Acceleration of slow autophagy flux induced by arabinofuranosyl cytidine improves its antileukemic effectiveness in M-NFS-60 cells.

Arabinofuranosyl cytidine (AraC) is an analog of deoxycytidine used as an anticancer drug for leukemic patients. The effective dose always produces severe complications. The present study investigated the modulation of autophagy and its impact on the cytotoxicity of AraC toward murine myelogenous leukemia cells (M-NFS-60). Autophagy was inhibited by NH4Cl or Bafilomycin A1 or enhanced by amino acid starvation, glucose starvation, mild hyperthermia (41 °C), or rapamycin (Rap). Cells were treated with different concentrations, 0 to 2 µM, of AraC in the presence or absence of autophagy modulators. AraC-induced apoptosis is combined with autophagy, especially at lower concentrations. This autophagy is characterized by a slow flux, as indicated by levels of LC3B II and P62 proteins. Inhibition of autophagy did not alter cleaved caspase 3 levels (c-casp.3) or cell viability measured by MTT assays. Conversely, acceleration of AraC-induced autophagy by co-treatment with autophagy inducers reduced cell viability and increased c-casp.3 and c-PARP levels. Further, c-PARP levels were reduced in the presence of caspase inhibitor, Z-VAD-FMK. Enhancement of slow autophagic flux induced by low concentrations of AraC significantly increased the cytotoxicity of AraC toward M-NFS-60 cells. Such coadministration of autophagy inducers might improve the efficacy of AraC treatment and reduce effective doses.

Melatonin inhibits breast cancer cell invasion through modulating DJ-1/KLF17/ID-1 signaling pathway.

Breast cancer is the most common neoplastic disorder diagnosed in women. The main goal of this study was to explore the effect of melatonin against breast cancer metastasis and compared this with the actions of taxol (a well-known chemotherapeutic drug), and the impact of their combination against breast cancer metastasis. Melatonin showed no cytotoxic effect while taxol showed antiproliferative and cytotoxic effects on MCF-7 and MDA-MB-231 cells. Furthermore, melatonin inhibited the generation of reactive oxygen species. Melatonin and taxol clearly decreased cell migration and invasion at low doses, especially those matching the normal physiological concentration at night. Melatonin and taxol markedly reduced DJ-1 and ID-1 and increased KLF17 messenger RNA and protein expression levels. The present results also showed that melatonin and taxol induced GSK3-ß nuclear and Snail cytosolic localization. These changes were accompanied by a concurrent rise in E-cadherin expression. The above data show that normal levels of melatonin may help in preventing breast cancer metastasis through inhibiting DJ-1/KLF17/ID-1 signaling pathway. The combination of melatonin and taxol is a potent candidate against breast cancer metastasis, better than using melatonin or taxol as a single drug.

Low doses of Paclitaxel repress breast cancer invasion through DJ-1/KLF17 signalling pathway.

Paclitaxel (taxol) is an important agent against many tumours, including breast cancer. Ample data documents that paclitaxel inhibits breast cancer metastasis while others prove that paclitaxel enhances breast cancer metastasis. The mechanisms by which paclitaxel exerts its action are not well established. This study focuses on the effect of paclitaxel, particularly the low doses on breast cancer metastasis and the mechanisms that regulate it. Current results show that, paclitaxel exerts significant cytotoxicity even at low doses in both MCF-7 and MDA-MB-231 cells. Interestingly, paclitaxel significantly inhibits cell invasion and migration, decreases Snail and increases E-cadherin mRNA expression levels at the indicated low doses. Furthermore, paclitaxel-inhibiting breast cancer metastasis is associated with down-regulation of DJ-1 and ID-1 mRNA expression level with a concurrent increase in KLF17 expression. Under the same experimental conditions, paclitaxel induces KLF17 and concurrently represses ID-1 protein levels. Our results show for the first time that paclitaxel inhibits breast cancer metastasis through regulating DJ-1/KLF17/ID-1 signalling pathway; repressed DJ-1 and ID-1 and enhanced KLF17 expression.

Melatonin administration ameliorates cadmium-induced oxidative stress and morphological changes in the liver of rat.

