NBCn1 and NHE1 expression and activity in ΔNErbB2 receptor-expressing MCF-7 breast cancer cells: Contributions to pHi regulation and chemotherapy resistance

Altered pH-regulatory ion transport is characteristic of many cancers; however, the mechanisms and consequences are poorly understood. Here, we investigate how a truncated, constitutively active ErbB2 receptor (ΔNErbB2) common in breast cancer impacts on the Na⁺/H⁺-exchanger NHE1 and the Na⁺,HCO₃⁻-cotransporter NBCn1 in MCF-7 human breast cancer cells and address the roles of these transporters in chemotherapy resistance. Upon ΔNErbB2 expression, mRNA and protein levels of NBCn1, yet not of NHE1, increased several-fold, and the localization of both transporters was altered paralleling extensive morphological changes. The rate of pH_i recovery after acid loading increased by 50% upon ΔNErbB2 expression. Knockdown and pharmacological inhibition confirmed the involvement of both NHE1 and NBCn1 in acid extrusion. NHE1 inhibition or knockdown sensitized ΔNErbB2-expressing cells to cisplatin-induced programmed cell death (PCD) in a caspase-, cathepsin-, and reactive oxygen species-dependent manner. NHE1 inhibition augmented cisplatin-induced caspase activity and lysosomal membrane permeability followed by cysteine cathepsin release. In contrast, NBCn1 inhibition attenuated cathepsin release and had no net effect on viability. These findings warrant studies of NHE1 as a potential target in breast cancer and demonstrate that in spite of their similar transport functions, NHE1 and NBCn1 serve different functions in MCF-7 cells.     

d-β-Hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress

Oxidative stress has an important role in neurodegenerative diseases and cerebral ischemic injury. It is reported that d-β-hydroxybutyrate (DβHB), the major component of ketone bodies, is neuroprotective in recent studies. Therefore, in the present work the neuroprotective effects of DβHB on H₂O₂-induced apoptosis mediated by oxidative stress was investigated. PC12 cells were exposed to H₂O₂ with different concentrations of H₂O₂ for different times after DβHB pretreatment. MTT assay, apoptotic rates, intracellular reactive oxygen species (ROS) level, GSH content, mitochondrial membrane potential (MMP) and caspase-3 activity were determined. The results showed that DβHB inhibited the decrease of cell viability induced by H₂O₂ in PC12 cells. DβHB decreased the apoptotic rates induced by H₂O₂. The changes of intracellular ROS, GSH, MMP and caspase-3 activity due to H₂O₂ exposure were partially reversed in PC12 cells. So DβHB inhibited the apoptosis of PC12 cells induced by H₂O₂ via inhibiting oxidative stress.     

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H₂O₂ in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H₂O₂ source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H₂O₂ is sensitive to intracellular H₂O₂ competitors: it decreases when H₂O₂ is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H₂O₂. Oxidation of the fluorescent probes following reaction of H₂O₂ with endogenous HRP overcomes most criticisms in their use for intracellular H₂O₂ detection. The method can be applied for direct determination in plate reader and is proposed to detect H₂O₂ generation in physio-pathological cell models.     

α-Difluoromethylornithine, ornithine decarboxylase inhibitor, antagonizes H2O2-induced cytotoxicity in HL-60 leukemia cells: Regulation of iron-dependent lysosomal damage

