Investigating AXIN1 gene polymorphisms in Turkish children with cryptorchidism: A pilot study.

INTRODUCTION: Cryptorchidism is one of the most common congenital anomalies in male children, occurring in 2-5% of full-term male infants. Both genetic and environmental factors are observed to play a role in its etiology. A study conducted in Japan identified the AXIN1 gene as being associated with cryptorchidism. OBJECTIVE: We aimed to conduct a pilot study on AXIN1 gene polymorphism in Turkish children with cryptorchidism, and whether AXIN1 gene polymorphism is a risk factor for cryptorchidism. STUDY DESIGN: Between January 2023 and December 2023, we have planned a prospective controlled study including 84 boys operated for cryptorchidism as study group, and 96 boys operated for circumcision as control group. The remaining blood samples of preoperative laboratory tests in ethylenediamine tetraacetic acid (EDTA) tubes were kept at -20 Co freezer for genomic studies. Patient demographics, physical examination and operative findings were recorded, study patients were grouped according to testis localization. After collecting all samples, genomic DNA isolation procedure was done, and analysis of the 3 polymorphisms (rs12921862, rs1805105 and rs370681) of AXIN1 gene was performed using conventional Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) method. Genotype and allele frequencies of each group was analyzed and compared. RESULTS: The most common location of cryptorchid testis was proximal inguinal (53%), followed by distal inguinal (25.3%), bilateral (13.3%), and intra-abdominal (8.4%). Regarding the 3 polymorphisms of AXIN1 gene, there was no significant difference between study and control groups, in terms of genotype and allele frequencies (P > 0.05). Eight haplotype blocks were estimated for 3 polymorphisms of AXIN1. However, no significant difference was observed between study and control groups regarding haplotype distributions (P > 0.05). In addition, the comparison of the localization of testis with AXIN1 gene polymorphism did not show any significant difference among cryptorchid testis groups (P > 0.05). DISCUSSION: The AXIN1 gene is located on chromosome 16p and its polymorphisms have been associated with various diseases. In a Chinese study, the rs370681 polymorphism was found to be associated with cryptorchidism. However, our results showed no association between the AXIN1 gene haplotypes for the studied polymorphisms and cryptorchidism. CONCLUSION: In this study we have investigated the AXIN1 gene polymorphism in Turkish children with cryptorchidism as a pilot study. Although we could not identify any difference as compared to control group, further research is necessary to uncover the underlying molecular mechanisms contributing to the development of cryptorchidism.

Evaluation of the parameters in predicting single-dose methotrexate therapy success for ectopic pregnancy.

AIM: Methotrexate has demonstrated efficacy in treating ectopic pregnancies. This study explores factors influencing treatment success, focusing on laboratory and ultrasonographic findings, particularly the day 4 to day 1 ß-hCG level ratio. METHODS: Retrospective cohort study was conducted within patients diagnosed with tubal ectopic pregnancy. Patients' characteristics, ultrasound findings, laboratory data, and ß-hCG levels (days 1, 4, 7), and operation findings were reviewed. Women's characteristics were investigated who were treated with single dose of MTX (50 mg/m2). Patients who were performed surgery after MTX treatment were identified as MTX treatment failure. RESULTS: Among 439 women, 259 underwent surgery due to acute symptoms. Of those treated with MTX, 143 experienced treatment success, while 37 underwent surgery after MTX (MTX failure). Comparative analysis revealed significant differences in ß-hCG levels on admission (1128 and 4125 mIU/mL) and the day 4 to day 1 ß-hCG ratio (0.91 and 1.25). The overall MTX success rate was 79%, reaching 93% and 89% for ß-hCG levels <1000 mIU/mL and <2000 mIU/mL, respectively. Success dropped to 50% with levels exceeding 5000 mIU/mL. ROC analysis identified a crucial 2255 mIU/mL cut-off for ß-hCG (sensitivity 70.3% and specificity 68.5%) and a day 4 to day 1 ß-hCG ratio of 95.5% (sensitivity 84.7%, specificity 72.5%, positive predictive value 75.4%) for predicting MTX success. CONCLUSION: Establishing a ß-hCG cutoff can reduce hospital stay. The day 4 to day 1 ß-hCG ratio holds promise as a widely applicable predictor for MTX success or for determining MTX administration on day 4.

In Silico Prediction of the Effects of Nonsynonymous Single Nucleotide Polymorphisms in the Human Catechol-O-Methyltransferase (COMT) Gene.