The oxidative status and the morphological changes of liver of rats exposed to cadmium (5 mg Cd/kg body weight subcutaneously) for 22 days and the protective role of melatonin (10mg/kg b.w.) against the toxicity of cadmium was studied. The concentration of malondialdehyde (MDA), as an indicator of lipid peroxidation, activity of the antioxidant enzyme superoxide dismutase (SOD) as well as the concentration of glutathione (GSH) was measured in the liver. The morphological changes were investigated using both light and electron microscopes. The exposure to Cd led to an increase of MDA levels and a decrease of both the activity of SOD and GSH concentration in the liver. In contrast, melatonin administration restored the previous changes to nearly the normal levels. Morphologically, Cd led to different histopathological changes such as loss of normal architecture of the parenchymatous tissue, cytoplasmic vacuolization, cellular degeneration and necrosis, congested blood vessels, destructed cristae mitochondria, fat globules, severe glycogen depletion, lipofuscin pigments, and collagenous fibers formation. Again, melatonin administration counteracts all changes and the tissue appears more or less normal. The rate of recovery was faster when melatonin was administered for treatment after the exposure to cadmium than if the animals left without any treatment. The results suggest that melatonin may be useful as an antioxidant in combating free radical-induced oxidative stress and tissue injury that is a result of cadmium toxicity.

Effect of chronic nicotine administration on the rat lung and liver: beneficial role of melatonin.

Cigarette smoking is common in societies worldwide and has been identified as injurious to human health. The current study was designed to investigate the protective effect of melatonin, a radical scavenger and antioxidant, on nicotine-induced oxidative stress and morphological changes in the lung and liver of the rats. Three groups of male rats (controls, nicotine-treated [0.5 mg/kg], and nicotine plus melatonin [10 mg/kg] were used in this study. Levels of lipid peroxidation (LPO) products, superoxide dismutase (SOD) and glutathione (GSH) activity were measured in the tissue homogenates. Immunohistochemical and histological changes were also examined. The results revealed an increase in LPO and decrease in both SOD and GSH activity in the lung and liver homogenates on nicotine-treated rats. Melatonin administration to nicotine-treated rats attenuated the increase in LPO products and restored the SOD activity and GSH levels. The immunohistochemical and histological examination demonstrated marked increase in the immunoreactivity of nitrotyrosine, a specific "footprint" of peroxynitrite, and tissue damage in the lung and liver of nicotine-administered animals. Again, melatonin treatment reduced both nitrotyrosine reactivity and tissue damage associated with nicotine administration. These results, along with previous observations, suggest that melatonin may be useful in combating free radical-induced oxidative stress and tissue injury that is a result of nicotine toxicity.

An autoradiographic study of cellular proliferaton, DNA synthesis and cell cycle variability in the rat liver caused by phenobarbital-induced oxidative stress: the protective role of melatonin.

The protective effect of melatonin against phenobarbital-induced oxidative stress in the rat liver was measured based on lipid peroxidation levels (malondialedyde and 4-hydroxyalkenals). Cellular proliferation, DNA synthesis and cell cycle duration were quantitated by the incorporation of (3)H-thymidine, detected by autoradiography, into newly synthesized DNA. Two experiments were carried out in this study, each on four equal-sized groups of male rats (control, melatonin [10 mg/kg], phenobabital [20 mg/kg] and phenobarbital plus melatonin). Experiment I was designed to study the proliferative activity and rate of DNA synthesis, and measure the levels of lipid peroxidation, while experiment II was for cell cycle time determination. Relative to the controls, the phenobarbital-treated rats showed a significant increase (P < 0.01) in the lipid peroxidation levels (30.7%), labelling index (69.4%) and rate of DNA synthesis (37.8%), and a decrease in the cell cycle time. Administering melatonin to the phenobarbital-treated rats significantly reduced (P < 0.01) the lipid peroxidation levels (23.5%), labelling index (38.2%) and rate of DNA synthesis (29.0%), and increased the cell cycle time. These results seem to indicate that the stimulatory effect of phenobarbital on the oxidized lipids, proliferative activity, kinetics of DNA synthesis and cell cycle time alteration in the liver may be one of the mechanisms by which the non-genotoxic mitogen induces its carcinogenic action. Furthermore, melatonin displayed powerful protection against the toxic effect of phenobarbital.

Role of melatonin in reducing hypoxia-induced oxidative stress and morphological changes in the liver of male mice.

