Cladribine enhances apoptotic cell death in lung carcinoma cells over-expressing DNase γ

Worldwide, lung cancer is the most common form of cancer and often has a poor prognosis. Establishment of effective therapies for lung cancer is a major concern in clinical cancer research. We compared the cytotoxic effects of chemotherapeutic agents including cisplatin, 5-fluorouracil, vinorelbine and cladribine, on a human lung cancer cell line, A549, and its derivative transfected with the DNase γ gene. We observed selective cytotoxicity of cladribine on the DNase γ-expressing sub-cell line of A549. Cladribine induces selective apoptosis in DNase γ-expressing A549 cells, which depends on activation of caspases. These results suggest that a combination therapy that includes cladribine along with the introduction of DNase γ has potential as a new therapeutic strategy for lung cancer.     

Protective effects of fucoidan against γ-radiation-induced damage of blood cells

Fucoidan, a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and Laminaria japonica, has a variety of biological activities, including antioxidant and antitumor activities. Here, we investigated the radioprotective effects of fucoidan on human monoblastic leukemia cell line U937. Further, animal tests were carried out using Balb/c mice in order to determine the radiation-induced changes in the counts of blood cells, including thrombocytes, erythrocytes, leukocytes and hematocrit. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wherein fucoidan (1, 10, and 100 μg/mL) was observed to improve recovery from damage caused by 8-Gy radiation in a dose dependent manner. The viability of U937 cells pre-treated with fucoidan also increased in a dose dependent manner. Furthermore, fucoidan at 100 mg/kg was found to protect against changes in the counts of blood cells as follows: on day 28 after irradiation, the thrombocyte count in the irradiated controls decreased to 45% compared with the non-irradiated controls, while that in the fucoidan-treated group was 60%. The hematocrit in the fucoidan-treated group recovered to 75% on day 28, while that in the irradiated control was 68%. The erythrocyte count in the irradiated controls consistently ranged from 64% to 67% throughout the experiment, but that in the fucoidan-treated group increased gradually, ranging from 75% to 80%. The mean number of survival days and 50-day actuarial survival rate increased dose dependently in the fucoidan-treated group. The mean number of survival days and the 50-day actuarial survival rate in this group was 16, 21, and 29 days and 12%, 20%, and 30% at fucoidan doses of 1, 10, and 100 mg/kg. The values of these parameters in the control group were 9 days and 0%, although the difference between the test and control groups was not statistically significant. Our results may prove valuable in the field of radioprotection.     

Intake of saffron reduces γ-radiation-induced genotoxicity and oxidative stress in mice

Saffron (SAF), the dried stigmas of Crocus sativus, is commonly used for flavoring and coloring food. Studies on bioactivity of SAF have demonstrated its in vivo antioxidant activity. The aim of our study was to assess the impact of SAF intake on γ-radiation (RAD) induced (a) chromosomal damage, (b) oxidative stress in liver and brain, and (c) histopathological effects in the intestinal cells and male germ cells in mice. Freeze-dried aqueous extract of SAF was used for the experiments. Our preliminary cell-free DNA nicking assay using pBR322 DNA revealed protective effects of freeze-dried SAF extract against hydroxyl radical induced DNA damage. For the in vivo investigations, freeze-dried SAF extract in distilled water was administered by gavage (40?mg/kg b.w.) to male Swiss albino mice for six consecutive days. On the sixth day, the animals were exposed to RAD (1 or 2?Gy) and sacrificed 24?h later to collect bone marrow cells for assessing chromosomal damage by measuring micronucleated polychromatic erythrocytes (MnPCEs). Liver and brain samples from animals exposed to 2?Gy RAD were used for evaluating lipid peroxidation and activity of antioxidant enzymes. The testis and intestine were used for histopathological analysis. Our results demonstrated significant protective effects of SAF against RAD-induced genotoxic damage. SAF pretreatment reduced the level of lipid peroxidation with concomitant increase in glutathione content and activity of glutathione S-transferase, glutathione peroxidase, and catalase. The histopathological analysis showed minimal impact of SAF on RAD-induced damage in the intestinal cells and male germ cells.     

