Fluoride-induced death of rat erythrocytes in vitro

Although fluoride (F) in low concentrations is essential for teeth and bone development, its excessive consumption causes numerous deleterious abnormalities in cellular metabolism and physiology often leading to cell death. The present study was performed to establish the toxic F effects inducing the death of rat erythrocytes in vitro. The cells were cultured in the presence of 0.5–16 mM NaF for 1, 5 and 24 h. The progression of erythrocyte death was monitored by cell viability (calcein assay), membrane integrity (hemolysis assay), alterations in the cell morphology (light microscopy) and size (flow cytometry forward scatter), plasma membrane scrambling (annexin V binding). To elucidate the molecular mechanisms underlying F-induced cell death, the cytosolic Ca²⁺ activity (Fluo-3 fluorescence) and ceramide formation (binding of FITC-labeled antibodies) were determined. Exposure of the rat erythrocytes to NaF considerably suppressed their viability and caused partial cell hemolysis within 24 h. The cells underwent dramatic morphological alterations resulted in appearance of shrunken echinocytes after 1 h and swollen spherocytes within 24 h. The development of NaF-induced erythrocyte death was accompanied by progressive PS externalization at the outer cell membrane, ∼45% of the cells were annexin V-positive in response to 16 mM NaF within 24 h with a small cell population exhibiting necrotic features. The cell death was preceded by considerable accumulation of the free cytosolic Ca²⁺, with statistically significant increase in the number of Fluo-3-positive erythrocytes observed as early as during 1-h incubation with 0.5 mM NaF. NaF also induced moderate ceramide formation. Overall, exposure of the rat erythrocytes to NaF triggers rapid progression of their death in a dose- and time-dependent manner, with appearance of apoptotic cells after 1 and 5 h and transition to necrosis within 24 h. An increase in intracellular [Ca²⁺] appears to be crucial mechanism implicated in development of NaF-induced apoptosis in rat erythrocytes.     

Fluoride-induced apoptosis in human epithelial lung cells (A549 cells): role of different G protein-linked signal systems

In the present study, possible mechanisms involved in fluoride-induced apoptosis in a human epithelial lung cell line (A549) were examined. Sodium fluoride (NaF) induced apoptosis in the A549 cells, with a maximum at 5-7.5 mM after 20 hours of exposure. The number of cells with plasma membrane damage (PI-positive cells) increased moderately up to 5 mM, but markedly at 7.5 mM. Deferoxamine (an Al3+ chelator) almost completely prevented these NaF-induced responses, which may suggest a role for G protein activation. The apoptotic effect was partially reduced by the PKA inhibitor H89. NaF induced a weak but sustained increase in PKC activity, whereas the PKC activator TPA induced a transient effect. TPA, which enhanced the NaF-induced PKC activity, was not apoptotic when added alone, but facilitated the NaF-induced apoptosis and the increase in PI-positive cells. PKC downregulation induced by TPA pretreatment almost completely prevented the NaF-induced apoptosis and the increase in PI-positive cells. Pretreatment with the PKC inhibitor GF109203X, which abolished the PKC activity after 3 hours, enhanced the NaF-induced apoptosis. KN93 (a CaM kinase II inhibitor) and W7 (a calmodulin inhibitor) seem to reduce the apoptotic effect of NaF, whereas BAPTA-AM (a Ca2+ chelator) was without effect. The tyrosine kinase inhibitor genistein also markedly reduced the NaF-induced apoptosis, whereas the PI-3 kinase inhibitor wortmannin augmented the response. In conclusion, the present results suggest that NaF induces an apoptotic effect and an increase in PI-positive A549 cells via similar mechanisms, involving PKC, PKA, tyrosine kinase and Ca2+-linked enzymes, whereas PI-3 kinase seems to exert a counteracting effect.     