Catechol-O-methyltransferase (COMT) enzyme performs transfer of methyl group to endogenous and exogenous catechol substrates. The COMT enzyme draws interest because of its association with psychiatric, neurological and cardiovascular diseases, and several cancers. Moreover, many prescribed drugs, supplements, and their metabolites are used as substrates of COMT enzyme. The human COMT gene has 226 nonsynonymous single nucleotide polymorphisms (nsSNPs) according to public databases. Uncovering of the molecular impacts of nsSNPs on COMT enzyme function and structure may provide standpoint on how COMT nsSNPs affect enzyme activity and contribute to disease development. Therefore, we aimed in this study to predict possible structural and functional damaging effects of all knowns nsSNPs in COMT gene by applying various bioinformatics tools. Two hundred and twenty-six nsSNPs were obtained from Ensembl, HGMD, ClinVar, and dbSNP databases. Twenty-eight nsSNPs were found to be high-risk changes for protein structure. Some of them were detected in extremely conserved sequences have functional and structural properties. Besides, high-risk nsSNPs were also uncovered to change properties of native COMT protein. Our findings demonstrated the significance of COMT high-risk nsSNPs on protein structure and function. We expect that our results will be helpful in future studies concerning experimental evaluation of the COMT gene polymorphisms and/or the association between COMT polymorphisms and disease development.

Effects of EF-24, RAD001, and paclitaxel on the expression profiles of apoptotic and anti-apoptotic genes.

CONTEXT: Cancer cells exert differential responses to chemotherapeutics and inhibitors. To the best of our knowledge, a few or no research has been performed until now to determine the effect of EF-24 and RAD001 on MDA-MB-231 breast cancer cells with regard to mRNA expression of apoptotic and anti-apoptotic genes. AIMS: In this study, we aimed to investigate the mRNA expression levels of apoptotic (caspase 2 [CASP2], CASP8, and CASP9) and anti-apoptotic (B-cell lymphoma 2 [BCL2] and BCL2-like protein 1 [BCL2L1]) genes after exposure to paclitaxel, EF-24, and RAD001 in MDA-MB-231 cells. MATERIALS AND METHODS: After treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to measure cell viability. mRNA expressions were analyzed using quantitative real-time polymerase chain reaction. RESULTS: Decrease in cell viability ratios was seen in a dose-dependent manner for all chemicals. MDA-MB-231 cells responded slightly different to paclitaxel, EF-24, and RAD001 at the transcriptional level of apoptotic and anti-apoptotic genes. CONCLUSIONS: Our results showed that response of these cells to paclitaxel, EF-24, and RAD001 was found different at the transcriptional level of apoptotic and antiapoptotic genes. Therefore, understanding transcriptional changes after these drug exposure may give us a change to figure out more realistic results of the apoptotic pathway inhibition.

Analysis of MMP-7 and TIMP-2 gene polymorphisms in coronary artery disease and myocardial infarction: A Turkish case-control study.

Matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) have a significant role in tissue remodeling related to cardiac function. In earlier studies, MMP-7 A-181G (rs11568818), C-153T (rs11568819), C-115T (rs17886546), and TIMP-2 G-418C (rs8179090) polymorphisms have been studied in various diseases. However, association between coronary artery disease (CAD) and these polymorphisms has been poorly studied. The goal of this study is to investigate the association of CAD and myocardial infarction (MI) with MMP-7 or TIMP-2 polymorphisms. This study included 122 CAD patients and 132 control individuals. DNA was extracted from whole blood. Polymerase chain reaction-restriction fragment length polymorphism and automated direct sequencing method were used for genotyping of these polymorphisms. No significant differences were found between MMP-7 A-181G, C-115T, and TIMP-2 G-418C polymorphism and CAD or MI in a Turkish population. Despite the fact that the genotypes of MMP-7 C-153T polymorphism had no significant differences among MI and control groups, allele frequencies of C-153T polymorphism were significantly different between the two groups. Our study is the first report to clarify the appreciable relationship between MMP-7 C-153T polymorphism and MI development in CAD patients. However, these findings also need to be confirmed in other populations so we can improve our knowledge about the genetic factors affecting the development of CAD.

Association of rs10757274 and rs2383206 Polymorphisms on 9p21 locus with Coronary Artery Disease in Turkish Population.