Oxygen deficiency during critical illness may cause profound changes in cellular metabolism and subsequent tissue and organ dysfunction. Thus, the present study was designed to determine the effects of hypoxia and reoxygenation on the levels of lipid peroxidation and the morphological changes in the liver of male mice as well as the protective role of melatonin as an antioxidant. Two experiments were carried out in this study. Experiment I includes three groups of mice (control, hypoxic, and hypoxic+melatonin) while the experiment II includes two groups (reoxygenated and reoxygenated+melatonin). The levels of oxidized lipids were measured and the morphological changes were investigated using light and electron microscopy. In experiment I, hypoxia strongly stimulated lipid peroxidation levels (88%) while melatonin administration inhibited this increase (69%). Severe morphological changes (necrosis, dilated congested blood vessels, collection of inflammatory cells, condensed heterochromatic with irregular outlines nuclei, and mitochondrial degeneration) were detected in the liver of hypoxic mice. In experiment II, reoxygenation inhibited the levels of oxidized lipids (42%) versus hypoxic mice and some morphological changes were detected. When melatonin was given before reoxygenation, it inhibited the levels of lipid peroxidation by 66% versus hypoxic mice. Also, melatonin enhanced the recovery profile by 41% when compared with mice that reoxygenated with room air only. All morphological alterations that detected in both hypoxic and reoxygenated mice were repaired when melatonin administered. These results indicate that hypoxia and reoxygenation induce severe alterations in the liver and that melatonin exerts beneficial role in restoring tissue alterations after subjection to hypoxia.

Aluminum-induced neurotoxicity and oxidative damage in rabbits: protective effect of melatonin.

OBJECTIVE: The present study was aimed to investigate: (1) the neurotoxic oxidative damage of orally administered aluminum chloride (AlCl3) in rabbits (Biochemical and morphopathological studies). (2) The effect of melatonin as an antioxidant and free radical scavenger on oxidative neuropathic changes. METHODS: Thirty-five male rabbits were divided into 4 groups (A, B, C [10 animals each] and D [5 animals]). Group A received AlCl3 (20 mg/l via drinking water for 3 months). Group B received AlCl3 for 3 months then administered with melatonin (10 mg/kg b.w. sc daily for 15 days). Group C received AlCl3 plus melatonin for 3 months. Group D received the solvent and served as control. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) as lipid peroxides as well as superoxide dismutase (SOD) as an antioxidant enzyme were measured. Aluminum residue in the brain tissue was measured spectrophotometerically. The morphopathological changes were also examined by light and electron microscopes. RESULTS: MDA and 4-HAD were significantly increased in group A versus those of controls while significantly decreased in groups B and C compared with those of A group. SOD run in an opposite manner. Aluminum concentration was significantly increased in groups A, B and C when compared with group D while it significantly decreased in groups B and C when compared with that of group A. The neuropathlogical examination in the animals of group A revealed atrophy and apoptosis of the neurons in cerebral cortex and hippocampus. This was associated with neurofibrillary degeneration as well as argyrophilic inclusion. Schwan cell degeneration and nerve fiber demylination were also encountered. The elaboration of lipid peroxidation products, inhibition of antioxidant enzymes and the morphopathological changes were minimized in the Al/Mel treated groups and markedly improved in Al+Mel treated group CONCLUSION: Chronic aluminum exposure in rabbits had dramatic encephalopathic morphopathological lesions. It enhances the lipid peroxidation production and inhibits the SOD enzyme. Melatonin had a good prophylactic effect as an antioxidant in aluminum encephalopathy.

Melatonin protects against lead-induced hepatic and renal toxicity in male rats.

The present study was designed to investigate the potential protective effect of melatonin against the hepatic and renal toxicity of lead in male rats. Three groups of animals were used in this study (control, lead acetate-treated (100 mg/kg), and lead acetate plus melatonin (10 mg/kg) for 30 days. Levels of lipid peroxidation (LPO) products, superoxide dismutase (SOD) activity, total glutathione (GSH), histopathological changes in the liver and kidneys were investigated. In addition, nuclear area (NA), nuclear volume (NV) and the ratio of nuclear volume/cellular volume (N/C) were measured in the liver. The results revealed increased LPO and decreased SOD, GSH, NA, NV and N/C in the studied organs of lead-treated rats. Histopathological observations showed severe damage in the liver and kidneys. Melatonin co-treatment to the lead-administered rats attenuated the increase of LPO and restored the activity of SOD and levels of GSH as well as the mean values of NA, NV and N/C. Also, the morphological damage in the liver and kidneys was reduced and the tissues appeared like those of controls. The present study suggests that melatonin may be useful in combating free radical-induced damage due to lead toxicity.