Recent studies indicate that reactive oxygen species, such as H₂O₂, can be generated by anti-cancer drugs, can damage cells, and then induce apoptotic cell death. In this study, we reported whether polyamines were capable of affecting apoptotic cell death triggered by H₂O₂ in leukemia cells or not. -Difluoromethylornithine treatment (DFMO, 3 mmol/L, 48 h), which depletes intracellular putrescine by inhibiting ornithine decarboxylase, reduced H₂O₂-induced cell death in the HL-60 leukemia cells. Cytotoxicity caused by H₂O₂ in putrescine-depleted cells was 50% lower than that in the control cells, as determined by propidium iodide, the annexin V and DNA fragmentation assays. Following putrescine (1 mmol/L) supplement, cell death induction caused by H₂O₂ was restored to a similar level as the DFMO-untreated control cells. It seems that this partly resulted from the intralysosomal iron-dependent oxidation of the cells because DFMO did not significantly affect the increment of enzymes related to oxidative-stress resistance. Putrescine depletion by DFMO treatment reduced the cellular iron uptake of the cells by about 70%. In parallel to the reduction of iron uptake, lysosomal damage (assayed by acridine orange relocalization or uptake test) in the DFMO-treated cells was far less than that in the control cells. Moreover, putrescine supplement also restored the iron uptake to the control cell levels. Pre-incubation with desferrioxamine (DFO), which chelates iron and forms a non-reactive Fe-DFO complex that is localized in the lysosomal compartment, inhibited H₂O₂-induced cell death. This work suggests that polyamines may play a critical role in apoptotic cell death triggered by H₂O₂ via the regulation of the iron-dependent instability of the lysosome.     

The ATM kinase inhibitor KU-55933 provides neuroprotection against hydrogen peroxide-induced cell damage via a γH2AX/p-p53/caspase-3-independent mechanism: Inhibition of calpain and cathepsin D

The role of the kinase ataxia-telangiectasia mutated (ATM), a well-known protein engaged in DNA damage repair, in the regulation of neuronal responses to oxidative stress remains unexplored. Thus, the neuroprotective efficacy of KU-55933, a potent inhibitor of ATM, against cell damage evoked by oxidative stress (hydrogen peroxide, H₂O₂) has been studied in human neuroblastoma SH-SY5Y cells and compared with the efficacy of this agent in models of doxorubicin (Dox)- and staurosporine (St)-evoked cell death. KU-55933 inhibited the cell death induced by H₂O₂ or Dox but not by St in undifferentiated (UN-) and retinoic acid-differentiated (RA)-SH-SY5Y cells, with a more pronounced effect in the latter cell phenotype. Furthermore, this ATM inhibitor attenuated the Dox- but not H₂O₂-induced caspase-3 activity in both UN- and RA-SH-SY5Y cells. Although KU-55933 inhibited the H₂O₂- and Dox-induced activation of ATM, it attenuated the toxin-induced phosphorylation of the proteins H2AX and p53 only in the latter model of cell damage. Moreover, the ATM inhibitor prevented the H₂O₂-evoked increases in calpain and cathepsin D activity and attenuated cell damage to a similar degree as inhibitors of calpain (MDL28170) and cathepsin D (pepstatin A). Finally, we confirmed the neuroprotective potential of KU-55933 against the H₂O₂- and Dox-evoked cell damage in primary mouse cerebellar granule cells and in the mouse hippocampal HT-22 cell line. Altogether, our results extend the neuroprotective portfolio of KU-55933 to a model of oxidative stress, with this effect not involving inhibition of the γH2AX/p-p53/caspase-3 pathway and instead associated with the attenuation of calpain and cathepsin D activity.     

H2O2-Induced Cell Death in Human Glioma Cells: Role of Lipid Peroxidation and PARP Activation

underlying mechanism of ROS-induced cell injury remains to be defined. This study was undertaken to examine the role of lipid peroxidation and poly (ADP-ribose) polymerase (PARP) activation in H₂O₂-induced cell death in A172 cells, a human glioma cell line. H₂O₂ induced a dose- and time-dependent cell death. The cell death was prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), H₂O₂ scavengers (catalase and pyruvate), and a hydroxyl radical scavenger (dimethylthiourea). Antioxidants N,N-diphenyl-p-phenylenediamine (DPPD) and Trolox had no effect on the H₂O₂-induced cell death. Lipid peroxidation did not increase in human glioma cells exposed to H₂O₂. The PARP inhibitor 3-aminobenzamide prevented the cell death induced by H₂O₂. The PARP activity was increased by H₂O₂ and the H₂O₂ effect was prevented by 3-aminobenzamide, dithiothreitol, and phenanthroline. The ATP depletion induced by H₂O₂ was prevented by catalase, dithiothreitol, phenanthroline, and 3-aminobenzamide, but not by DPPD. These results indicate that the H₂O₂-induced cell death is mediated by PARP activation but not by lipid peroxidation in human glioma cells.     

