Redox homeostasis in human renal cells that had been treated with amphotericin B in combination with selected 1,3,4-thiadiazole derivatives.

BACKGROUND: The use of amphotericin B (AmB) in the therapy of systemic mycosis is associated with strong side effects, including nephrotoxicity, and hepatotoxicity. Therefore, agents that can reduce the toxic effects of AmB while acting synergistically as antifungal agents are currently being sought. 1,3,4-thiadiazole derivatives are promising compounds that have an antifungal activity and act synergically with AmB. Such combinations might allow the dose of AmB, which is essential for preventing patients from having serious side effects, to be decreased. This might result from the antioxidant properties of 1,3,4-thiadiazoles. Thus, the aim of the study was to investigate redox homeostasis in human renal proximal tubule epithelial cells (RPTEC) after they had been treated with AmB in combination with 1,3,4-thiadiazole derivatives. METHODS: Cellular redox homeostasis was assessed by investigating the total antioxidant capacity (TAC) of cells, the malondialdehyde (MDA) concentration, and the activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). TAC was measured using an ABTS method. The MDA concentration, and the activity of SOD, GPX, and CAT were determined spectrophotometrically using commercially available assays. Additionally, the antioxidant defense system-related gene expression profile was determined using oligonucleotide microarrays (HG-U133A 2.0). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to confirm the microarray results. RESULTS: Amphotericin B and selected 1,3,4-thiadiazole derivatives had a significant effect on the total antioxidant capacity of the RPTEC cells, and the activity of the antioxidant enzymes. We also revealed that the effect of thiadiazoles on the SOD and CAT activities is dependent on the treatment of RPTEC cells with AmB. At the transcriptional level, the expression of several genes was affected by the studied compounds and their combinations. CONCLUSIONS: The results confirmed that thiadiazoles can stimulate the RPTEC cells to defend against the oxidative stress that is generated by AmB. In addition, together with the previously demonstrated synergistic antifungal activity, and low nephrotoxicity, these compounds have the potential to be used in new therapeutic strategies in the treatment of fungal infections.

Static Magnetic Field Reduces the Anticancer Effect of Hinokitiol on Melanoma Malignant Cells-Gene Expression and Redox Homeostasis Studies.

BACKGROUND: Melanoma malignant is characterized by a high mortality rate, accounting for as much as 65% of deaths caused by skin cancer. A potential strategy in cancer treatment may be the use of natural compounds, which include hinokitiol (ß-Thujaplicin), a phenolic component of essential oils extracted from cypress trees. Many studies confirm that a high-induction SMF (static magnetic field) has anticancer effects and can be used as a non-invasive anticancer therapy in combination with or without drugs. AIM: The aim of this experiment was to evaluate the effect of a static magnetic field on melanoma cell cultures (C32 and COLO 829) treated with hinokitiol. METHODS AND RESULTS: Melanoma cells were exposed to a static magnetic field of moderate induction and hinokitiol. The research included determining the activity of the antioxidant enzymes (SOD, GPx, and CAT) and MDA concentration as well as the gene expression profile. CONCLUSION: Hinokitiol disturbs the redox homeostasis of C32 and COLO 829 melanoma malignant cells. Moreover, a static magnetic field has a protective effect on melanoma malignant cells and abolishes the anticancer effect of hinokitiol.

Kinin Receptors and Kinin-Related Gene Expression in Astrocytic Brain Tumors.

Kinins are a set of peptides present in tissues that are involved in the inflammatory response and cancer progression. However, studies showing the expression of kinin receptors in human glioma samples are still incomplete and contradictory. The aim of the present study was to ascertain the expression of BDKRB1 and BDKRB2 genes, as well as the level of B1R and B2R proteins in human gliomas, depending on the degree of malignancy. Additionally, representative kinin-dependent genes with altered expression were indicated. The expression profile of kinin-dependent genes was determined using oligonucleotide microarray technique. In addition, RT-qPCR was used to assess the expression level of selected differentiating genes. The location of kinin receptors in brain gliomas was assessed using immunohistochemical methods. The oligonucleotide microarray method was used to identify 12 mRNA IDs of kinin-related genes whose expression was upregulated or downregulated in gliomas of different grades. In immunohistochemically stained samples, the concentrations of BR1 and BR2 proteins, measured by optical density, were statistically significantly higher in grade G3 vs. G2 and G4 vs. G3. Increased expression of kinin receptors BDKRB1 and BDKRB2 in brain gliomas, depending on the degree of malignancy, suggests the involvement of kinins and their receptors in the disease's pathogenesis. Quantitative assessment of mRNA BDKRB1, PRKAR1A, MAP2K, and EGFR in patients with brain tumors may hold diagnostic and therapeutic significance.