Melatonin supplementation restores cellular proliferation and DNA synthesis in the splenic and thymic lymphocytes of old rats.

OBJECTIVES: In this study we investigated the effect of melatonin treatment on the proliferative activity, the rate of DNA synthesis and the histopathological changes of splenic and thymic lymphocytes in old rats. METHODS: Two subgroups of old rats (25-months-old) were used in this study. One subgroup was given melatonin in the drinking water (250-300 mg/day/rat) for 3 months while the second subgroup was given water containing diluent. A third group consisted of young rats (3-months-old) which served as an additional control. RESULTS: A (3)H-thymidine autoradiographic investigation showed a reduction in both the proliferative activity and the rate of DNA synthesis in splenic and thymic lymphocytes in old rats. In addition, light and electron microscopy showed severe histopathological changes in these cells from diluent-treated old rats. Melatonin administration increased the proliferative activity and the rate of DNA synthesis in the lymphocytes of both the spleen and thymus of the old animals. Also, histopathological changes were partially reversed by melatonin treatment with the tissues appearing similar to those in the young rats. CONCLUSION: The stimulation of the lymphocyte activity by melatonin is a beneficial response, especially in old rats, since aging results in an inhibition in lymphocytic functions.

Prophylactic effect of melatonin in reducing lead-induced neurotoxicity in the rat.

Oxidative stress is a likely molecular mechanism in lead neurotoxicity. Considering the antioxidant properties of melatonin, this study investigated the neuroprotective potential of melatonin in the hippocampus and corpus striatum of rats treated with lead. Three groups of male rats (control, lead acetate-treated [100 mg/kg], and lead acetate plus melatonin [10 mg/kg] for 21 consecutive days) were used. Levels of products of lipid peroxidation (LPO), glutathione (GSH) and superoxide dismutase (SOD) activity were measured in brain homogenates. Histological changes in the pyramidal cells of the hippocampus and the putamen of the corpus striatum were examined. The results documented increased LPO and decreased GSH and SOD activity in the brain homogenates of lead-treated rats. Histological observations revealed severe damage and a reduction in neuronal density in the hippocampus and corpus striatum. When melatonin was given to lead-treated rats, it almost completely attenuated the increase in LPO products and restored GSH levels and SOD activity. Also, the morphological damage was reduced and neuronal density was restored by melatonin. Considering the ease with which melatonin enters the brain, these results, along with previous observations, suggest that melatonin may be useful in combating free radical-induced neuronal injury that is a result of lead toxicity.

Inhibition of 2-nitropropane-induced cellular proliferation, DNA synthesis and histopathological changes by melatonin.

OBJECTIVES: 2-Nitropropane (2-NP) is mutagenic in a number of short-term mutagenicity assays in vitro and in vivo, and is a potent hepatocarcinogen in rats. Many studies have determined that differences in the metabolism and disposition of the chemicals that produce mutagenicity were not responsible for their observed carcinogenic differences, but that carcinogenicity correlated with the ability of the respective isomer to induce cell proliferation in the target organ. METHODS: Three groups of male rats (control, 2-NP-treated [4 mmol/kg] and 2-NP + melatonin [10 mg/kg]) were used in the current study. Cell proliferation was quantitated by incorporation of 3H-thymidine, detected by autoradiography, into newly synthesized DNA. Histopatholgical study was carried out to investigate the morphological changes. RESULTS: Twenty four hours after 2-NP administration, there was an increase in the labelling index (LI) and grain count per labelled nucleus (GC/N) in the hepatocytes of 2-NP-injected rats versus those of control animals. The increase was 69.5% in LI and 29.4% in GC/N. Melatonin treatment, 30 minutes preceding 2-NP, reduced the increase in LI (44.4%) and GC/N (20.7%) when compared with 2-NP-treated rats. Histopathology revealed multiple focal areas of necrosis in the liver following 2-NP injection. In the lung, there was a mucinous degeneration of the bronchial epithelium. Melatonin treatment restored the histopathological changes in both the liver and lung and they are more or less normal. CONCLUSION: Overall, these results seem to indicate that the stimulatory effect of 2-NP on the cellular proliferation and the rate of DNA synthesis in the liver may be one of mechanisms by which the carcinogen induces its carcinogenic action. Also, melatonin treatment strongly protects the studied organs against the toxic effect of 2-NP.

