Yasemin Aksoy

Atorvastatin causes regression of endometriotic implants in a rat model.

Endometriotic implants were induced surgically in female Wistar albino rats, which were randomly divided into three groups. The rats in group I (n=10) and group II (n=9) were given 2.5 mg/kg/day intraperitoneal and oral atorvastatin, respectively, for 28 days. Group III (n=9) was given no medication (control). The mean volume and weight of explants in group I were significantly lower (both P < 0.05) compared with group III. Histopathological score of the implants was significantly lower in groups I and II, when compared with group III (P < 0.01 and P < 0.05, respectively). There were significant reductions in explant concentrations of vascular endothelial growth factor and matrix metalloproteinase 9 in group I (P < 0.01 and P < 0.001, respectively) and group II (both P < 0.01) compared with group III while staining due to tissue inhibitor of metalloproteinase 2 was significantly higher in group I (P < 0.01) and group II (P < 0.01) compared with group III. Moreover, explant concentration of superoxide dismutase was significantly increased in groups I and II compared with group III (both P < 0.05). In conclusion, atorvastatin causes significant regression of endometriotic implants in rats. Moreover, intraperitoneal atorvastatin seems to be more effective than oral atorvastatin.

Metformin regresses endometriotic implants in rats by improving implant levels of superoxide dismutase, vascular endothelial growth factor, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinase-9

OBJECTIVE We sought to test if metformin could regress endometriotic explants in rats. STUDY DESIGN After inducing endometriotic implants and randomization of female Wistar albino rats, they were given 25 and 50 mg/kg/day of oral metformin in group A (n = 9) and B (n = 8), respectively, for 28 days. Group C (n = 9) was given saline as placebo. RESULTS Mean volume, weight, and histologic score of implants in groups A (P < .01, P < .05, and P < .05, respectively) and B (P < .01, P < .05, and P < .05, respectively) were significantly lower than in group C. The activity of superoxide dismutase and tissue inhibitor of metalloproteinase-2 staining in groups A (P < .05 and P < .01, respectively) and B (P < .01 and P < .01, respectively) was significantly higher than in the control group. Moreover, there were more significant reductions in implant levels of vascular endothelial growth factor and matrix metalloproteinase-9 in groups A (both P < .001) and B (both P < .001) than in group C. CONCLUSION Metformin causes regression of endometriotic implants in rats.

FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1.

OBJECTIVE Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. METHODS We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. RESULTS We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. CONCLUSION AND PRACTICAL IMPLICATIONS In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.

Synthesis and Biological Evaluation of 2-Substituted-5-(4-nitrophenylsulfonamido)benzoxazoles as Human GST P1-1 Inhibitors, and Description of the Binding Site Features

Glutathione‐S‐transferases (GSTs) are enzymes involved in cellular detoxification by catalyzing the nucleophilic attack of glutathione (GSH) on the electrophilic center of numerous of toxic compounds and xenobiotics, including chemotherapeutic drugs. Human GST P1‐1, which is known as the most prevalent isoform of the mammalian cytosolic GSTs, is overexpressed in many cancers and contributes to multidrug resistance by directly conjugating to chemotherapeutics. It is suggested that this resistance is related to the high expression of GST P1‐1 in cancers, thereby contributing to resistance to chemotherapy. In addition, GSTs exhibit sulfonamidase activity, thereby catalyzing the GSH‐mediated hydrolysis of sulfonamide bonds. Such reactions are of interest as potential tumor‐directed prodrug activation strategies. Herein we report the design and synthesis of some novel sulfonamide‐containing benzoxazoles, which are able to inhibit human GST P1‐1. Among the tested compounds, 2‐(4‐chlorobenzyl)‐5‐(4‐nitrophenylsulfonamido)benzoxazole (5 f) was found as the most active hGST P1‐1 inhibitor, with an IC50 value of 10.2 μM, showing potency similar to that of the reference drug ethacrynic acid. Molecular docking studies performed with CDocker revealed that the newly synthesized 2‐substituted‐5‐(4‐nitrophenylsulfonamido)benzoxazoles act as catalytic inhibitors of hGST P1‐1 by binding to the H‐site and generating conjugates with GSH to form S‐(4‐nitrophenyl)GSH (GS–BN complex) via nucleophilic aromatic substitution reaction. The 4‐nitrobenzenesulfonamido moiety at position 5 of the benzoxazole ring is essential for binding to the H‐site and for the formation of the GST‐mediated GSH conjugate.

New sample preparation method for the capillary electrophoretic determination of adenylate energy charge in human erythrocytes

The first step in the separation of adenine nucleotides from different types of tissues or cells is deproteinization. Several sample preparation methods successfully used for a number of tissues or cells failed to work with erythrocytes. Use of strong acids or bases for deproteinization resulted in a low yield due to the hydrolysis of adenine nucleotides. Moreover, the neutralization of these acids or bases increased the ionic strength, resulting in broad and overlapping peaks. In neutral salt precipitation methods, saturated salts caused clogging of the capillaries. A new deproteinization procedure method was developed. The samples were deproteinized by heating of erythrocytes in boiling distilled water at 95 degrees C for 5 min. The denatured proteins were removed by centrifugation and membrane filtration. The adenine nucleotides were then separated using a polyacrylamide coated capillary. Depending on the type, diameter, length of the capillary and the voltage applied, an average of 16.50 min was sufficient for the separation of adenine nucleotides. All adenine nucleotides were clearly resolved and gave very sharp peaks. The amount of each adenine nucleotide was calculated from the areas under the peaks and AEC values were calculated using the integrator software. The AEC value of 0.91+/-0.04 (n=10) obtained for healthy persons was in good agreement with the literature value of 0.85-0.95. These reported method for sample preparation and capillary electrophoresis is simple, fast and inexpensive compared to the previously reported sample preparation, HPLC and enzymatic methods for the determination of AEC.