BACKGROUND AND OBJECTIVES: Genetic predisposition is an important risk factor for coronary artery disease (CAD). In this study, we aimed to evaluate the impact of rs10757274 and rs2383206 polymorphisms in chromosome 9p21 on presence and severity of CAD in a Turkish population. SUBJECTS AND METHODS: A total of 646 patients who underwent coronary angiography were included in this study. Coronary vessel score and Gensini score were calculated to assess the angiographic severity of CAD. Alleles of AA, AG, and GG were determined for rs10757274 (polymorphism-1) and rs2383206 (polymorphism-2) polymorphisms located in chromosome 9p21 from the blood samples. RESULTS: There was a significant difference between the alleles in polymorphism-1 in the presence of coronary artery disease (38.9% in AA, 48.0% in GG and 56.4% in AG, p=0.017). However, there was no difference between the alleles in polymorphism-2. According to vessel scores, there was a significant difference between the alleles in polymorphism-1 (AA 0.71±1.04, GG 0.88±1.07, AG 1.06±1.12, p=0.018). In polymorphism-2, vessel scores did not show a difference between the alleles. In polymorphism-1, there was a significant difference in Gensini score (p=0.041). Gensini scores did not differ between the alleles in polymorphism-2 (p>0.05 for all). In multivariate analyses, none of the alleles was an independent factor for presence of CAD. CONCLUSION: The presence of rs10757274 polymorphism including AG allele in chromosome 9p21 was related to CAD. However, this relationship was not independent of other cardiovascular risk factors.

HDAC inhibitors, MS-275 and salermide, potentiates the anticancer effect of EF24 in human pancreatic cancer cells.

Histone deacetylases (HDACs) play a major role in the regulation of chromatin structure and gene expression by changing acetylation status of histone and non-histone proteins. MS-275 (entinostat, MS) is a well-known benzamide-based HDACI and Salermide (SAL), a reverse amide compound HDACI, have antiproliferative effects on several human cancer cells. In this study, we aimed to investigate the effects of HDACIs (MS and SAL) alone and/or combined use with EF24 (EF), a novel synthetic curcumin analog, on human pancreatic cancer cell line (BxPC-3). In vitro, BxPC-3 cells were exposed to varying concentrations of MS, SAL with or without EF, and their effects on cell viability, acetylated Histone H3 and H4 levels, cytotoxicity, and cleaved caspase 3 levels, and cell cycle distribution were measured. The viability of BxPC-3 cells decreased significantly after treatment with EF, MS and SAL treatments. MS and SAL treatment increased the acetylation of histone H3 and H4 in a dose dependent manner. MS and SAL alone or combined with EF were increased the number of cells in G1 phase. In addition, treatment with agents significantly decreased the ratio of cell in G2/M phase. There were significant dose-dependent increases at cleaved Caspase 3 levels after MS treatment but not after SAL treatment. Our results showed that HDAC inhibitors (MS and SAL), when combined with EF, may effectively reduce pancreatic cancer cell (BxPC-3) progression and stop the cell cycle at G1 phase. Further molecular analyses are needed to understand the fundamental molecular consequences of HDAC inhibition in pancreas cancer cells.

Reduced BCL2 and CCND1 mRNA expression in human cervical cancer HeLa cells treated with a combination of everolimus and paclitaxel.

AIM OF THE STUDY: Cervical cancer is the second most common malignancy in women worldwide. Everolimus displays direct effects on growth and proliferation of cancer cells via inhibition of mammalian target of rapamycin (mTOR) protein, which is known to be associated with drug resistance. In this study, we aimed to investigate the effects of everolimus, gemcitabine, and paclitaxel in terms of cell viability and mRNA expression levels of GRP78, CCND1, CASP2, and BCL2 genes. MATERIAL AND METHODS: HeLa cells were treated with different doses of everolimus, gemcitabine, and paclitaxel. Cell viability was assessed using MTT assay, and obtained dose response curves were used for the calculations of inhibitory concentration (IC) values. At the end of the treatment times with selected doses, RNA isolation and cDNA synthesis were performed. Finally, GRP78, CCND1, CASP2, and BCL2 genes mRNA expression levels were analysed using quantitative PCR. RESULTS: The IC50 value of everolimus was 0.9 µM for 24-hour treatment. Moreover, the IC50 value of gemcitabine and paclitaxel was found to be around 18.1 µM and 7.08 µM, respectively. Everolimus, gemcitabine, and paclitaxel treatments alone did not change the GRP78, CCND1, BCL2 and CASP2 mRNA expression levels significantly. However, combined treatment of everolimus and paclitaxel significantly reduced BCL2 and CCND1 mRNA expression (p < 0.05). In contrast, this combination did not change GRP78 and CASP2 mRNA expression levels (p > 0.05). CONCLUSIONS: Down-regulation of CCND1 and BCL2 expression may be an important mechanism by which everolimus increases the therapeutic window of paclitaxel in cervical cancers.