Melatonin reduces oxidative damage and increases survival of mice infected with Schistosoma mansoni.

The tropical parasite Schistosoma mansoni causes granulomatous inflammation after its eggs lodge in hepatic portal capillaries. In vitro studies indicate that the host's response involves the production of reactive oxygen species, although whether this occurs in vivo at the site of the infection is unknown. The role of oxidative processes in mice infected with S. mansoni was investigated in the current study using the antioxidant melatonin. In Experiment 1, the survival rate of infected mice with and without daily melatonin (10 mg/kg) administration was determined. After 56 d, 25 of 25 infected mice that were diluent treated had died. In contrast, 22 or 25 infected mice (88%) given melatonin were still alive at 56 d. Of these 22 surviving mice, melatonin injections were continued in 11 while the 11 others were switched to diluent. Within 10 d, 11 of 11 diluent-injected mice that were infected with S. mansoni were dead while 6 of 11 melatonin-treated mice survived. In Experiment 2, S. mansoni-infected mice were treated for 30 d with either melatonin or diluent. Uninfected, untreated mice served as controls. In these mice, the levels of lipid peroxidation (LPO) products, vitamin E, nitric oxide (NO), glutathione (GSH), and superoxide dismutase (SOD) activity in the liver, kidney, and spleen were measured. In the serum, cholesterol levels and liver damage (alkaline phosphatase (ALP), aspartate transaminases (AST), total protein, and albumin) were monitored. In addition, peroxynitrite anion (ONOO(-)) in the liver and kidney and inducible nitric oxide synthase (iNOS) in the spleen were immunocytochemically localized. Also, histopathological changes in the liver, kidney, and spleen were examined. The results documented increased LPO and NO levels and decreased vitamin E, GSH, and SOD activity in the liver, kidney, and spleen of S. mansoni-infected mice. Also, there was an increase in serum cholesterol and evidence of liver damage in the infected mice. Immunohistochemical results indicated positive staining of ONOO(-) in the liver and kidney and positive iNOS staining in the spleen of S. mansoni-infected mice. Histopathological observations revealed granuloma formation in the liver with eosinophil infiltration, a large number of megakaryocytes in the spleen, and degeneration with necrotic cells in some tubules of the kidney cortex in the infected mice. Melatonin administration after S. mansoni infection prevented most of the previously described changes. These results suggest that oxidative processes occur at the site of inflammation and are involved in the damaging effects of schistosomiasis and indicate that free radicals may be a major component of the disease. Likewise, melatonin, presumably due to its antioxidant and free radical scavenging activity, is highly protective against the pathological changes associated with schistosomiasis.

Melatonin protects against oxidative stress induced by the kidney carcinogen KBrO(3).

OBJECTIVES: Free radical scavengers can protect against the genotoxicity induced by chemical carcinogens by decreasing oxidative stress. The protective effect of the antioxidant melatonin was studied in the kidney and liver of rats treated with the kidney-specific carcinogen potassium bromate (KBrO(3)). The major endpoint of oxidative damage measured in this report was lipid peroxidation. METHODS: Four groups of male rats (controls, melatonin-injected [10 mg/kg x4], KBrO(3)-injected [100 mg/kg], and melatonin+-KBrO(3)) were used in the current study. The concentrations of malondialdehyde (MDA) were assayed as an index of oxidatively damaged lipid in the kidney and liver. RESULTS: Twenty-four hours after KBrO(3) administration, MDA levels were significantly increased in the kidney while the increase in the liver was not statistically significant compared to levels in control rats. The percentage increases in lipid peroxidation products were 32.8% and 12.6% for the kidney and liver, respectively. In rats given melatonin 30 minutes before KBrO(3), and three more times after KBrO(3) (i.e., every 6 hours), the increase in MDA levels was reduced in the kidney. Histopathological examination demonstrated marked changes in the structure of the kidney and slight changes in the liver. In the kidney, microscopic examination revealed atypical tubules, atypical hyperplasia, hyaline droplet degeneration, necrotic changes and stratified squamous cell metaplasia. Again, melatonin treatment inhibited the tissue damage associated with KBrO(3) administration. CONCLUSION: These results show that melatonin as an antioxidant and free radical scavenger can prevent oxidative stress induced by the carcinogen KBrO(3).