Phenolic acids and a static magnetic field change the expression of transforming growth factor ß isoforms in amelanotic melanoma cells.

BACKGROUND: Melanoma is an aggressive type of cancer that can metastasize to numerous other organs. TGFß is one of the key signaling pathways in melanoma progression. Previous studies on various types of cancer have shown that both: polyphenols and a static magnetic field (SMF) can be potential chemopreventive/therapeutic agents. Therefore, the aim of the study was to evaluate the effect of a SMF and selected polyphenols on the transcriptional activity of TGFß genes in melanoma cells. METHODS AND RESULTS: Experiments were performed on the C32 cell line treated with caffeic or chlorogenic acids, and with simultaneous exposure to a moderate-strength SMF. The RT-qPCR method was used to determine the mRNA level of genes encoding the TGFß isoforms and their receptors. The concentration of the TGFß1 and TGFß2 proteins were also measured in the cell culture supernates. The first response of C32 melanoma cells to both factors is the reduction of TGFß levels. Then, mRNA level of these molecules returned to values close to pre-treatment level by the end of experiment. CONCLUSION: Our study results demonstrate the potential of polyphenols and a moderate-strength SMF to support cancer therapy by altering TGFß expression, which is a very promising topic for the diagnosis and treatment of melanoma.

The MAP2K2 Gene as Potential Diagnostic Marker in Monitoring Adalimumab Therapy of Psoriatic Arthritis.

BACKGROUND: MAP kinases are some of the cascades that are specialized in the cell's response to external stimuli. Their impaired functioning can be observed during the course of psoriatic arthritis. Currently, the best-known class of biological drugs is the inhibitors of the proinflammatory cytokine TNF-α, including adalimumab. OBJECTIVE: The aim of this study was to assess changes in the expression of MAP kinase genes in patients with psoriatic arthritis treated with adalimumab, as well as to determine which of the analyzed transcripts could be used as a diagnostic or therapeutic target. METHODS: An analysis was performed on the total RNA extracted from PBMCs of patients with psoriatic arthritis before and after three months of adalimumab therapy as well as from a control group. Changes in the expression of the mitogen-activated protein kinase genes were assessed using the HG-U133A 2.0 oligonucleotide microarray method, while the obtained results were validated using the real-time RT-qPCR method. RESULTS: Using the oligonucleotide microarray method, 14 genes coded for proteins from the MAPK group were identified with at least a two-fold change of expression in the control group and during adalimumab therapy. Validation of the results confirmed a statistically significant decrease in the transcriptional activity of the MAP2K2 gene in the group of patients three months after the administration of adalimumab relative to the control group. CONCLUSION: Adalimumab therapy alters the expression of MAPK-coding genes. The assessment of the number of MAP2K2 mRNA molecules can potentially be used in diagnostic analyses or in monitoring adalimumab therapy.

Effect of Antibiotic Amphotericin B Combinations with Selected 1,3,4-Thiadiazole Derivatives on RPTECs in an In Vitro Model.

4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol (C1) and 4-[5-(naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl] benzene1,3-diol (NTBD) are representative derivatives of the thiadiazole group, with a high antimycotic potential and minimal toxicity against normal human fibroblast cells. The present study has proved its ability to synergize with the antifungal activity of AmB. The aim of this work was to evaluate the cytotoxic effects of C1 or NTBD, alone or in combination with AmB, on human renal proximal tubule epithelial cells (RPTECs) in vitro. Cell viability was assessed with the MTT assay. Flow cytometry and spectrofluorimetric techniques were used to assess the type of cell death and production of reactive oxygen species (ROS), respectively. The ELISA assay was performed to measure the caspase-2, -3, and -9 activity. ATR-FTIR spectroscopy was used to evaluate biomolecular changes in RPTECs induced by the tested formulas. The combinations of C1/NTBD and AmB did not exert a strong inhibitory effect on the viability/growth of kidney cells, as evidenced by the negligible changes in the apoptotic/necrotic rate and caspase activity, compared to the control cells. Both NTBD and C1 displayed stronger anti-oxidant activity when combined with AmB. The relatively low nephrotoxicity of the thiadiazole derivative combinations and the protective activity against AmB-induced oxidative stress may indicate their potential use in the therapy of fungal infections.