Cancer cell death by programmed necrosis?

A recent paper by Zong et al. describes how alkylating agents kill cells by a process they term "programmed necrosis," induced by excessive activation of PARP resulting in degradation of cytosolic NAD(+) and inhibition of glycolysis. We argue that it is not obvious whether chemotherapy in patients can induce sufficient NAD(+) loss to affect glycolysis; that the "programmed" nature of the necrosis requires more evidence; and that there are mechanisms making cancer cells hypersensitive to DNA damage other than their high rate of aerobic glycolysis.     

NFκB inhibitors induce cell death in glioblastomas

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy.     

Is cisplatin-induced cell death always produced by apoptosis?

It is generally accepted that DNA damage and subsequent induction of apoptosis may be the primary cytotoxic mechanism of cisplatin and other DNA-binding antitumor drugs (Fisher,1994). Because the final step of apoptosis is characterized by morphological changes in the nucleus, the death signals of the execution phase must be transmitted from the cytoplasm to the nucleus. Thus, the recognition and processing of cisplatin-induced DNA damage through"classic" apoptosis, requires that a nuclear signal, generated at the initiation phase, be transmitted to the cytoplasm to be processed through the effector and execution phases. At the end of the execution phase, the apoptotic signal must come back to the nucleus to produce internucleosomal DNA degradation. Therefore, the induction of apoptosis from detection and subsequent processing of cisplatin-induced DNA damage seems to be a long and complex process of cell death. However, because cisplatin is a nonspecific drug and reacts not only with DNA but also with proteins,we cannot rule out the possibility that in some cases of cisplatin-induced apoptosis, an easier process of initiation, such as damage to cytoplasmic proteins, may take place (Pérez, 1998). Thus, damage to proteins is worth considering as a factor contributing to cisplatin-induced apoptosis. Moreover, it is possible that cisplatin damage to proteins could induce apoptosis at the execution phase level. In fact, initiation of apoptosis at the execution phase (activation of caspases) has been previously reported for the cell killing produced by cytotoxic T lymphocytes (Golstein et al., 1991). Although apoptosis and necrosis are conceptually distinct forms of cell death with very different morphological and biochemical characteristics, these two types of demise may occur simultaneously in tissues or cell cultures exposed to the same insult (Eguchi et al., 1997, Zhan et al., 1999). In fact, both types of cell death have been found in the same population of cisplatin-treated cells (Pestell et al., 2000). Moreover, it has been hypothesized that in a tissue or cell population,apoptosis and necrosis might be two extremes of a continuum of possible types of cell demise. Individual cell death would be decided by factors such as the availability of energy and the metabolic condition of the cell (Leist et al., 1997). Thus, some cells might die as a result of an unfinished apoptotic program. In fact, in L1210 leukemic cells, cisplatin-induced cell death seems to be the result of a defective apoptotic program that lacks some morphological and biochemical characteristics attributed to apoptosis (Segal-Bendirdjian and Jacquemin-Sablon, 1995). In addition, at high doses, cisplatin could damage molecules involved in cellular energy supply (i.e., ATP) and also proteins directly or indirectly involved in the apoptotic process (i.e., p53, Bax, Bcl-2, and caspases), leading to necrotic cell death. In fact, in cisplatin-resistant keratinocytes transformed by H-ras oncogene, a high dose of cisplatin (312 microM) induces characteristic features of necrotic cell death(Pérez et al., 1999). Thus, depending on the level of cellular damage induced by cisplatin, necrosis could take place either directly or as a consequence of an unfinished apoptotic program. In summary, a growing body of evidence suggests that cisplatin-induced cell death does not always come from "classic"apoptosis. Depending on both cisplatin dose and cellular status, cisplatin may also induced cell death by a defective apoptotic program or even by necrosis. Elucidation of the conditions under which the apoptotic program induced by cisplatin as well as other antitumor drugs is totally or partially executed may have important implications for the outcome of cancer chemotherapy.     