The effect of N-acetylcysteine and calcium hydroxide on TNF-α and TGF-β1 in lipopolysaccharide-activated macrophages

OBJECTIVE The present study was designed to evaluate the pro- and anti-inflammatory effects of NAC and calcium hydroxide (Ca(OH)2) on lipopolysaccharide-stimulated human macrophage cell lines. DESIGN THP-1 human monocyte precursor cells were differentiated into macrophage adherent cells. Cell cytotoxicity was measured by flow cytometry analysis. NAC and Ca(OH)2 were applied in the presence or absence of lipopolysaccharides (LPS) for time periods of 4, 8, and 24h. Protein and mRNA levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta1 (TGF-β1) were determined using ELISA and qRT-PCR. The data were statistically analyzed by three-way ANOVA followed by Bonferroni test at α=0.05. RESULTS In LPS-stimulated cell lines, while the TNF-α protein and mRNA levels were reduced in the first 4h, only the TGF-β1 mRNA levels increased in the 24th hour following treatment with Ca(OH)2 and NAC when compared with the control group (p<0.001). In LPS-unstimulated cells, the TNF-α protein level was significantly decreased by NAC and Ca(OH)2 at the 4th hour. Additionally, while the TGF-β1 mRNA levels were significantly reduced, the protein level of TGF-β1 was increased at the 24th hour. CONCLUSIONS It was concluded that NAC, similar to Ca(OH)2, has anti-inflammatory properties and might be considered an alternate candidate therapeutical agent to Ca(OH)2.

CD26/dipeptidyl-peptidase IV and adenosine deaminase serum levels in psoriatic patients treated with cyclosporine, etanercept, and psoralen plus ultraviolet A phototherapy.

Objective  The aim of this study is to determine serum levels of soluble forms of CD26/dipeptidyl‐peptidase IV (DPP‐IV) and adenosine deaminase (ADA), thought to be markers of T‐cell activation, and changes in their levels in response to cyclosporine, etanercept, and psoralen plus ultraviolet A (PUVA) treatments with respect to disease activity.

The determination of matrix metalloproteinase 9 activity and gene expression levels in Behcet’s disease patients with aneurysmal complications

The information concerning aneurysmal progress in Behcet’s disease is still insufficient, while researches in the role of matrix metalloproteinases (MMPs) in aneurysmal formation are rapidly expanding. The goal of the present study is to investigate the role of metalloproteinase 9 (MMP-9) in vascular complications which is observed in 10% of Behcet’s disease patients. Three groups have been studied; patients with Behcet’s disease, patients with Behcet’s disease who have vascular problems (vasculo-Behcet’s), and patients with abdominal aortic aneurysm (AAA). The third group was used as a control. The activity and gene expression levels of MMP-9 in plasma have been determined. We showed that compared to AAA patients there was no difference in the MMP-9 activity in Behcet’s disease patients (vascular and non-vascular). We also evaluated the gene expression level and activity of MMP-9 for every patient. The increase in the gene expression level for MMP-9 could only be detected at two patients. One of them was Behcet’s, the other was AAA patient. It is surprising that MMP levels of these patients were different. While the patient with Behcet’s had low protein level, another patient with AAA had high of MMP-9 level. This result suggested to us that the relationship between gene expression and active protein level is not correlated. It is not sufficient alone to determine MMPs levels for evaluating the pathogenesis. At the same time gene expression and the level of active protein should be assessed together.

THE EFFECTS OF OXIDATIVE STRESS ON THE REDOX SYSTEM OF THE HUMAN ERYTHROCYTE

Erythrocytes are a group of cells which do not contain a nucleus, mitochondria and other cytoplasmic organelles. Due to the lack of a nucleus and other organelles they can not synthesize proteins and their energy metabolism depends solely on anaerobic glycolysis. The function of these highly differentiated cells is to exchange respiratory gasses between the lung and the tissues. The gas transporting protein, hemoglobin, constitutes 95% of the erythrocyte proteins. Hemoglobin consists of a nonprotein heme group and the protein, globulin. Heme contains iron. For a functional protein this iron must be in the ferrous (Fe2+) state (Mathews and van Holde, 1996; Stryer, 1988).

Age-related Changes in the Alkaline Phosphatase Activity of Healthy and Inflamed Human Dental Pulp

INTRODUCTION Alkaline phosphatase (ALP) plays an important role in inducing mineralization events in the dental pulp. This study investigated and compared the ALP levels in healthy and inflamed pulp in young and old human pulp. METHODS Tissue samples were collected from young (<30 years) and old (>60 years) donors. In both age groups, healthy human pulp (n = 18) were collected from extracted wisdom teeth. For reversible and irreversible pulpitis, pulp samples (n = 18 each) were obtained during endodontic treatment. ALP activity was assessed by spectrophotometry and immunhistochemistry. RESULTS Regardless of age, reversible pulpitis group samples showed a slight elevation in ALP activity compared with normal healthy pulp. In elderly patients, ALP expression with irreversible pulpitis was significantly higher than those with a healthy pulp (P < .05). CONCLUSIONS In the hyperemic state, both the young and old pulp shows a slight increase in ALP activity, whereas in irreversible pulpitis, only the old pulp shows significantly elevated ALP levels. Such an increase may trigger calcification events, which may eventually cause difficulties in endodontic treatment procedures in elderly individuals.