Cytoprotective effect of arjunolic acid in response to sodium fluoride mediated oxidative stress and cell death via necrotic pathway

The present study was conducted to investigate the role of arjunolic acid (AA) against sodium fluoride (NaF)-induced cytotoxicity and necrotic cell death in murine hepatocytes. Dose-dependent studies suggest that incubation of hepatocytes with NaF (100mM) for 1h significantly decreased the cell viability as well as intracellular antioxidant power. Besides, NaF administration increased the activities of the membrane leakage enzymes and accumulation of intracellular reactive oxygen species; decreased the activities of the antioxidant enzymes, the glutathione (GSH) and total thiol contents; and elevated the level of oxidised glutathione (GSSG), lipid peroxidation end products as well as protein carbonyl content. In addition to the oxidative impairments, fluoride exposure caused hepatic cell death mainly via the necrotic pathway as supported by the flowcytometric and DNA fragmentation analyses. Incubation with AA (100 microg/ml) both prior to and in combination with NaF almost normalized the altered activities of antioxidant indexes. AA treatment enhanced the cellular antioxidant capability and protected hepatocytes against NaF-induced cytotoxicity and necrotic death. The cytoprotective activity of AA was found to be comparable to that of a known antioxidant, vitamin C. Combining, data suggest that AA plays a protective role against NaF-induced cellular damage and prevents hepatocytes from necrotic death.     

Regulation of Bax by c-Jun NH2-terminal kinase and Bcl-xL in vinblastine-induced apoptosis

Microtubule inhibitors, such as vinblastine, are widely used in cancer chemotherapy. Vinblastine exerts its antitumor effect by inducing apoptosis. In KB-3 cells, we have shown previously that vinblastine activates c-Jun NH₂-terminal protein kinase (JNK) and causes Bax mitochondrial translocation and activation. In this study, we sought to test the hypothesis that JNK and Bcl-xL act as positive and negative regulators, respectively, of Bax translocation. The JNK inhibitor SP600125 inhibited vinblastine-induced JNK activation and in concert inhibited Bax mitochondrial translocation, Bax oligomerization, and Bax activation. Furthermore, the JNK inhibitor blocked vinblastine-induced apoptosis. The ability of vinblastine to induce Bax translocation and the inhibitory effect of SP600125 were confirmed in cells stably expressing GFP-Bax. However, if transiently overexpressed, Bax localized to the mitochondria, and this was associated with loss of viability and subsequent cell death. If Bcl-xL was co-expressed with Bax, the cells readily tolerated Bax overexpression. Indeed, physical interaction between Bcl-xL and Bax but not Bak was demonstrated by co-immunoprecipitation. These findings provide novel insight into the role of Bax and its regulation in vinblastine-induced apoptosis.     

The role of the IRE1 pathway in excessive iodide- and/or fluoride-induced apoptosis in Nthy-ori 3-1 cells in vitro

Excessive iodide and fluoride coexist in the groundwater in many regions, causing a potential risk to the human thyroid. To investigate the mechanism of iodide- and fluoride-induced thyroid cytotoxicity, human thyroid follicular epithelial cells (Nthy-ori 3-1) were treated with different concentrations of potassium iodide (KI), with or without sodium fluoride (NaF). Cell morphology, viability, lactate dehydrogenase (LDH) leakage, apoptosis, and expression of inositol-requiring enzyme 1 (IRE1) pathway-related molecules were assessed. Results showed 50 mM of KI, 1 mM of NaF, and 50 mM of KI +1 mM of NaF changed cellular morphology, decreased viability, and increased LDH leakage and apoptosis. Elevated expression of binding protein (BiP), IRE1, and C/EBP homologous protein (CHOP) mRNA and protein, as well as spliced X-box-binding protein-1 (sXBP-1) mRNA, were observed in the 1 mM NaF and 50 mM KI +1 mM NaF groups. Collectively, excessive iodide and/or fluoride is cytotoxic to the human thyroid. Although these data do not manifest iodide could induce the IRE1 pathway, the cytotoxicity followed by exposure to fluoride alone or in combination with iodide may be related to IRE1 pathway-induced apoptosis. Furthermore, exposure to the combination of excessive iodide and fluoride may cause interactive effects on thyroid cytotoxicity.     