Lycopene: An antioxidant and radioprotector against γ-radiation-induced cellular damages in cultured human lymphocytes

The present study aimed to evaluate the radioprotective effect of lycopene, a naturally occurring dietary carotenoid on γ-radiation-induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), hydroperoxides (HP), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analyzed by cytokinesis blocked micronucleus assay (CBMN), dicentric aberration (DC) and translocation frequency. The γ-radiation at different doses (1, 2 and 4 Gy) resulted in a significant increase in the number of micronuclei (MN), DC, translocation frequency, TBARS and HP level, whereas the levels of GSH and antioxidant enzymes were significantly decreased when compared with normal control. The maximum damage to lymphocytes was observed at 4 Gy irradiation. Lycopene pretreatment (1, 5 and 10 μg/ml) significantly decreased the frequency of MN, DC and translocation when compared with γ-radiation control. The levels of TBARS, HP were also decreased and activities of SOD, CAT and GPx were significantly increased along with GSH levels when compared with γ-radiation control. The dose of 5 μg/ml of lycopene was found to be more effective than the other two doses. Thus, our result shows that pretreatment with lycopene offers protection to normal lymphocytes against γ-radiation-induced cellular damage.     

Modulatory effects of curcumin on γ-radiation-induced cellular damage in primary culture of isolated rat hepatocytes

Ionizing radiation is known to induce oxidative stress through generation of reactive oxygen species (ROS) resulting in imbalance of the pro-oxidant and antioxidant in the cells, which is suggested to culminate in cell death. The present work was aimed to evaluate the radioprotective effect of curcumin, a yellow pigment of turmeric on γ-radiation-induced toxicity in primary cultures of isolated rat hepatocytes. Hepatocytes were isolated from the liver of rats by collagenase perfusion. The cellular changes were estimated using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH), ceruloplasmin, vitamins A, E and C and uric acid. The comet assay is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells was exposed under γ-radiation. The increase in the severity of DNA damage was observed with the increase dose (1, 2 and 4Gy) of γ-radiation in cultured hepatocytes. TBARS were increased significantly, whereas the levels of GSH, vitamins C, E and A, ceruloplasmin, uric acid and antioxidant enzymes were significantly decreased in γ-irradiated hepatocytes. The maximum damage to hepatocytes was observed at 4Gy irradiation. On pretreatment with curcumin (1, 5 and 10μg/ml) showed a significant decrease in the levels of TBARS and DNA damage. The antioxidant enzymes were increased significantly along with the levels of GSH, vitamins A, E and C, uric acid and ceruloplamin. The maximum protection of hepatocytes was observed at 10μg/ml of curcumin pretreatment. Thus, pretreatment with curcumin helps in protecting the hepatocytes against γ-radiation-induced cellular damage and can be developed as an effective radioprotector during radiotherapy in near future.     

BDE-99 congener induces cell death by apoptosis of human hepatoblastoma cell line – HepG2

Polybrominated Diphenyl Ethers (PBDEs) are an important class of flame retardants with a wide range of toxic effects on biotic and abiotic systems. The toxic mechanisms of PBDEs are still not completely understood because there are several different congeners with different chemical and biological characteristics. BDE-99 is one of these, widely found in the environment and biological samples, showing evidence of neurotoxic and endocrine disruption activities, but with little information about its action mechanism described in the current literature. This work investigated the effects of BDE-99 on the HepG2 cell line in order to clarify its toxic mechanism, using concentrations of 0.5–25 μM (24 and 48 h). Our results showed that BDE-99 could cause cell death in the higher concentrations, its activity being related to a decrease in mitochondrial membrane potential and an accumulation of ROS. It was also shown that BDE-99 induced the exposure of phosphatidylserine, caspases 3 and 9 activation and DNA fragmentation in HepG2 cells, without causing the release of LDH. Thus it was shown that BDE-99 could cause HepG2 cell death by apoptosis, suggesting its toxicity to the human liver.     

