Viability and oxidative response of human colorectal HCT-116 cancer cells treated with visfatin/eNampt in vitro.

Visfatin/eNampt is a novel adipokine, secreted by visceral and subcutaneous fat, which could be involved in the development of obesity-associated cancer. Only few studies revealed reactive oxygen species (ROS)-dependent action of visfatin in endothelial cells, myotubes and melanoma cells. The potential pro-apoptotic properties of visfatin/eNampt in human colorectal HCT-116 cells remain unknown. The aim of the study was to examine the effects of visfatin/eNampt on cell viability along with the determination of apoptosis/necrosis extent and ROS level in HCT-116 cells. Additionally antioxidant enzymes' activities (i.e catalase (CAT), gluthatione peroxidase (GSH-Px)), and lipid peroxidation intensity in HCT-116 cells line was evaluated. Viability of HCT-116 cells was decreased after visfatin/eNampt treatment for 24 hours. The number of apoptotic cells in tested cells treated with increasing visfatin/eNampt concentrations (10, 100, 250 ng/ml) was elevated compared to untreated cells (6.4%, 9.7%, 16% vs. 3.2%; respectively). After 24 hours in the visfatin/eNampt treated group (10 - 100 ng/ml) CAT and GSH-Px activities significantly increased and this observation was accompanied by the decrease of ROS level when compared to the control group. Interestingly ROS level (using DCF detection technique) and lipid peroxidation ratio were increased in cells stimulated by visfatin/eNampt in concentration of 250 ng/ml along with the decreased activity of selected antioxidant enzymes when compared to remaining study groups, including control. We concluded that visfatin/eNampt induces decrease of cell viability and apoptosis boost in human colorectal cancer HCT-116 cells line. Visfatin/eNampt affected the level of ROS as well as antioxidant capacity, however the association of ROS level and apoptosis rate was not linear. The role for visfatin/eNampt in cancer redox status in vitro may provide a greater insight into the association between fat derived visfatin/eNampt and its endocrine action in colorectal carcinoma cells.

Influence of melatonin supplementation on serum antioxidative properties and impact of the quality of life in multiple sclerosis patients.

The relationship between the prevalence of multiple sclerosis (MS) and sunlight's ultraviolet radiation was proved. Oxidative stress plays a role in the pathogenic traits of MS. Melatonin possesses antioxidative properties and regulates circadian rhythms. Several studies have reported that the quality of life is worse in patients with MS than in healthy controls, with a higher prevalence of sleep disturbances, depression and fatigue. The aim of study was to evaluate 5 mg daily melatonin supplementation over 90 days on serum malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity and its' influence on impact of the quality of life of MS patients. A case-control prospective study was performed on 102 MS patients and 20 controls matched for age and sex. The EDSS, MRI examinations and Multiple Sclerosis Impact Scale (MSIS-29) questionnaire was completed. Marked increase in serum MDA concentration in all MS patients groups was observed and after melatonin treatment decreased significantly in interferons-beta and glatiramer acetate-treated groups, but not in mitoxantrone-treated group. A significant increase in SOD activity compared to controls only in glatiramer acetate-treated group was observed. After 3 months melatonin supplementation the SOD activity increased compared to initial values in interferons beta-treated groups. A significant increase in both MSIS-29-PHYS and MSIS-29-PSYCH items mean scores only in the MX group as compared to other groups was observed. There were no significant differences in mean MSIS-29-PHYS was observed before and after melatonin therapy. Melatonin supplementation caused a decrease in mean MSIS-29-PSYCH scores compared to initial values in interferons beta-treated groups. Finding from our study suggest that melatonin can act as an antioxidant and improves reduced quality in MS patients.

Exogenous administration of visfatin affects cytokine secretion and increases oxidative stress in human malignant melanoma Me45 cells.