Novel cytotoxicity test based on menadione-catalyzed H2O2 productivity for food safety evaluation

Menadione-catalyzed H₂O₂ production by viable cells was proportional to viable cell number, and the assay of this H₂O₂ production was applied to the cytotoxicity test of 17 substances which were used for international validation of fixed-dose procedure as an alternative to the classical LD₅₀ test. The cytotoxicity of substances tested was observed 4 h after the incubation with animal cells, and the viability was determined in 10 min according to menadione-catalyzed H₂O₂ production assay. IC₅₀ of each substance required for 50% inhibition of menadione-catalyzed H₂O₂ production was similar among HepG2, HuH-6KK, HUVE, Vero, Intestine407, NIH/3T3 and Neuro-2a cells. Twelve substances, 3 substances and 2 substances showed the difference of one, two and three orders in the magnitude between LD₅₀ and IC₅₀, respectively. These results show that menadione-catalyzed H₂O₂ production assay is useful for the rapid detection of toxic compounds having the basal cytotoxicity common to various cells, but is unfit for the detection of organ-specific toxic compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Participation of autophagy in renal ischemia/reperfusion injury

Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation, and it results in renal tubular epithelial cells undergoing cell death. We observed an increase in autophagosomes in the tubular epithelial cells of I/R-injured mouse models, and in biopsy specimens from human transplanted kidney. However, it remains unclear whether autophagy functions as a protective pathway, or contributes to I/R-induced cell death. Here, we employed the human renal proximal tubular epithelial cell line HK-2 in order to explore the role of autophagy under hypoxia (1% O₂) or activation of reactive oxygen species (500 μM H₂O₂). When compared to normoxic conditions, 48 h of hypoxia slightly increased LC3-labeled autophagic vacuoles and markedly increased LAMP2-labeled lysosomes. We observed similar changes in the mouse IR-injury model. We then assessed autophagic generation and degradation by inhibiting the downstream lysosomal degradation of autophagic vacuoles using lysosomal protease inhibitor. We found that autophagosomes increased markedly under hypoxia in the presence of lysosomal protease inhibitors, thus suggesting that hypoxia induces high turnover of autophagic generation and degradation. Furthermore, inhibition of autophagy significantly inhibited H₂O₂-induced cell death. In conclusion, high turnover of autophagy may lead to autophagic cell death during I/R injury.     

Role of ERK in Hydrogen Peroxide-Induced Cell Death of Human Glioma Cells

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. Activation of extracellular signal-regulated kinase (ERK) in oxidative stress remains controversial. In some cellular systems, the ERK activation is associated with protection against oxidative stress, while in other system, the ERK activation is involved in apoptotic cell death. The present study was undertaken to examine the role of ERK activation in H₂O₂-induced cell death of human glioma (A172) cells. H₂O₂ resulted in a time- and dose-dependent cell death, which was largely attributed to apoptosis. H₂O₂ treatment caused marked sustained activation of ERK. The ERK activation and cell death induced by H₂O₂ was prevented by catalase, the hydrogen peroxide scavenger, and U0126, an inhibitor of ERK upstream kinase MEK1/2. Transient transfection with constitutive active MEK1, an upstream activator of ERK1/2, increased H₂O₂-induced cell death, whereas transfection with dominant-negative mutants of MEK1 decreased the cell death. The ERK activation and cell death caused by H₂O₂ was inhibited by antioxidants (N-acetylcysteine and trolox), Ras inhibitor, and suramin. H₂O₂ produced depolarization of mitochondrial membrane potential and its effect was prevented by catalase and U0126. Taken together, these findings suggest that growth factor receptor/Ras/MEK/ERK signaling pathway plays an active role in mediating H₂O₂-induced apoptosis of human glioma cells and functions upstream of mitochondria-dependent pathway to initiate the apoptotic signal.     