Programmed cell death in human pathogenic fungi – a possible therapeutic target

ABSTRACT

Introduction: Diseases caused by pathogenic fungi are increasing because of antibiotic overuse, the rise of immunosuppressive therapies, and climate change. The limited variety of antimycotics and the rapid adaptation of pathogenic fungi to antifungal agents serve to exacerbate this issue. Unfortunately, about 1.6 million people are killed by fungal infections annually.

Areas covered: The discovery of the small antimicrobial proteins produced by microorganisms, animals, humans, and plants will hopefully overcome challenges in the treatment of fungal infections. These small proteins are highly stable and any resistance to them rarely evolves; therefore, they are potentially good candidates for the treatment and prevention of infections caused by pathogenic fungi. Some of these proteins target the programmed cell death machinery of pathogenic fungi; this is potentially a novel approach in antimycotic therapies. In this review, we highlight the elements of apoptosis in human pathogenic fungi and related model organisms and discuss the possible therapeutic potential of the apoptosis-inducing, small, antifungal proteins.

Expert opinion: Small antimicrobial proteins may establish a new class of antimycotics in the future. The rarity of resistance and their synergistic effects with other frequently used antifungal agents may help pave the way for their use in the clinic.     

Evodiamine induces tumor cell death through different pathways： apoptosis and necrosis

AIM: To study the different death pathways in human cervical cancer HeLa and melanoma A375-S2 cells initiated by evodiamine. METHODS: Viability of evodiamine-induced HeLa and A375-S2 cells was measured by MTT assay. Apoptotic cells with condensed or fragmented nuclei were visualized by Hoechst 33258 staining. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Proportion of cell death through apoptotic and necrotic pathways was determined by LDH activity-based cytotoxicity assays. Cell cycle distribution was observed by flow cytometry. RESULTS: Evodiamine induced HeLa and A375-S2 cell death dose- and time-dependently.Caspase-3 and -8 were activated in apoptosis induced by evodiamine 15 μmol/L. However, over 24- h incubation of A375-S2 cells, evodiamine 15 μmol/L initiated necrosis related to p38 and ERK (extracellular signal-regulated kinases)activities. Evodiamine-induced HeLa cell death was preceded by an accumulation of cells at the G2/M phase of the cell cycle, but there was no significant effect of evodiamine on A375-S2 cell cycle. CONCLUSION: Evodiamineinduces caspase-3,8-dependent apoptosis in HeLa cells which is related to G2/M arrest of the cell cycle. On the other hand, in A375-S2 cells, evodiamine initiates caspase-3,8-mediated apoptosis at early stages and the induction of MAPK-mediated necrosis at later stages of cell culture.     

Polyphenolic fraction of Pilea microphylla (L.) protects Chinese hamster lung fibroblasts against γ-radiation-induced cytotoxicity and genotoxicity

Present study was designed to compare cytoprotective and antigenotoxic activity of the polyphenolic fraction of Pilea microphylla (PM1) with that of its active polyphenolic constituents against γ-radiation in V79 cells. PM1 was standardized with respect to the polyphenols present by RP-HPLC. It was evaluated for its free radical scavenging potential using Fenton reaction-induced DNA damage and lipid peroxidation. Further, PM1 was subjected against γ-radiation-induced cytotoxicity and genotoxicity in V79 cells.PM1 significantly reduced free radical-mediated calf thymus DNA damage and lipid peroxidation. Among the concentrations tested (12.5, 25 and 50 μg/ml) for radioprotection, PM1 at 25 μg/ml exhibited maximum protection. Further, when compared with constituent polyphenols viz., rutin, quercetin and chlorogenic acid (concentrations equivalent to that present in PM1-25 μg/ml), a combination of polyphenols was found most effective in preventing γ-radiation-induced cytotoxicity and genotoxicity. To conclude, radioprotection is possibly a synergistic effect of the phytochemicals present in the herbal extract, rather than any single component.     