Visfatin has recently been established as a novel adipokine that is predominantly expressed in visceral fat. Recombinant visfatin has immunomodulating properties, which can activate human leukocytes in vitro to induce cytokine production (IL-1ß, TNF-α, and IL-6). Only few studies have investigated the effect of visfatin on prostate, breast, ovarian cancer as well as astrocytoma cell biology. There have been no studies on the cytokine secretion in human melanoma cells in response to visfatin stimulation along with intracellular protein kinases inhibitors. ELISA assay was performed in supernatants of Me45 cells stimulated with visfatin in the presence or the absence of specific pharmacological inhibitors of the indicated protein kinases (p38, MEK 1, PI3k and JAK kinase) and nuclear factor kappa B (NK-κB) inhibitor. Intracellular reactive oxygen species level was measured in 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA)-loaded cells using a fluorescent measurement system. For determination of NF-κB activation, activated NF-κB p65 subunit was determined using an EZ-TFA-detect chemiluminescent transcription factor assay. We report that visfatin led to the significant increase in IL-6 and IL-8 level in culture supernatants of human malignant melanoma Me45 cells. Additionally visfatin resulted in the increase of the intracellular reactive oxygen species level. PI3k and NF-κB pathways were activated upon visfatin stimulation. The results may reflect the fact that PI3k pathway stimulation by visfatin may further lead to NF-κB activation and pro-inflammatory response.

Antioxidative enzymes activity and malondialdehyde concentration during mitoxantrone therapy in multiple sclerosis patients.

Mitoxantrone (MX) is approved for the treatment of aggressive relapsing-remitting, secondary-progressive and progressive-relapsing form of multiple sclerosis (MS). The mechanism of its action is multiaxial, however, it is not free from side effects. The causes of the side effects are still unknown and require further investigation. The aim of this study was to investigate the influence of MX therapy on enzymatic parameters of endogenous antioxidative status: manganese and copper/zinc superoxide dismutase (MnSOD, Cu/ZnSOD), catalase (CAT), glutathione peroxidase (GSH-Px) and lipid peroxidation marker--malondialdehyde (MDA) in blood serum and cerebrospinal fluid (CSF) in patients suffering from MS. After the MX therapy serum and the CSF MDA concentrations increased significantly. We reported that MnSOD activities decrease in serum and the CSF, while, surprisingly, the serum Cu/ZnSOD activity increases after the MX therapy. We also noted a marked decrease in CSF CAT and GSH-Px activity after the MX treatment. Our results strongly suggest the influence of MX therapy on oxidation/antioxidation status of serum and the CSF. These findings open up new opportunities for a better understanding of underlying physiopathological events in MS and provide a new insight into MX's mechanisms of action, especially its potent side effects.

Antioxidant enzyme activity and malondialdehyde concentration in the plasma and erythrocytes of patients with urticaria induced by nonsteroidal anti-inflammatory drugs.

BACKGROUND: It has been suggested that oxidative stress is a crucial event in some forms of urticaria. AIM: To evaluate the blood oxidant/antioxidant profile of patients suffering from urticaria induced by nonsteroidal anti-inflammatory drugs (NSAIDs). METHODS: We measured the activity of the antioxidant enzymes copper-zinc superoxide dismutase (Cu/ZnSOD), glutathione peroxidase (GSH-Px), and catalase (CAT), and the levels of malondialdehyde (a marker of lipid peroxidation) in the plasma and erythrocytes of 12 females with NSAID-induced urticaria and in 19 healthy controls. RESULTS: The enzyme activity in plasma (CuZn/SOD) and in erythrocytes (CuZn/SOD, GSH-Px, and CAT) did not differ significantly between urticaria patients and controls. Moreover, the levels of malondialdehyde in plasma and erythrocytes did not differ significantly between the 2 groups. CONCLUSIONS: It seems that processes associated with urticaria induced by NSAIDs may not modify antioxidant enzyme activity and may not enhance lipid peroxidation in peripheral blood.

Influence of local peripheral temporary ischaemia on biochemical and histological effects in small intestine and serum of rats following abdominal irradiation.