Fluoride-induced apoptosis in epithelial lung cells involves activation of MAP kinases p38 and possibly JNK.

Exposure to fluorides can induce inflammatory reactions, cell cycle arrest, and apoptosis in different experimental systems. Fluorides are known G-protein activators, but less is known about fluoride effects downstream of G-protein activation. The aim of this study was to elucidate whether the induction of apoptosis by fluorides and inhibition of proliferation is mediated by MAP kinases in primary rat lung, alveolar type 2 cells and the human epithelial lung cell line A549. Sodium fluoride (NaF) induced apoptosis in both cell types but at different concentrations, with the primary cells being more sensitive to NAF: Proliferation of the type 2 cells and A549 cells was inhibited in the presence of NAF: NaF induced a prolonged activation of MAP kinase ERK. NaF also activated p38 and JNK in A549 cells for several hours (maximally 6-fold and 3-fold increase, respectively). Inhibition of ERK with the MEK1,2 inhibitor PD98059 increased apoptosis 2-fold, whereas the inhibitor of p38, SB202190, decreased the level of apoptotic cells by approximately 40%. SB202190 also inhibited apoptosis by almost 40% when ERK activity was reduced in the presence of PD98059. Neither PD98059 nor SB202190 did affect the NaF-induced inhibition of proliferation. These observations indicate that activation of MAP kinases p38 and possibly JNK are involved in NaF-induced apoptosis of epithelial lung cells, whereas ERK activation seems to counteract apoptosis in epithelial lung cells. In contrast, activation of ERK and p38 are not involved in NaF-induced inhibition of cell proliferation.     

Fluoride-induced c-Fos expression in MC3T3-E1 osteoblastic cells

Excessive systemic exposure to fluoride leads to disturbances of bone homeostasis. c-Fos is known to be essential in bone development by affecting osteoblast and osteoclast differentiation. In this study, we examined the effects of fluoride exposure on c-Fos expression and its regulatory signaling pathways in MC3T3-E1 mouse osteoblast cell line. c-fos mRNA level, c-Fos protein level and c-Fos DNA-binding activity were markedly increased, with a peak at 2 or 4?h, in MC3T3-E1 cells exposed to sodium fluoride (NaF). Fra-1 protein, another member of Fos family, was also elevated, whereas FosB and Fra-2 proteins remained unchanged. NaF further induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), ERK5, c-Jun NH₂-terminal kinase and p38. NaF-induced expression of c-Fos protein was markedly suppressed with U0126, the inhibitor of both activated and non-activated forms of MAPK/ERK kinase 1/2 (MEK1/2) and BIX02189, the MEK5 inhibitor, but partially with SP600125, the JNK inhibitor and SB203580, the p38 inhibitor. Therefore, ERK1/2 and ERK5 signal transduction pathways are important for accumulating c-Fos. siRNA targeting against the mouse c-fos gene further enhanced NaF-induced up-regulation of osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, suggesting that c-Fos might negatively regulate OPG expression induced by fluoride in osteoblastic cells.     

Involvement of both mitochondrial- and death receptor-dependent apoptotic pathways regulated by Bcl-2 family in sodium fluoride-induced apoptosis of the human gingival fibroblasts