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H₂O₂) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H₂O₂ accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H₂O₂ production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H₂O₂ accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells.     

Protective effects of sulfated chitooligosaccharides against hydrogen peroxide-induced damage in MIN6 cells

Sulfated chitooligosaccharides (COS-S) with different degrees of substitution (DS) were obtained by the chlorosulfuric acid/pyridine method. Protective effects of COS-S against hydrogen peroxide (H₂O₂)-induced damage were investigated in pancreatic β-cells MIN6 cell line. The cell viability, morphology, insulin contents, malondialdehyde (MDA) inhibition, lactate dehydrogenase (LDH) release and the levels of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidise (GPx) were evaluated under oxidative damage by 150 μM H₂O₂ for 6 h. COS-S did not show any harmful or inhibitory effect on cell growth at concentrations ranging from 0.1 to 0.5 mg/ml. While COS-S could enhance the cell viability, decrease the production of ROS, and reduce the MDA level as well as LDH level in oxidative damaged β-cells by being an antioxidant. The underlining mechanisms of protective effects of COS-S are partly due to the enhancement of antioxidant enzyme activity and inhibition of intracellular ROS production, along with suppressing MIN6 cell apoptosis subsequent to the amelioration of ROS. Moreover, increased DS might contribute to the defense mechanisms against H₂O₂-induced oxidative damage in MIN6 cells. These results indicated that the antioxidant properties of COS-S hold great potential for the oxidative diseases treatment, and the sulfate content of polysaccharides made great role in regulating antioxidant activities.     

Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured <Emphasis Type="Italic">in vitro</Emphasis>

Hydrogen peroxide (H₂O₂) is known to induce cell cycle arrest and apoptosis in various somatic cell types cultured in vitro. We hypothesize that this reactive oxygen species (ROS) could modulate cell cycle and induce morphological features characteristics of apoptosis in oocytes cultured in vitro. To test this hypothesis, immature and mature oocytes were cultured in medium containing various doses of H₂O₂ with or without caspase-3 inhibitor for various times. The treatment of H₂O₂ induced germinal vesicle break down (GVBD) in all immature oocytes followed by initiation of shrinkage. Some of immature oocytes (but not mature oocytes) also showed membrane blebbing. On the other hand, H₂O₂ treatment inhibited first polar body emission in mature oocytes just prior to initiation of shrinkage. The cytoplasmic granulation and fragmentation into apoptotic bodies were observed in mature oocytes during later stages of H₂O₂ treatment. The shrinkage was induced by H₂O₂ in a dose- and time-dependent manner in both immature and mature oocytes. Although, H₂O₂-induced degeneration was observed in both immature and mature oocytes after 2.0 hrs of treatment, immature oocytes were more susceptible to undergo quick shrinkage, membrane blebbing and degeneration. Co-addition of caspase-3 inhibitor prevented shrinkage and degeneration of both immature and mature oocytes except membrane blebbing that was observed at higher doses of H₂O₂ after 1.0 hr of culture. Treatment of H₂O₂ induced bax protein expression (3 times), DNA fragmentation and caspase-3 activity (2.5 times) in oocytes undergoing morphological apoptotic changes. These findings clearly suggest that H₂O₂ induced GVBD in immature oocytes, inhibited first polar body extrusion in mature oocytes prior to initiation of morphological changes characteristic of apoptosis such as shrinkage, membrane blebbing and cytoplasmic fragmentation prior to degeneration.     