The local temporary ischaemia effect on radiation-induced lipid peroxidation, superoxide dismutase isoenzyme activities, and intestinal crypt number was estimated in male WAG-strain rats in vivo. The animals were irradiated in the abdomen area with doses of 2 Gy for ten consecutive days using a Philips 60Co source. The calculated dose rate was 0.595 Gy/min. Local temporary ischaemia was induced by clamping the tail base before each irradiation. The parameters evaluated were: TBA-RS level and enzymatic activities of CuZnSOD, MnSOD in serum and jejunum. The number of jejunum crypts was assigned as a histopathologic parameter. The results showed a clear protection by ischaemic preconditioning for crypt survival. The difference in the number of crypts in irradiated animals with and without local temporary ischaemia was statistically significant (Student's t-test P < 0.05). Also, significant enhancement of TBA-RS was observed in the serum of irradiated animals. Local temporary ischaemia application diminished the concentration of radiation- induced TBA-RS. The differences in the levels of TBA-RS in the serum were statistically significant (ANOVA P < 0.002). In contrast, there was no evident effect on the level of TBA-RS in tissue homogenates in any investigated groups. Some fluctuation of CuZnSOD isoenzyme activity in intestinal tissue was noted; however, the differences were not significant. Local temporary ischaemia had no influence on Mn- SOD activity in serum, and in both irradiated groups the behaviour of this isoenzyme was similar. Also, there were no differences in MnSOD activity measured in tissue homogenates. These findings support results of our previous in vivo studies, suggesting that local temporary ischaemia can prevent oxidative effects of fractionated radiotherapy.

Influence of melatonin on cell proliferation, antioxidative enzyme activities and lipid peroxidation in 3T3-L1 preadipocytes--an in vitro study.

Melatonin, acting via MT1, MT2 and MT3 membrane receptors, influences central and peripheral regulatory mechanisms of energy homeostasis in mammals. In peripheral tissues, it evokes the pro-proliferative effect in a number of normal cells. Moreover, this hormone inhibits lipolysis in subcutaneous adipocytes in vitro and reduces free oxygen metabolites-induced damage acting directly, as a free radical scavenger, and indirectly, by stimulation of antioxidative enzyme activities. The aim of the study was to examine the effects of melatonin on cell proliferation, antioxidative enzyme activities and malondialdehyde (MDA) concentration in 3T3-L1 preadipocyte cell culture. We found that melatonin (10(-3) and 10(-6) M/L) stimulated cell proliferation in dose- and time-depending manner, and this effect was inhibited by a relatively selective MT2 receptor antagonist - luzindole (10(-4) M/L). Melatonin, increased activities of manganese containing and copper-zinc containing superoxide dismutase (MnSOD and Cu/ZnSOD) isoenzymes, catalase, glutathione reductase and glutathione peroxidase after 24 h of incubation. In contrast, after 48 h of incubation, activities of all studied enzymes were lower than in the control group. There were no changes in MDA concentrations after 24 h of incubation, whereas, in melatonin-treated media, after 48 h of the experiment, MDA level was significantly decreased. Our results demonstrate that melatonin, acting via MT2 receptors, stimulates proliferation of 3T3-L1 preadipocytes and this action could be due to the enhancement in antioxidative enzyme activities and attenuation of lipid peroxidation by this indole.

Effect of extremely low frequency of electromagnetic fields on cell proliferation, antioxidative enzyme activities and lipid peroxidation in 3T3-L1 preadipocytes--an in vitro study.