Association of the BsmI, ApaI, TaqI, Tru9I and FokI Polymorphisms of the Vitamin D Receptor Gene with Nephrolithiasis in the Turkish Population.

PURPOSE: To analyze the relationship between nephrolithiasis and vitamin D receptor (VDR) gene BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236), Tru9I (rs757343) and FokI (rs2228570) polymorphisms in a study group from the Turkish population. MATERIALS AND METHODS: Ninety-eight patients with calcium oxalate kidney stones and 70 controls were enrolled in this study. Five polymorphisms of the VDR gene were studied using the Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) method. RESULTS: For all polymorphisms, genotype frequencies were in line with Hardy-Weinberg equilibrium in the patients and controls. For the BsmI polymorphism, allele frequency distribution was found to differ significantly between the patients and the controls (P < .05). The "B" allele was found to increase the risk of nephrolithiasis by approximately 1.5-fold (odds ratio = 1.55, 95% confidence interval: 1.00-2.40; P = .048). However, we did not find any statistically significant differences in the allele and genotype frequencies for the ApaI, TaqI, Tru9I and FokI polymorphisms. Proportionally, the "BAt" and "baT" haplotypes were more common than other haplotypes in the cases and controls, respectively. For the haplotypes of the BsmI and TaqI polymorphisms, the "bT" haplotype frequency was found to be common in both the patients and the controls. However, we did not find statistically significant differences between the cases and the controls for either the BsmI / ApaI / TaqI or the BsmI/TaqI haplotypes. Moreover, no relationship was identified between family history and development of stone disease. CONCLUSION: The "B" allele of the BsmI polymorphism of the VDR gene may increase stone development risk. Further investigations are needed to improve our knowledge regarding the genetic factors affecting urinary stone development.

Bioinformatic Analysis of GJB2 Gene Missense Mutations.

Gap junction beta 2 (GJB2) gene is the most commonly mutated connexin gene in patients with autosomal recessive and dominant hearing loss. According to Ensembl (release 74) database, 1347 sequence variations are reported in the GJB2 gene and about 13.5% of them are categorized as missense SNPs or nonsynonymous variant. Because of the high incidence of GJB2 mutations in hearing loss patients, revealing the molecular effect of GJB2 mutations on protein structure may also provide clear point of view regarding the molecular etiology of deafness. Hence, the aim of this study is to analyze structural and functional consequences of all known GJB2 missense variations to the Cx26 protein by applying multiple bioinformatics methods. Two-hundred and eleven nonsynonymous variants were collected from Ensembl release 74, Leiden Open Variation Database (LOVD) and The Human Gene Mutation Database (HGMD). A number of bioinformatic tools were utilized for predicting the effect of GJB2 missense mutations at the sequence, structural, and functional levels. Some of the mutations were found to locate highly conserved regions and have structural and functional properties. Moreover, GJB2 mutations were also found to affect Cx26 protein at the molecular level via loss or gain of disorder, catalytic site, and post-translational modifications, including methylation, glycosylation, and ubiquitination. Findings, presented here, demonstrated the application of bioinformatic algorithms to predict the effects of mutations causing hearing impairment. I expect, this type of analysis will serve as a start point for future experimental evaluation of the GJB2 gene mutations and it will also be helpful in evaluating other deafness-related gene mutations.

Does hyaluronic acid decrease the apoptotic effect of bupivacaine?

OBJECTIVES: In this study, we aimed to study the anti-apoptotic effects of hyaluronic acid on the apoptotic effects of bupivacaine in cultured rat chondrocytes in a time and dose-dependent manner. MATERIAL AND METHODS: The rat chondrocytes were treated with 7.69 µM, 76.9 µM, and 384.5 µM bupivacaine and 50 µg/ml hyaluronic acid concentrations for six, 24, and 48 hours. At the end of the treatment period, cells were stained with mixture of acridine orange and ethidium bromide. Apoptosis was evaluated using a fluorescence microscope. RESULTS: A significant protective effect of hyaluronic acid on chondrocytes against bupivacaine exposure at 7.69 µM and 76.9 µM concentrations, particularly was observed. There was also a significant protective effect in the exposure time at six and 24 hours for 7.69 µM and 76.9 µM bupivacaine doses. CONCLUSION: Our study results show that hyaluronic acid against chondrotoxicity of bupivacaine may have a protective effect in a time and dose-dependent manner.