The Apoptotic Effect of Caffeic or Chlorogenic Acid on the C32 Cells That Have Simultaneously Been Exposed to a Static Magnetic Field.

The induction of apoptosis is one of the main goals of the designed anti-cancer therapies. In recent years, increased attention has been paid to the physical factors such as magnetic fields and to the natural bioactive compounds and the possibilities using them in medicine. Hence, the aim of this study was to evaluate the anti-tumor effect of caffeic or chlorogenic acid in combination with a moderate-strength static magnetic field on C32 melanoma cells by assessing the effect of both factors on the apoptotic process. The apoptosis of the C32 cells was evaluated using a flow cytometry analysis. The expression of the apoptosis-associated genes was determined using the RT-qPCR technique. The caspase activity and the concentration of the oxidative damage markers were also measured. It was found that phenolic acids and a static magnetic field trigger the apoptosis of the C32 cells and also affect the expression of the genes encoding the apoptosis regulatory proteins. In conclusion, our study indicated that both of the phenolic acids and a static magnetic field can be used supportively in the treatment of melanoma and that caffeic acid is more pro-apoptotic than chlorogenic acid.

The Influence of Betulin and Its Derivatives EB5 and ECH147 on the Antioxidant Status of Human Renal Proximal Tubule Epithelial Cells.

Betulin and its derivatives, 28-propyne derivative EB5 and 29-diethyl phosphonate analog ECH147, are promising compounds in anti-tumor activity studies. However, their effect on kidney cells has not yet been studied. The study aimed to determine whether betulin and its derivatives-EB5 and ECH147-influence the viability and oxidative status of human renal proximal tubule epithelial cells (RPTECs). The total antioxidant capacity of cells (TEAC), lipid peroxidation product malondialdehyde (MDA) level, and activity of antioxidant enzymes (SOD, CAT, and GPX) were evaluated. Additionally, the mRNA level of genes encoding antioxidant enzymes was assessed. Cisplatin and 5-fluorouracil were used as reference substances. Betulin and its derivatives affected the viability and antioxidant systems of RPTECs. Betulin strongly reduced TEAC in a concentration-dependent manner. All tested compounds caused an increase in MDA levels. The activity of SOD, CAT, and GPX, and the mRNA profiles of genes encoding antioxidant enzymes depended on the tested compound and its concentration. Betulin showed an cisplatin-like effect, indicating its nephrotoxic potential. Betulin derivatives EB5 and ECH147 showed different impacts on the antioxidant system, which gives hope that these compounds will not cause severe consequences for the kidneys in vivo.

The role of miR-370 and miR-138 in the regulation of BMP2 suppressor gene expression in colorectal cancer: preliminary studies.

PURPOSE: Colorectal cancer (CRC) is the fourth-most common cancer worldwide and the second most common cancer cause of death in the world. The components of the TGFß-signalling pathway, which are often affected by miRNAs, are involved in the regulation of apoptosis and cell cycle. Therefore, in the current study, the expression of BMP2 gene in CRC tissues at different clinical stages compared to the non-tumour tissues has been assessed. Moreover, the plasma BMP2 protein concentration in the same group of CRC patients has been validated. Due to the constant necessity to conduct further research of the correlation between specific miRNAs and mRNAs in CRC, in silico analysis has been performed to select miRNAs that regulate BMP2 mRNA. METHODS: The cDNA samples from tumor and non-tumor tissue were used in a qPCR reaction to determine the mRNA expression of the BMP2 gene and the expression of selected miRNAs. The concentration of BMP2 protein in plasma samples was also measured. RESULTS: It was indicated that BMP2 was downregulated in CRC tissue. Moreover, miR-370 and miR-138 expression showed an upward trend. Decreased BMP2 with accompanied increasing miR-370 and miR-138 expression was relevant to the malignant clinicopathological features of CRC and consequently poor patient prognosis. CONCLUSION: Our data suggest that miR-370 with its clear expression in plasma samples may be a potential diagnostic marker to determine the severity of the disease in patients at a later stage of colorectal cancer.