Sodium fluoride (NaF) has been shown to be cytotoxic and produces inflammatory responses in humans. However, the cellular mechanisms underlying the NaF-induced cytotoxicity in periodontal tissues are unclear. This study examined whether or not NaF induces apoptosis in human gingival fibroblasts (HGF), and its underlying mechanisms by monitoring various apoptosis-associated processes. NaF reduced the cell viability of HGF in a dose- and time-dependent manner. NaF increased TUNEL-positive cell and induced apoptosis with concomitant chromatin condensation and DNA fragmentation in HGF. In addition, NaF increased the level of cytochrome c released from the mitochondria into the cytosol, enhanced the caspase-9, -8 and -3 activities, the cleavage of poly (ADP-ribose) polymerase (PARP), and up-regulated the voltage-dependent anion channel (VDAC) 1. However, NaF did not affect the production of reactive oxygen species (ROS) which is a strong apoptotic inducer. Furthermore, NaF up-regulated the Fas-ligand (Fas-L), a ligand of death receptor. Bcl-2, a member of the anti-apoptotic Bcl-2 family, was down-regulated, whereas the expression of Bax, a member of the pro-apoptotic Bcl-2 family, was unaffected in the NaF-treated HGF. These results suggest that NaF induces apoptosis in HGF through both the mitochondria-mediated pathways regulated by the Bcl-2 family and death receptor-mediated pathway.     

Effects of non-dioxin-like polychlorinated biphenyl congeners (PCB 101, PCB 153 and PCB 180) alone or mixed on J774A.1 macrophage cell line: modification of apoptotic pathway

Non-dioxin-like polychlorinated biphenyls (PCBs) are stable and lipophilic chemicals that persist in the environment and tend to bioaccumulate in the food chains. In the present study, we have investigated the effect of PCBs 101, 153, and 180 on macrophage J774A.1 by assessing cell viability and apoptotic cell death. We have combined morphological techniques and biochemical ones to establish the relevance of apoptosis in macrophage cell death induced by PCBs, alone or in combination. Treatment with the examined PCBs caused the loss of cell viability and accelerated apoptosis in a concentration-dependent manner. Moreover, a synergistic effect on cell death and apoptosis was evidenced for all PCBs at concentrations which were inactive alone. The apoptosis induced by PCBs involved the increase of caspase-3 activity. Also, Bcl-2 and Bax proteins were assessed to elucidate the apoptosis machinery induced in macrophage cultures by PCBs. Our results indicate that the increase in PCB-induced apoptosis correlates with a reduction in the expression of antiapoptotic Bcl-2 and an increase in the expression of proapoptotic Bax. Interestingly, concentrations of PCBs inactive by themselves induce apoptosis when PCBs are combined. In conclusion, our findings suggest that, although less toxic than dioxin like congeners, the examined non-dioxin-like PCBs are equally dangerous as immunotoxic pollutants, also considering their presence as mixtures at higher levels than dioxin-like PCBs in biotic and abiotic matrices.     

Cellular and molecular mechanisms of bromate-induced cytotoxicity in human and rat kidney cells

The mechanisms of bromate (BrO₃⁻)-induced toxicity in Normal Rat Kidney (NRK) and human embryonic kidney 293 (HEK293) cells were investigated. BrO₃⁻ (added as KBrO₃) induced concentration-dependent decreases in 3-(4, dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) staining after 48 h. BrO₃⁻-induced necrosis based on tandem increases in annexin V and PI staining. Cell cycle analysis demonstrated that BrO₃⁻ also induced G2/M arrest and nuclear fragmentation, prior to alterations in MTT staining or annexin V and PI staining. Immunoblot analysis demonstrated that the G2/M arrest correlated to induction of phosphorylated (p)-p53, p21, cyclin B1 and p-cdc2. Further, BrO₃⁻ induced time-dependent increases in the activity of the mitogen activated protein kinases p38 and ERK1/2. Treatment of cells with the p38 inhibitor SB202190, but not the ERK1/2 inhibitor PD98059, partially reversed BrO₃⁻-induced G2/M arrest and decreased BrO₃⁻-induced p-p53, p21 and cyclin B1 expression. In addition, BrO₃⁻ treatment induced reactive oxygen species (ROS) based on increases in CM-H₂DCFDA fluorescence. The antioxidant ascorbic acid inhibited BrO₃⁻-induced p38 activation, G2/M arrest, p-p53, p21 and cyclin B1 expression; however, ascorbic acid had no effect on BrO₃⁻-induced formation of 8-OHdG, a marker of DNA oxidative damage, whose increases preceded cell death by 24 h. These data suggest that ROS mediated MAPK activation is involved in the molecular mechanisms of BrO₃⁻-induced cell cycle arrest, which occurs independently of 8-OH-dG production. The similar mode of action in both NRK and HEK293 cells suggests that the mechanisms of BrO₃⁻-induced renal cell death are model-independent.     