Biphasic effects of H2O2 on BKCa channels

Abstract

The inhibitory or activating effect of H₂O₂ on large conductance calcium and voltage-dependent potassium (BK_Ca) channels has been reported. However, the mechanism by which this occurs is unclear. In this paper, BK_Ca channels encoded by mouse Slo were expressed in HEK 293 cells and BK_Ca channel activity was measured by electrophysiology. The results showed that H₂O₂ inhibited BK_Ca channel activity in inside-out patches but enhanced BK_Ca channel activity in cell-attached patches. The inhibition by H₂O₂ in inside-out patches may be due to oxidative modification of cysteine residues in BK_Ca channels or other membrane proteins that regulate BK_Ca channel function. PI3K/AKT signaling modulates the H₂O₂-induced BK_Ca channel activation in cell-attached patches. BK_Ca channels and PI3K signaling pathway were involved in H₂O₂-induced vasodilation and H₂O₂-induced vasodilation by PI3K pathway was mainly due to modulation of BK_Ca channel activity.     

Peroxidase mediated hydrogen peroxide production in barley roots grown under stress conditions

All applied metals (Co, Al, Cu, Cd) and NaCl inhibited barley root growth. No root growth inhibition was caused by drought exposure, in contrast to cold treatment. 0.01 mM H₂O₂ stimulated root growth and GA application did not affect root growth at all. Other activators and inhibitors of H₂O₂ production (SHAM, DTT, 10 mM H₂O₂, 2,4-D) inhibited root growth. Loss of cell viability was most significant after Al treatment, followed by Cd and Cu, but no cell death was induced by Co. Drought led to slight increase in Evans blue uptake, whereas neither NaCl nor cold influenced this parameter. DTT treatment caused slight increase in Evans blue uptake and significant increases were detected after 2,4-D and 10 mM H₂O₂ treatment, but were not induced by others stressors. Metal exposure increased guaiacol-POD activity, which was correlated with oxidation of NADH and production of H₂O₂. Exposure to drought caused a minor change in NADH oxidation, but neither H₂O₂ production nor guaiacol-POD activity was increased. Cold and NaCl application decreased all monitored activities. Increase in NADH oxidation and guaiacol-POD activity was caused by 10 mM H₂O₂ and 0.01 mM 2,4-D treatment, which also caused enhancement of H₂O₂ production. Slight inhibition of all activities was caused by 0.01 mM H₂O₂, GA, DTT; more pronounced inhibition was detected after SHAM treatment. The role of H₂O₂ production mediated by POD activity in relation to root growth and cell viability under exposure to some abiotic stress factors is discussed.     

Assessment of cell death studies by monitoring hydrogen peroxide in cell culture

Hydrogen peroxide (H₂O₂) has been widely used to study the oxidative stress response. However, H₂O₂ is unstable and easily decomposes into H₂O and O₂. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H₂O₂ concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H₂O₂, therefore acting as a protection against cell death by accidental necrosis.     

Vasoactive intestinal peptide reduces oxidative stress in pancreatic acinar cells through the inhibition of NADPH oxidase

Vasoactive intestinal peptide (VIP) attenuates experimental acute pancreatitis (AP) by inhibition of cytokine production from inflammatory cells. It has been suggested that reactive oxygen species (ROS) as well as cytokines play pivotal roles in the early pathophysiology of AP. This study aimed to clarify the effect of VIP on the oxidative condition in pancreas, especially pancreatic acinar cells (acini). Hydrogen peroxide (H₂O₂)-induced intracellular ROS, assessed with CM-H₂DCFDA, increased time- and dose-dependently in acini isolated from rats. Cell viability due to ROS-induced cellular damage, evaluated by MTS assay, was decreased with ≥100 μmol/L H₂O₂. VIP significantly inhibited ROS production from acini and increased cell viability in a dose-dependent manner. Expression of antioxidants including catalase, glutathione reductase, superoxide dismutase (SOD) 1 and glutathione peroxidase was not altered by VIP except for SOD2. Furthermore, Nox1 and Nox2, major components of NADPH oxidase, were expressed in pancreatic acini, and significantly increased after H₂O₂ treatment. Also, NADPH oxidase activity was provoked by H₂O₂. VIP decreased NADPH oxidase activity, which was abolished by PKA inhibitor H89. These results suggested that VIP affected the mechanism of ROS production including NADPH oxidase through induction of a cAMP/PKA pathway. In conclusion, VIP reduces oxidative stress in acini through the inhibition of NADPH oxidase. These results combined with findings of our previous study suggest that VIP exerts its protective effect in pancreatic damage, not only through an inhibition of cytokine production, but also through a reduction of the injury caused by oxidative stress.     