The effect of a static magnetic field and baicalin or baicalein interactions on amelanotic melanoma cell cultures (C32).

BACKGROUND: Baicalin and baicalein have antioxidant, anti-inflammatory, hepatoprotective and anti-cancer properties. However, it is not known how a static magnetic field will modify these properties. Therefore, the aim of our study was to evaluate the simultaneous exposure of melanoma cells to flavones and the static magnetic fields that are generated by permanent magnets on the gene expression and the activity of the antioxidant enzymes that are associated with the antioxidant defense system. METHODS AND RESULTS: Melanoma cells that had been treated with baicalin or baicalein were subjected to a static magnetic fields with a moderate induction. The static magnetic field was emitted by permanent magnets and the cell cultures were carried out in special test chambers. The research included determining the activity of the antioxidant enzymes (superoxide dismutase, glutathione peroxidase and catalase) as well as the gene expression profile. The addition of the flavones to the cell cultures at a concentration of 50 µmol/L resulted increase in the expression of the SOD1, SOD2 and GPX1 genes compared to the nontreated cell cultures. Simultaneous exposure of the melanoma cells to static magnetic field and baicalin or baicalein reduced their mRNA levels compared to the cultures to which only baicalin or baicalein had been added. The change in gene expression was accompanied by changes at the protein level associated with an increase in the activity of antioxidant enzymes. CONCLUSION: We showed that baicalin or baicalein have anticancer properties by disturbing the redox homeostasis in melanoma cells and also increases the antioxidant system gene expression. There was also an antagonistic interaction between the studied flavones and the static magnetic field, which cause a decrease in the anticancer effects of baicalin or baicalein.

The impact of the co-exposure of melanoma cells to chlorogenic acid and a moderate-strength static magnetic field.

A static magnetic field (SMF) or the bioactive compounds that are found in foods are potential agents that can be used to support cancer therapy. Therefore, the aim of our study was to assess the impact of the SMF that are induced by neodymium magnets on the culture growth and antioxidant status of melanoma cells that had been treated with chlorogenic acid (CGA). The melanoma cells, the control and those that had been treated with CGA, were put in special magnetic test chambers that generated a 0.7 T magnetic field. The mRNA levels of the antioxidant enzymes were analyzed using RT-qPCR. The activity of SOD, GPx, and CAT was measured in the cell lysates. While the expression and activity of the antioxidant enzymes was inhibited relative to the untreated cells as a result of the CGA treatment (1 mmol/L), it was not after the CGA treatment in combination with an SMF. The demonstrated cytotoxicity of CGA (1 mmol/L) and its inhibition of the antioxidant enzymes suggests the usefulness of phenolic compounds as a supporting pharmacological treatment for melanoma. PRACTICAL APPLICATIONS: Phenolic acids and their derivatives, which are the bioactive components of the human diet, are signal molecules that transfer information from the external environment that affects the level of gene expression in cells. This study suggests the usefulness of phenolic compounds as a supporting pharmacological treatment for melanoma and seems to be important for the development of experimental oncology.

Adalimumab changes the expression profile of selected BCL-2 family genes.

Biological drugs are an alternative to treatment of psoriasis and psoriatic arthritis. Adalimumab is a representative of the anti-TNF group. The underlying of this disease is a cellular homeostasis disorder-apoptosis. Many proteins are involved in the apoptosis induction pathways, including those from the BCL-2 family. The aim of the study was to perform a transcriptional analysis of the genes coding selected proteins from the BCL-2 family in patients treated with adalimumab therapy, and to determine the direction of these changes. The test materials were peripheral blood mononuclear cells. The cells were obtained from 20 patients with psoriatic arthritis who were being treated with adalimumab (study group) and 20 healthy volunteers (control). The gene expression profile was determined using the real-time quantitative reverse transcription polymerase chain reaction technique. Statistically significant changes were observed in the expression level of the BNIP3, BNIP3L, and BCL2L1 genes (p < .05) during a 24-month observation of therapy. We indicated that adalimumab therapy has an impact on the expression of the analyzed genes, which may constitute a new class of molecular markers for assessing the effectiveness of a therapy. It appears that the BNIP3L gene could be used as a potential diagnostic marker of psoriasis.