4＇-Chloro-3,5-dihydroxystilbene, a resveratrol derivative, induces lung cancer cell death

Aim:

To examine the antitumor effect of 4′-chloro-3,5-dihydroxystilbene, a resveratrol derivative, on lung adenocarcinoma A549 cells.

Methods:

The cytotoxic IC₅₀ was determined by direct cell counting. Flow cytometry, monodansylcadaverine (MDC) staining, transfection, Western blot and a proteasome activity assay were used to study the cellular mechanism of 4′-chloro-3,5-dihydroxystilbene. A xenograft nude mouse model was used to analyze the antitumor effect in vivo.

Results:

4′-Chloro-3,5-dihydroxystilbene induced a rapid and persistent increase in the intracellular reactive oxygen species in the cells, but the cell death could not be inhibited by two antioxidant agents. The derivative caused sub-G₁ formation, a decrease in the mitochondria membrane potential and poly (ADP-ribose) polymerase degradation, and the caspase inhibitor Z-VAD-FMK could partially prevent cell death. It also induced a significant increase in intracellular acidic vacuoles, LC3-II formation and intracellular GFP-LC3 aggregation. An autophagic inhibitor partially reversed cell death. Additionally, 4′-chloro-3,5-dihydroxystilbene induced the accumulation of ubiquitinated conjugates and inhibited proteasome activity in cells. In an in vivo study, 4′-chloro-3,5-dihydroxystilbene retarded tumor growth in nude mice.

Conclusion:

These data suggest that the resveratrol derivative 4′-chloro-3,5-dihydroxystilbene could be developed as an anti-tumor compound.     

The β-carboline alkaloid harmol induces cell death via autophagy but not apoptosis in human non-small cell lung cancer A549 cells

β-Carboline alkaloids are naturally occurring plant substances that have a wide spectrum of neuropharmacological, psychopharmacological, and antitumor effects. Recently, we have demonstrated that harmol, a β-carboline alkaloid, induces apoptosis by caspase-8 activation independently from Fas/Fas ligand interaction in human non-small cell lung cancer (NSCLC) H596 cells. Here, we found that harmol induces autophagy and cell death in human NSCLC A549 cells. Although harmol induced cell death in A549 cells in a significant dose- and time-dependent manner, it did not induce caspase-3, caspase-8, or caspase-9 activity. Furthermore, cleavage of poly-(ADP-ribose)-polymerase was not induced in A549 cells by harmol treatment. Autophagy, but not apoptosis, was detected by electron microscopy in A549 cells treated with 70 μM harmol. Pretreatment of A549 cells with 3-methyladenine, an autophagy inhibitor, as well as small interfering RNA (siRNA)-mediated knockdown of LC3, both suppressed harmol-induced cell death. These suggest that the induction of autophagy by harmol precedes cell death. The cytotoxicity of some anticancer agents is reportedly linked to autophagy induction. The 2 major autophagy regulatory pathways are the Akt/mammalian target of rapamycin (mTOR) pathway and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Although harmol treatment showed no effect on the Akt/mTOR pathway, it transiently activated the ERK1/2 pathway. However, inhibition of the ERK1/2 pathway using the mitogen-activated protein kinase (MEK)/ERK inhibitor U0126 partially suppressed autophagy. Therefore, although activation of the ERK1/2 pathway might be related to harmol-induced autophagy, another major pathway may also be involved in A549 cells.     

Pharmacological modulation of cell death in atherosclerosis: a promising approach towards plaque stabilization?

Despite tremendous advances over the last 15 years in identifying vulnerable atherosclerotic plaques, the incidence of death and disability caused by such lesions still remains the number one health threat in developed countries. Therefore, new systemic or focal therapies aimed at decreasing the overall burden of disease, and a change to a more benign phenotype, are needed. Because cell death is a prominent feature of advanced atherosclerotic plaques with a major impact on plaque destabilization, an increasing number of compounds targeting the apoptotic or autophagic machinery in atherosclerosis are being explored, predominantly at the preclinical level. This review will provide an overview of these compounds, with a focus on both inhibition and stimulation of cell death, to prevent acute coronary syndromes and sudden cardiac death.     