Synthesis, biological evaluation and molecular docking studies of trans-indole-3-acrylamide derivatives, a new class of tubulin polymerization inhibitors.

In this study, we synthesized a series of trans-indole-3-acrylamide derivatives (3a-k) and investigated their activity for inhibition of cell proliferation against five human cancer cell lines (HeLa, MCF7, MDA-MB-231, Raji and HL-60) by MTT assay. Compound 3e showed significant antiproliferative activity against both the Raji and HL-60 cell lines with IC50 values of 9.5 and 5.1 µM, respectively. Compound 3e also exhibited moderate inhibitory activity on tubulin polymerization (IC50=17 µM). Flow cytometric analysis of cultured cells treated with 3e also demonstrated that the compound caused cell cycle arrest at the G2/M phase in HL-60 and HeLa cells. Moreover, 3e, the most active compound, caused an apoptotic cell death through the activation of caspase-3. Docking simulations suggested that 3e binds to the colchicine site of tubulin.

The epigenetically regulated effects of Wnt antagonists on the expression of genes in the apoptosis pathway in human bladder cancer cell line (T24).

The epigenetic suppression of Wnt antagonists (sFRPs, DKKs, and WIF-1) causes the activation of both ß-catenin and target genes, which play an important role in cell proliferation, metastasis, and angiogenesis. This study is aimed to investigate, on transcriptional and protein levels, the synergic effects of unaccompanied and/or combined use of 5-aza-2'-deoxycytidine (DAC, 5-aza-dC), trichostatin A (TSA), and gemcitabine+cisplatin chemotherapeutic agents on the apoptotic pathway of human bladder cancer cell line T24. The anti-tumor effects of gemcitabine (0-500 nM), cisplatin (0-10 µM), DAC (10 µM), and TSA (300 nM) alone and/or together on T24 cells were determined by WST-1. ELISA method was used to analyze the effects of unaccompanied and combined use of gemcitabine+cisplatin, DAC, and TSA on cell proliferation and determine the cytotoxic and apoptotic dosages at the level of H3 histone acetylation. Methylation-specific PCR was used to evaluate methylation profiles of Wnt antagonist gene (WIF-1). In the case of unaccompanied and/or combined use of specified drugs, the variations in the expression levels of CTNNB1, GSK3ß, c-MYC, CCND1, CASP-3, CASP-8, CASP-9, BCL2L1, and WIF-1 genes were determined by quantitative real-time PCR. Our results indicate that through inhibition of DNA methylation, expression of ß-catenin and Wnt antagonist re-activation and expressions of canonical Wnt/ß-catenin pathway target genes, c-myc and cyclin D1 (CCND1), have decreased. In addition, DAC, TSA, and gemcitabine+cisplatin combination caused an increase in GSK3ß mRNA levels, which in turn significantly decreased CCND1 mRNA levels. Moreover, BCL2L1, an anti-apoptotic gene, was downregulated significantly. Meanwhile, both CASP-3 mRNA and active caspase-3 protein levels increased with respect to control (p<0.01). The results revealed that use of quadruplicate gemcitabine+cisplatin+DAC+TSA combination led to a reduced inhibition of canonical Wnt/ß-catenin pathway and reduced cell proliferation. Our findings may offer a new approach to consider in the treatment of bladder cancer.

Effects of AG490 and S3I-201 on regulation of the JAK/STAT3 signaling pathway in relation to angiogenesis in TRAIL-resistant prostate cancer cells in vitro.

