hypodiploids, mitochondrial dysfunctions, and the activation of PARP, and caspase cascades. These MeHg-induced apoptotic-related signals could be remarkably reversed by antioxidant NAC. MeHg also increased the phosphorylation of ERK1/2 and p38, but not JNK. Pharmacological inhibitors NAC, PD98059, and SB203580 attenuated MeHg-induced cytotoxicity, ERK1/2 and p38 activation, MMP loss, and caspase-3 activation in Neuro-2a cells. Taken together, these results suggest that the signals of ROS-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic pathways that are involved in MeHg-induced neurotoxicity.

Inorganic arsenic causes cell apoptosis in mouse cerebrum through an oxidative stress-regulated signaling pathway.

Arsenic pollution is a major public health problem worldwide. Inorganic arsenic (iAs) is usually more harmful than organic ones. iAs pollution increases the risk of human diseases such as peripheral vascular disease and cancer. However, the toxicological effects of iAs in the brain are mostly unclear. Here, we investigated the toxic effects and possible mechanisms of iAs in the cerebrum of mice after exposure to iAs (0.5 and 5 ppm (mg/l) As(2)O(3), via the drinking water), which was the possible human exposed dose via the ingestion in iAs-contaminated areas, for 6 consecutive weeks. iAs dose-dependently caused an increase of LPO production in the plasma and cerebral cortex. iAs also decreased the reduced glutathione levels and the expressions of NQO1 and GPx mRNA in the cerebral cortex. These impairments in the cerebral cortex caused by iAs exposure were significantly correlated with the accumulation of As. Moreover, iAs induced the production of apoptotic cells and activation of caspase-3, up-regulation of Bax and Bak, and down-regulation of Mcl-1 in the cerebral cortex. Exposure to iAs also triggered the expression of ER stress-related genes, including GRP78, GRP94, and CHOP. Meanwhile, an increase of p38 activation and dephosphorylation of ERK1/2 were shown in the cerebral cortex as a result of iAs-exposed mice. These iAs-induced damages and apoptosis-related signals could be significantly reversed by NAC. Taken together, these results suggest that iAs-induced oxidative stress causes cellular apoptosis in the cerebrum, signaling of p38 and ERK1/2, and ER stress may be involved in iAs-induced cerebral toxicity.

Arsenic induces pancreatic ß-cell apoptosis via the oxidative stress-regulated mitochondria-dependent and endoplasmic reticulum stress-triggered signaling pathways.

Arsenic (As), a ubiquitous toxic metal, is an important environmental and industrial pollutant throughout the world. Inorganic As (iAs) is usually more harmful than organic ones and with a high risk of diabetes incidence by exposure. However, the toxicological effects of iAs on growth and function of pancreatic ß-cells still remain unclear. Here, we found that iAs significantly decreased insulin secretion and cell viability, and increased ROS and MDA formation in pancreatic ß-cell-derived RIN-m5F cells. iAs also induced the increases in sub-G1 hypodiploids, annexin V-Cy3 binding, and caspase-3 activity in RIN-m5F cells, indicating that iAs could induce ß-cell apoptosis. Moreover, iAs induced MAPKs activation, mitochondria dysfunction, p53 up-regulation, Bcl-2 and Mdm-2 down-regulation, PARP, and caspase cascades, which displayed features of mitochondria-dependent apoptotic signals. In addition, exposure of RIN-m5F cells to iAs, could trigger ER stress as indicated by the enhancement in ER stress-related molecules induction (such as GRP78, GRP94, CHOP, and XBP1), procaspase-12 cleavage, and calpain activation. The iAs-induced apoptosis and its-related signalings could be effectively reversed by antioxidant N-acetylcysteine. We next observed that exposure of mice to iAs in drinking water for 6 consecutive weeks significantly decreased decreased the plasma insulin, elevated glucose intolerance and plasma lipid peroxidation, and induced islet cells apoptosis, which accompanied with arsenic accumulation in the whole blood and pancreas. N-acetylcysteine effectively antagonized the iAs-induced responses in mice. Taken together, these results suggest that iAs-induced oxidative stress causes pancreatic ß-cells apoptosis via the mitochondria-dependent and ER stress-triggered signaling pathways.