1-β-D-Arabinosylcytosine and 5-azacytidine induce internucleosomal DNA fragmentation and cell death in thymocytes

Incubation of mouse thymocytes with arabinosylcytosine or 5-azacytidine induced dose-dependent internucleosomal DNA cleavage followed by cell death. This process was RNA- and protein synthesis-dependent, since DNA fragmentation and cell death was inhibited by actinomycin D and cycloheximide. The results suggest that the cytidine analogs induce apoptosis, a programmed cell death, in thymocytes. The DNA cleavage induced by arabinosylcytosine and 5-azacytidine was inhibited by deoxycytidine and cytidine, respectively, suggesting that phosphorylation of these antimetabolites is required to induce DNA cleavage. DNA fragmentation was unaffected by the addition of aphidicolin or 3-aminobenzamide, indicating that DNA cleavage is not due to the inhibition of DNA synthesis or repair. Other antimetabolites, including methotrexate, fluoropyrimidines and thiopurines, failed to induce DNA fragmentation. Arabinosylguanine induced DNA fragmentation similar to that produced by the cytidine analogs, suggesting similarly to the selective sensitivity of T lymphocytes to deoxyguanosine toxicity. The precise mechanism by which DNA cleavage is induced remains unclear, but the present study shows that certain antimetabolites act on cells not only by inhibiting proliferation, but by inducing apoptosis with internucleosomal DNA fragmentation.     

[6]-Gingerol attenuates β-amyloid-induced oxidative cell death via fortifying cellular antioxidant defense system

β-Amyloid (Aβ) is involved in the formation of senile plaques, the typical neuropathological marker for Alzheimer’s disease (AD) and has been reported to cause apoptosis in neurons via oxidative and/or nitrosative stress. In this study, we have investigated the neuroprotective effect and molecular mechanism of [6]-gingerol, a pungent ingredient of ginger against Αβ_25-35-induced oxidative and/or nitrosative cell death in SH-SY5Y cells. [6]-Gingerol pretreatment protected against Aβ_25-35-induced cytotoxicity and apoptotic cell death such as DNA fragmentation, disruption of mitochondrial membrane potential, elevated Bax/Bcl-2 ratio, and activation of caspase-3. To elucidate the neuroprotective mechanism of [6]-gingerol, we have examined Aβ_25-35-induced oxidative and/or nitrosative stress and cellular antioxidant defense system against them. [6]-Gingerol effectively suppressed Aβ_25-35-induced intracellular accumulation of reactive oxygen and/or nitrogen species and restored Aβ_25-35-depleted endogenous antioxidant glutathione levels. Furthermore, [6]-gingerol treatment up-regulated the mRNA and protein expression of antioxidant enzymes such as γ-glutamylcysteine ligase (GCL) and heme oxygenase-1 (HO-1), the rate limiting enzymes in the glutathione biosynthesis and the degradation of heme, respectively. The expression of aforementioned antioxidant enzymes seemed to be mediated by activation of NF-E2-related factor 2 (Nrf2). These results suggest that [6]-gingerol exhibits preventive and/or therapeutic potential for the management of AD via augmentation of antioxidant capacity.     

Inhibition of calpain prevents NMDA-induced cell death and β-amyloid-induced synaptic dysfunction in hippocampal slice cultures

Background and purpose:

Alzheimer''s disease (AD) is a multifactorial, neurodegenerative disease, which is in part caused by an impairment of synaptic function, probably mediated by oligomeric forms of amyloid-β (Aβ). While the Aβ pathology mainly affects the physiology of neurotransmission, neuronal decline is caused by excitotoxic cell death, which is mediated by the NMDA receptor. A comprehensive therapeutic approach should address both Aβ-induced synaptic deficits, as well as NMDA receptor-mediated neurodegeneration, via one molecular target. This study was designed to test whether calpain could be involved in both pathological pathways, which would offer a promising avenue for new treatments.