Impact of fluoride and a static magnetic field on the gene expression that is associated with the antioxidant defense system of human fibroblasts.

Fluoride cytotoxicity has been associated with apoptosis, oxidative stress, general changes in DNA and RNA and protein biosynthesis, whereas the results of studies on the effect of SMF on antioxidant activity of cells are contradictory. Therefore, the aim of our study was to evaluate the simultaneous exposure of human cells to fluoride SMF that are generated by permanent magnets on the expression profile of the genes that are associated with the antioxidant defense system. Control fibroblasts and fibroblasts that had been treated with fluoride were subjected to the influence of SMF with a moderate induction. In order to achieve our aims, we applied modern molecular biology techniques such as the oligonucleotide microarray. Among the antioxidant defense genes, five (SOD1, PLK3, CLN8, XPA, HAO1), whose expression was significantly altered by the action of fluoride ions and the exposure to SMF were normalized their expression was identified. We showed that fluoride ions cause oxidative stress, whereas exposure to SMF with a moderate induction can suppress their effects by normalizing the expression of the genes that are altered by fluoride. Our research may explain the molecular mechanisms of the influence of fluoride and SMF that are generated by permanent magnets on cells.

Influence of static magnetic fields up to 700 mT and dihydrochalcones on the antioxidant response in fibroblasts.

The effects of a static magnetic field (SMF) and the dihydrochalcones phloretin and phloridzin on the redox homeostasis of fibroblasts were investigated. The aim of the present study was to determine the redox homeostasis of fibroblasts that were simultaneously exposed to a static magnetic field and the dihydrochalcones phloretin and phloridzin. The fibroblasts were cultured for 72 h in special magnetic test chambers at different moderate intensities (0.4, 0.55 and 0.7 T). In this report, the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione transferase (GST); the concentrations of malondialdehyde (MDA), adenosine triphosphate (ATP) and total antioxidant status were measured using commercially available kits. We did not observe any impairment in the redox balance in cells in fibroblasts that were only exposed to static magnetic fields of different intensities or In fibroblast cultured with dihydrochalcones and exposed to static magnetic field increase the SOD, GPx, GST activities and MDA concentration. Our investigations revealed that the activities of SOD, GPx, GST and the concentration of MDA that were determined for the fibroblasts that were cultured with dihydrochalcones were higher in the presence of a static magnetic field. Our results indicated that exposure to SMF (0.7 T) with dihydrochalcones induces oxidative stress in fibroblasts.

Transforming growth factor ß-related genes in human retinal pigment epithelial cells after tacrolimus treatment.

BACKGROUND: The transforming growth factor ß (TGFß) family plays an important role in the pathogenesis of many diseases, including fibrotic pathologies of the eyes. The difficulties of surgical procedures contribute to the search for new treatment strategies for proliferative vitreoretinopathy. Therefore, the aim of this study was to investigate the expression profile of TGFß isoforms, their receptors, and TGFß-related genes in human retinal pigment epithelial cells (RPE) after tacrolimus (FK-506) treatment in the presence or absence of lipopolysaccharide (LPS)-induced inflammation. METHODS: The expression profile was analyzed using oligonucleotide microarrays and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) techniques. RESULTS: Analysis using oligonucleotide microarrays revealed 20 statistically significant differentially expressed TGFß-related genes after LPS treatment in relation to control cells, and after tacrolimus and LPS treatment in relation to LPS-treated cells. Moreover, our results showed that mRNA levels for TGFß2 and TGFßR3 after tacrolimus treatment, and for TGFßR3 after tacrolimus and LPS treatment in RPE cells were decreased. In turn, in the presence of LPS-induced inflammation, TGFß2 mRNA level was increased. CONCLUSIONS: These results can be important in regard to the treatment of proliferative vitreoretinopathy, pathogenesis of which is associated with processes regulated by TGFß, such as inflammation, proliferation, epithelial-mesenchymal transition (EMT), and fibrosis.