Pyrrolidine dithiocarbamate (PDTC)/Cu complex induces lung epithelial cell apoptosis through mitochondria and ER-stress pathways.

Pyrrolidine dithiocarbamate (PDTC) is widely used in pesticides, fungicides, insecticides, and herbicides. Copper (Cu) is a toxic heavy metal in the environment, and an essential trace metal element in the body, which is involved in many biological processes as a catalytic cofactor. The present study is designed to investigate the cellular toxicity of PDTC, CuCl(2), and PDTC/Cu complex exposure in lung alveolar epithelial cells that serve primary structural and functional roles in the lungs. The results showed that PDTC or CuCl(2) alone did not affect cell viability, but PDTC/Cu complex significantly decreased lung alveolar epithelial cell viability. PDTC/Cu complex also significantly increased intracellular copper concentration, but PDTC or CuCl(2) alone had low levels of copper. PDTC/Cu complex dramatically enhanced the JNK protein phosphorylation and ERK protein phosphorylation proteins. PDTC/Cu complex did not affect the p38 protein phosphorylation. PDTC/Cu complex was capable of activating the apoptosis-related caspases including caspase-9, caspase-7, and caspase-3, which could be reversed by the addition of JNK inhibitor SP600125 or transfection of MAPK8 short hairpin RNA. PDTC/Cu complex also increased cytosolic cytochrome c and decreased mitochondrial transmembrane potential. The Bcl-2 mRNA and protein expressions were decreased in lung epithelial cells treated with PDTC/Cu complex, which could be reversed by SP600125. Furthermore, PDTC/Cu complex could trigger the expressions of ER stress-associated signaling molecules including Grp78, Grp94, caspase-12, ATF4, and CHOP, which could be reversed by SP600125. Taken together, these results indicate that exposure to PDTC/Cu complex induces cytotoxicity and apoptosis in alveolar epithelial cells via the mitochondria- and ER-stress-related signaling pathways.

Methylmercury chloride induces alveolar type II epithelial cell damage through an oxidative stress-related mitochondrial cell death pathway.

Mercury, one of the widespread pollutants in the world, induces oxidative stress and dysfunction in many cell types. Alveolar type II epithelial cells are known to be vulnerable to oxidative stress. Alveolar type II epithelial cells produce and secrete surfactants to maintain morphological organization, biophysical functions, biochemical composition, and immunity in lung tissues. However, the precise action and mechanism of mercury on alveolar type II epithelial cell damage remains unclear. In this study, we investigate the effect and possible mechanism of methylmercury chloride (MeHgCl) on the human lung invasive carcinoma cell line (Cl1-0) and mouse lung tissue. Cl1-0 cells were exposed to MeHgCl (2.5-10 microM) for 24-72 h. The results showed a decrease in cell viability and an increase in malondialdehyde (MDA) level and ROS production at 72 h after MeHgCl exposure in a dose-dependent manner. Caspase-3 activity, sub-G1 contents and annexin-V binding were dramatically enhanced in Cl1-0 cells treated with MeHgCl. MeHgCl could also activate Bax, release cytochrome c, and cleave poly(ADP-Ribose) polymerase (PARP), and decrease surfactant proteins mRNA levels. Moreover, in vivo study showed that mercury contents of blood and lung tissues were significantly increased after MeHgCl treatment in mice. The MDA levels in plasma and lung tissues were also dramatically raised after MeHgCl treatment. Lung tissue sections of MeHgCl-treated mice showed pathological fibrosis as compared with vehicle control. The mRNA levels of proteins in apoptotic signaling, including p53, mdm-2, Bax, Bad, and caspase-3 were increased in mice after exposure to MeHgCl. In addition, the mRNA levels of surfactant proteins (SPs), namely, SP-A, SP-B, SP-C, and SP-D (alveolar epithelial cell functional markers) were significantly decreased. These results suggest that MeHgCl activates an oxidative stress-induced mitochondrial cell death in alveolar epithelial cells.