Characterization of Hydrogen Peroxide Toxicity in Cultured Rat Forebrain Neurons

We investigated the ability of hydrogen peroxide (H₂O₂) to cause apoptotic cell death in cultured rat forebrain neurons and the potential mechanisms by which oxidative stress triggers delayed neuronal death. H₂O₂ (25 M for 5 min) reduced cell viability to 34.5 ± 8.3% of untreated controls 20 h after exposure, and resulted in a significant proportion of neurons which exhibited apoptotic nuclear morphology. Using single cell fluorescence assays, we measured H₂O₂-induced changes in DNA strand breaks, 27 dichlorofluorescin fluorescence, reduced glutathione, intracellular free Ca²⁺, and mitochondrial membrane potential. DNA strand breaks in response to H₂O₂ were not evident immediately following exposure, but were increased 12h and 20h after exposure. Millimolar concentrations of H₂O₂ caused increases in the fluorescence of the oxidant-sensitive fluorescent dye, 27-dichlorofluorescin. H₂O₂ treatment decreased reduced glutathione following 30 minutes of exposure using the fluorescent indicator, 5-chloromethylfluorescein diacetate, and increased intra-neuronal free Ca²⁺ levels in a subpopulation of neurons. Mitochondrial membrane potential, measured by rhodamine 123 localization was unaffected by 25 H₂O₂, while higher concentrations of H₂O₂ (10 or 30 mM) depolarized mitochondria. These studies demonstrate that H₂O₂ is a potent and effective neurotoxin that produces oxidative stress, as well as apoptotic neuronal death     

Induction of the intrinsic apoptosis pathway in insulin-secreting cells is dependent on oxidative damage of mitochondria but independent of caspase-12 activation

Pro-inflammatory cytokine-mediated beta cell apoptosis is activated through multiple signaling pathways involving mitochondria and endoplasmic reticulum. Activation of organelle-specific caspases has been implicated in the progression and execution of cell death. This study was therefore performed to elucidate the effects of pro-inflammatory cytokines on a possible cross-talk between the compartment-specific caspases 9 and 12 and their differential contribution to beta cell apoptosis. Moreover, the occurrence of ROS-mediated mitochondrial damage in response to beta cell toxic cytokines has been quantified. ER-specific caspase-12 was strongly activated in response to pro-inflammatory cytokines; however, its inhibition did not abolish cytokine-induced mitochondrial caspase-9 activation and loss of cell viability. In addition, there was a significant induction of oxidative mitochondrial DNA damage and elevated cardiolipin peroxidation in insulin-producing RINm5F cells and rat islet cells. Overexpression of the H₂O₂ detoxifying enzyme catalase effectively reduced the observed cytokine-induced oxidative damage of mitochondrial structures. Taken together, the results strongly indicate that mitochondrial caspase-9 is not a downstream substrate of ER-specific caspase-12 and that pro-inflammatory cytokines cause apoptotic beta cell death through activation of caspase-9 primarily by hydroxyl radical-mediated mitochondrial damage.     

Calpain mediates caspase-dependent apoptosis initiated by hydrogen peroxide in pancreatic acinar AR42J cells

Abstract

Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H₂O₂-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H₂O₂ induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H₂O₂-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.     

Exposure of human intestinal epithelial cells and primary human hepatocytes to trypsin-like serine protease inhibitors with potential antiviral effect

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 μM in HIEC-6. These inhibitors did not cause elevations in extracellular H₂O₂ levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 μM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 μM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.