Roles of p38 and JNK mitogen-activated protein kinase pathways during cantharidin-induced apoptosis in U937 cells

Cantharidin is an active compound from blister beetles traditionally used for the treatment of cancer. It is known to exert its antitumor activity by inducing apoptosis in cancer cells. However, its signaling pathway still remains unclear. Therefore, we investigated the roles of the mitogen-activated protein kinases (MAPKs) and the tumor suppressor gene, p53, during cantharidin-induced apoptosis in U937 human leukemic cells. Cantharidin effectively activated ERK-1/2, p38 and JNK in U937 cells in a time- and dose-dependent manner. Cantharidin also exhibited a strong cytotoxicity and induced apoptosis in U937 cells. For the evaluation of the role of MAPKs, PD98059, SB202190 and SP600125 were used as MAPK inhibitors for ERK-1/2, p38 and JNK. PD98059 did not affect cantharidin-induced cytotoxicity and apoptosis, whereas SB202190 and SP600125 significantly interfered with cytotoxic and apoptotic activities induced by cantharidin. Cantharidin alone induced the apoptosis by phosphorylation of p53, up-regulation of downstream target genes, MDM2 and p21 and also cleaved caspase-3, whereas SB202190 and SP600125 caused the down-regulation of p53, MDM-2, p21 and cleaved caspase-3 after a co-treatment with cantharidin. Similarly, SB202190 and SP600125 significantly disturbed the caspase-3 activity after a co-treatment with cantharidin by colorimetric assay. Taken together, these results suggest that cantharidin can induce apoptosis by activation of p38 and JNK MAP kinase pathways associated with p53 and caspase-3.     

Study of cytotoxic and apoptogenic properties of saffron extract in human cancer cell lines

Saffron (dried stigmas of Crocus sativus L.) has been used as a spice, food colorant and medicinal plant for millennia. In this study cytotoxic effect of saffron extract was evaluated in HepG2 and HeLa cell lines. Meanwhile role of apoptosis and ROS were explored. Malignant and non-malignant cells (L929) were cultured in DMEM medium and incubated with different concentrations of ethanolic saffron extract. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). ROS was measured using DCF-DA by flow cytometry analysis. Saffron could decrease cell viability in malignant cells as a concentration and time-dependent manner. The IC50 values against HeLa and HepG2 were determined 800 and 950 μg/ml after 48 h, respectively. Saffron induced a sub-G1 peak in flow cytometry histogram of treated cells compared to control indicating apoptotic cell death is involved in saffron toxicity. This toxicity was also independent of ROS production. It might be concluded that saffron could cause cell death in HeLa and HepG2 cells, in which apoptosis or programmed cell death plays an important role. Saffron could be also considered as a promising chemotherapeutic agent in cancer treatment in future.     

Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death

Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC₅₀ (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolate (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC₅₀ (concentration at which 50% of maximal cell death is inhibited) of 0.6 μM and 0.4 μM, respectively. In contrast, MTHF (up to 10 μM) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS.     

Transient activation of protein kinase C contributes to fluoride-induced apoptosis of rat erythrocytes