The exposure to extremely low frequency electromagnetic field (ELF-MF, frequencies less than 200-300 Hz) can alter the transcription and translation of genes, influence the cell proliferation rate and affect enzyme activities. Moreover, the hypothesis that ELF-MF increases free oxygen metabolites generation has been proposed. Since recent in vivo studies suggest that electric and magnetic fields are able to affect adipose cells metabolism. The aim of the study was to examine the effects of ELF-MF (frequency of basic impulse 180-195 Hz, induction 120 microT) on cell proliferation, antioxidative enzyme activities and malondialdehyde (MDA) concentration in 3T3-L1 preadipocyte cell culture. We found that ELF-MF application lasting 36 minutes daily failed to influence cell count after 24h and 48 h of incubation. After 24 h, in the ELF-MF treated group, manganese- and copper-zinc-containing superoxide dismutase (MnSOD and Cu/ZnSOD) isoenzymes media activities were decreased, catalase activity was increased, whereas there were no significant differences in glutathione peroxidase (GSH-Px) and glutathione reductase (GSSG-Rd) activities in comparison to the control. After 48 h of incubation, all enzyme activities were reduced, except for GSSG-Rd, in which no changes were noticed. MDA concentration at 24 h after incubation with the exposure to ELF-MF was significantly higher in comparison to the control, without ELF-MF. After 48 h of incubation, MDA levels were significantly lower in both groups with no differences between the groups without and with ELF-MF. We conclude that ELF-MF influences antioxidative enzyme activities and increases lipid peroxidation in 3T3-L1 preadipocyte cultures.

Influence of extremely-low-frequency magnetic field on antioxidative melatonin properties in AT478 murine squamous cell carcinoma culture.

Effects of melatonin, extremely-low-frequency magnetic field (ELF-MF), and their combination on AT478 murine squamous cell carcinoma line were studied. Manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (Cu/ZnSOD), and glutathione peroxidase (GSH-Px) were used as markers of cells antioxidative status, and malondialdehyde (MDA) level was used as a marker of lipid peroxidation. After melatonin treatment, antioxidative enzyme activities were increased and MDA level was decreased. Application of ELF-MF on treated cells caused an increase of both superoxide dismutases activity and MDA level, but influence of ELF-MF on GSH-Px activity was negligible. All enzyme activity in culture medium containing melatonin (10(-3), 10(-4), 10(-5) M) after exposure to ELF-MF were significantly diminished compared to cells treated only with melatonin. Also MDA levels after combined treatment with melatonin and ELF-MF were significantly decreased. Observed changes were statistically significant (p<0.05). These results strongly suggest that ELF-MF attenuates antioxidative actions of melatonin on cellular level.

Influence of thyroxine on serum soluble interleukin-2 receptor alpha levels in thyroid disorders.

BACKGROUND: Activation of cell-mediated immunity by soluble interleukin-2 receptor alpha (sIL-2Ralpha) release is well documented. The aim of this study was to measure serum concentrations of sIL-2Ralpha in patients with autoimmune and non-autoimmune thyroid disorders in different stages of thyroid function, before and after administration of l-thyroxine (l-T4) and its discontinuation as well as before and during methimazole administration. MATERIALS AND METHODS: The study included 80 females: 16 with Graves' disease, 15 with Hashimoto's thyroiditis and subclinical hypothyroidism, 14 with Hashimoto's thyroiditis with fibrosis and clinical hypothyroidism, 20 after subtotal thyroidectomy following nodular non-toxic goitre and 15 healthy controls. Patients were examined at two different time points. Serum concentrations of sIL-2Ralpha were measured with the use of enzyme immunoassay technique. RESULTS: Souble IL-2Ralpha serum concentration increased in patients with untreated Graves' disease and decreased after methimazole treatment. In Hashimoto's thyroiditis, the sIL-2Ralpha level was within the normal range, in Hashimoto's thyroiditis with clinical hypothyreosis it was low and after l-T4 administration it increased in both patient groups. After thyroidectomy, patients treated with l-T4, had increased levels of sIL-2Ralpha which decreased after discontinuation of therapy. There were a significant positive correlation between sIL-2Ralpha and free thyroxine in patients with (i). Graves' disease both before and after methimazole administration, (ii). Hashimoto's thyroiditis (with subclinical hypothyroidism) both before and after l-T4 therapy, (iii). Hashimoto's thyroiditis with fibrosis and (iv). overt hypothyroidism before l-T4 administration and in individuals during long-term l-T4 treatment (after subtotal thyroidectomy). CONCLUSION: Serum sIL-2Ralpha concentration in autoimmune thyroid diseases depends on thyroid function. In both autoimmune and non-autoimmune thyroid diseases, thyroxine stimulates the release of sIL-2Ralpha.