Experimental approach:

Application of the specific, water-soluble calpain inhibitor A-705253 was used to inhibit calpain in hippocampal slice cultures. We examined whether inhibition of calpain would prevent Aβ-induced deficits in neurotransmission in CA1, as well as NMDA-induced neuronal cell death.

Key results:

A-705253 dose-dependently prevented excitotoxicity-induced neurodegeneration at low nanomolar concentrations, determined by propidium iodide histochemistry. Inhibition of the NMDA receptor similarly protected from neuronal damage. Caspase staining indicated that calpain inhibition was protective by reducing apoptosis. Electrophysiological analysis revealed that inhibition of calpain by A-705253 also fully prevented Aβ oligomer-induced deficits in neurotransmission. The protective effect of calpain was compared to the clinically available NMDA receptor antagonist memantine, which was also effective in this model.

Conclusions and implications:

We suggest that inhibition of calpain exhibits a promising strategy to address several aspects of the pathology of AD that may go beyond the available therapeutic intervention by memantine.     

Uranyl nitrate-exposed rat alveolar macrophages cell death: influence of superoxide anion and TNF α mediators

Uranium compounds are widely used in the nuclear fuel cycle, military and many other diverse industrial processes. Health risks associated with uranium exposure include nephrotoxicity, cancer, respiratory, and immune disorders. Macrophages present in body tissues are the main cell type involved in the internalization of uranium particles. To better understand the pathological effects associated with depleted uranium (DU) inhalation, we examined the metabolic activity, phagocytosis, genotoxicity and inflammation on DU-exposed rat alveolar macrophages (12.5-200 μM). Stability and dissolution of DU could differ depending on the dissolvent and in turn alter its biological action. We dissolved DU in sodium bicarbonate (NaHCO₃ 100 mM) and in what we consider a more physiological vehicle resembling human internal media: sodium chloride (NaCl 0.9%). We demonstrate that uranyl nitrate in NaCl solubilizes, enters the cell, and elicits its cytotoxic effect similarly to when it is diluted in NaHCO₃. We show that irrespective of the dissolvent employed, uranyl nitrate impairs cell metabolism, and at low doses induces both phagocytosis and generation of superoxide anion (O₂⁻). At high doses it provokes the secretion of TNFα and through all the range of doses tested, apoptosis. We herein suggest that at DU low doses O₂⁻ may act as the principal mediator of DNA damage while at higher doses the signaling pathway mediated by O₂⁻ may be blocked, prevailing damage to DNA by the TNFα route. The study of macrophage functions after uranyl nitrate treatment could provide insights into the pathophysiology of uranium‐related diseases.     

Streptomyces spores from mouldy houses induce nitric oxide, TNFα and IL-6 secretion from RAW264.7 macrophage cell line without causing subsequent cell death

The current view is that only bacterial lipopolysaccharide (LPS) and gamma interferon (IFNγ) are able to alone activate macrophages to secrete nitric oxide (NO), probably a causative agent of cell death. Moreover, some cytokines and gram positive pathogens together with IFNγ induce NO-production. Surprisingly, spores of Streptomyces sp., which are mesophilic gram-positive bacteria found in mouldy houses, stimulated RAW264.7 macrophages to produce pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6), and induced the expression of inducible NO-synthase (iNOS) with a subsequent NO-production. However, the Streptomyces spores did not kill NO-producing macrophages, as did both LPS and gram negative bacteria Pseudomonas fluorescens, strong inducers of cytokine- and NO-production. These results imply that Streptomyces sp., induced cytokine and NO-secretion, may play a role in the responses evoked by exposure to these microbes. Moreover, factors other than, or in addition to NO, are necessary for cytotoxicity in murine macrophages.