Role of PKC in fluoride-induced apoptosis of rat erythrocytes was studied in vitro and in vivo. Treatment of erythrocytes with 5 mM NaF for 1–24 h caused progressive accumulation of cytosolic Ca²⁺ and PS exposure at outer membrane surface. After 1 h, these processes were suppressed by PKC inhibitors staurosporine, GF 109203X and chelerythrine, but increased by PKC activator PMA. Following 24 h, NaF-induced Ca²⁺ uptake and PS externalization were partly prevented by PMA or staurosporine, but not by GF 109293X and chelerythrine. Application of PP inhibitor OA augmented NaF-induced cell responses within 1 h, but not after 24 h. Incubation of erythrocytes with 0.1–10 mM NaF for 1 h produced a dose-dependent PKCα translocation from cytosol to membranes with appearance of active PKM fragment. 24 h NaF exposure led to complete loss of cytosolic PKCα and proteolysis of membrane PKCα. Besides, NaF weakly stimulated membrane PKCζ, although its subcellular distribution was not altered. Thus, transient PKCα activation/translocation positively contributes to NaF-induced apoptosis in vitro. Consumption of 2–20 ppm fluoride by the rats for 12 months also induced dose-dependent PKCα translocation to membranes and activation of membrane PKCζ, what indicates that PKC stimulation is an important physiological mechanism of fluoride toxicity.     

Antitumor effects of emodin on LS1034 human colon cancer cells in vitro and in vivo: Roles of apoptotic cell death and LS1034 tumor xenografts model

Emodin, an active natural anthraquinone derivative, is found in the roots and rhizomes of numerous Chinese medicinal herbs and exhibits anticancer effects on many types of human cancer cell lines. The aim of this study investigated that emodin induced apoptosis of human colon cancer cells (LS1034) in vitro and inhibited tumor nude mice xenografts bearing LS1034 in vivo. In in vitro study, emodin induced cell morphological changes, decreased the percentage of viability, induced G2/M phase arrest and increased ROS and Ca²⁺ productions as well as loss of mitochondrial membrane potential (ΔΨ_m) in LS1034 cells. Emodin-triggered apoptosis was also confirmed by DAPI staining and these effects are concentration-dependent. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9 and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. Emodin induced the productions of ROS and Ca²⁺ release, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in tumor nude mice xenografts bearing LS1034. Overall, the potent in vitro and in vivo antitumor activities of emodin suggest that it might be developed for treatment of colon cancer in the future.     

Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK

Since hepatocellular carcinoma remains a major challenging clinical problem in many parts of the world including Eastern Asia and Southern Africa, it is imperative to develop more effective chemopreventive and chemotherapy agents. Herein, we present an investigation regarding the anticancer potential of luteolin, a natural flavonoid, and the mechanism of its action in human hepatoma HepG2 cells. Using DNA fragmentation assay and nuclear staining assay, it showed that luteolin induced apoptosis of HepG2 cells. Luteolin induced the cytosolic release of cytochrome c and activated CPP32. We found that Bax and Bak translocated to mitochondria apparently, whereas Fas ligand (FasL) was unchanged after a treatment with luteolin for 3 h. In addition, it showed that c-Jun NH2-terminal kinase (JNK) was activated after the treatment of luteolin for 3-12 h. Further investigation showed that a specific JNK inhibitor, SP600125, reduced the activation of CPP 32, the mitochondrial translocation of Bax, as well as the cytosolic release of cytochrome c that induced by luteolin. Finally, the apoptosis induced by luteolin was suppressed by a pretreatment with SP600125 via evaluating annexin V-FITC binding assay. These data suggest that luteolin induced apoptosis via mechanisms involving mitochondria translocation of Bax/Bak and activation of JNK.     

Elevated glutathione level does not protect against chronic alcohol mediated apoptosis in recombinant human hepatoma cell line VL-17A over-expressing alcohol metabolizing enzymes - Alcohol dehydrogenase and Cytochrome P450 2E1

Chronic consumption of alcohol leads to liver injury. Ethanol-inducible Cytochrome P450 2E1 (CYP2E1) plays a critical role in alcohol mediated oxidative stress due to its ability to metabolize ethanol. In the present study, using the recombinant human hepatoma cell line VL-17A that over-expresses the alcohol metabolizing enzymes - alcohol dehydrogenase (ADH) and CYP2E1; and control HepG2 cells, the mechanism and mode of cell death due to chronic ethanol exposure were studied. Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells. Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells. Chronic ethanol mediated cell death in VL-17A cells was predominantly apoptotic, with increased oxidative stress as the underlying mechanism. Chronic ethanol exposure of VL-17A cells resulted in 1.1- to 2.5-fold increased levels of ADH and CYP2E1. Interestingly, the level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation to the persistent and over-whelming oxidative stress. In conclusion, the increased GSH level may not be sufficient enough to protect VL-17A cells from chronic alcohol mediated oxidative stress and resultant apoptosis.     

Activation of distinct P2Y receptor subtypes stimulates insulin secretion in MIN6 mouse pancreatic β cells

Extracellular nucleotides and their receptor antagonists have therapeutic potential in disorders such as inflammation, brain disorders, and cardiovascular diseases. Pancreatic β cells express several purinergic receptors, and reported nucleotide effects on insulin secretion are contradictory. We studied the effect of P2Y receptors on insulin secretion and cell death in MIN6, mouse pancreatic β cells. Expression of P2Y₁ and P2Y₆ receptors was revealed by total mRNA analysis using RT-PCR. MIN6 cells were stimulated in the presence of 16.7 mM glucose with or without P2Y₁ and P2Y₆ agonists, 2-MeSADP and Up₃U, respectively. Both the agonists increased insulin secretion with EC₅₀ values of 44.6 ± 7.0 nM and 30.7 ± 12.7 nM respectively. The insulin secretion by P2Y₁ and P2Y₆ agonists was blocked by their selective antagonists MRS2179 and MRS2578, respectively. Binding of the selective P2Y₁ receptor antagonist radioligand [¹²⁵I]MRS2500 in MIN6 cell membranes was saturable (K_D 4.74 ± 0.47 nM), and known P2Y₁ ligands competed with high affinities. Inflammation and glucose toxicity lead to pancreatic β cell death in diabetes. Flow cytometric analysis revealed that Up₃U but not 2-MeSADP protected MIN6 cells against TNF-α induced apoptosis. Overall, the results demonstrate that selective stimulation of P2Y₁ and P2Y₆ receptors increases insulin secretion that accompanies intracellular calcium release, suggesting potential application of P2Y receptor ligands in the treatment of diabetes.     

The JNK,ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine,doxorubicin, and etoposide

Assessment of specific apoptosis and survival pathways implicated in anticancer drug action is important for understanding drug mechanisms and modes of resistance in order to improve the benefits of chemotherapy. In order to better examine the role of mitogen-activated protein kinases, including JNK and ERK, as well as the tumor suppressor p53, in the response of tumor cells to chemotherapy, we compared the effects on these pathways of three structurally and functionally distinct antitumor agents. Drug concentrations equal to 50 times the concentration required to reduce cell proliferation by 50% were used. Vinblastine, doxorubicin, or etoposide (VP-16) induced apoptotic cell death in KB-3 carcinoma cells, with similar kinetic profiles of PARP cleavage, caspase 3 activation, and mitochondrial cytochrome c release. All three drugs strongly activated JNK, but only vinblastine induced c-Jun phosphorylation and AP-1 activation. Inhibition of JNK by SP600125 protected cells from drug-induced cytotoxicity. Vinblastine caused inactivation of ERK whereas ERK was unaffected in cells exposed to doxorubicin or VP-16. Inhibition of ERK signaling by the MEK inhibitor, U0126, potentiated the cytotoxic effects of vinblastine and doxorubicin, but not that of VP-16. Vinblastine induced p53 downregulation, and chemical inhibition of p53 potentiated vinblastine-induced cell death, suggesting a protective effect of p53. In contrast, doxorubicin and VP-16 induced p53, and inhibition of p53 decreased drug-induced cell death, suggesting a pro-apoptotic role for p53. These results highlight the differential roles played by several key signal transduction pathways in the mechanisms of action of key antitumor agents, and suggest ways to specifically potentiate their effects in a context-dependent manner. In addition, the novel finding that JNK activation can occur without c-Jun phosphorylation or AP-1 activation has important implications for our understanding of JNK function.