The aim of the present study was to analyze the molecular mechanisms involved in blocking the signaling pathway and the effects of this on the progression of prostate cancer (CaP) cells in vitro. LNCaP human CaP cell line was stimulated with interleukin-6 (IL-6) in the presence/absence of Janus kinase (JAK) 2 (AG490), signal transducer and activator of transcription 3 [(STAT3) S3I-201] inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Cytotoxic activity, the activation of phosphorylated (p)-STAT3 protein, caspase (CASP) 3 activity at protein level, vascular endothelial growth factor (VEGF) A, VEGFC, vascular endothelial growth factor receptor 2, STAT3, matrix metalloproteinase-2, myeloid cell leukemia sequence 1 (MCL-1), CASP8 and CASP9 messenger RNA (mRNA) levels were determined. Morphology and apoptosis were confirmed by DAPI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. IL-6 rapidly induced the phosphorylation of STAT3 in a dose- and time-dependent manner with a peak expression at 3 h at a concentration of 25 ng/ml. In addition, AG490 (50 µM) and S3I-201 (300 µM) inhibited STAT3 activation. Western blotting results revealed that p-STAT3 protein expression decreased significantly with AG490 and S3I-201 treatment in LNCaP cells. AG490 and S3I-201 induced the downregulation of VEGFA, MCL-1 and STAT3 and the upregulation of CASP8 and CASP9 mRNA transcription levels. In addition, the inhibitors increased the level of CASP3 protein. Combinations of AG490- and S3I-201-TRAIL did not result in an increase in this effect. Parallel results were found by DAPI staining and TUNEL assay. To the best of our knowledge, this is the first study to investigate the possible clinical use of AG490 or S3I-201, together with the reduced use of chemotherapeutic agents with high cytotoxicity, for their ability to exert an apoptotic effect, targeting the JAK/STAT3 pathway.

Quantitative measurement of m-RNA levels to assess expression of cyclooxygenase-II, inducible nitric oxide synthase and 12-lipoxygenase genes in middle ear cholesteatoma.

To assess expression of three main inflammatory genes, COX-II, ALOX-12 and i-NOS, quantitatively at transcriptional level in cholesteatoma matrix tissue. Ten patients who have chronic otitis media with primary acquired cholesteatoma were included in this study. Tissue samples obtained from cholesteatoma matrix and external ear canal skin (control tissue). Expression of the targeted genes (COX-II, i-NOS and LOX-12) was assessed using real-time quantitative polymerase chain reaction (RT-PCR) technique. The amount of COX2 mRNA was significantly higher in cholesteatoma matrix at transcriptional level (p = 0.038). There was no statistically significant difference regarding expression of iNOS and LOX12 mRNA levels (p > 0.05). There is a significant overexpression of the mRNA of COX-II in cholesteatoma matrix, which indicates a difference between the normal skin and cholesteatoma matrix at molecular level. COX-II gene overexpression seems to be associated with pathogenesis of cholesteatoma. This molecular change is similar to the molecular abnormalities observed in some benign and malignant neoplasms. Invasive and locally destructive nature of cholesteatoma may be due to COX-II overexpression. Absence of an increase in the gene expressions of i-NOS and LOX-12 in cholesteatoma matrix suggests that these mediators may not be related with the pathogenesis and evolution of cholesteatoma.

Bupivacaine and levobupivacaine induce apoptosis in rat chondrocyte cell cultures at ultra-low doses.

PURPOSE: Osteoarthritis (OA) is characterized by chondrocyte apoptosis and necrosis which play a key role during the progression of OA. Intra-articular administration of bupivacaine is a practical and effective way of postoperative pain control following various joint surgeries. 0.25 % bupivacaine showed to be safe in terms of chondrocyte toxicity. Around 200 nM of bupivacaine was shown to be effective for peripheral nerve block. This study aims to observe the possible cytotoxic effects of bupivacaine and its enantiomer levobupivacaine on chondrocyte cell culture at 7.69, 76.9, and 384.5 µM or at 0.0125, 0.0025, and 0.00025 % concentrations, respectively. METHODS: Chondrocytes were isolated from rat articular cartilage after incubating with collagenase in RPMI-1640 medium. Cells were treated with bupivacaine and levobupivacaine at 7.69, 76.9, and 384.5 µM concentrations for 6, 24, and 48 h. Treated chondrocytes were stained with acridine orange and ethidium bromide and examined under a fluorescence microscope at a 490 nm excitation wavelength for apoptotic changes. RESULTS: Study results suggest that both bupivacaine and levobupivacaine have dose-dependent chondrocyte toxicity, and this is significantly lesser at 7.69 µM dose. There was no significant difference in terms of chondrocyte apoptosis, (p > 0.05). CONCLUSIONS: Clinicians should be skeptic for the serious long-term side effects of bupivacaine and its analogs, even at ultra-low doses.

DNA methyltransferase inhibitor-mediated apoptosis in the Wnt/ß-catenin signal pathway in a renal cell carcinoma cell line.

The Wnt signaling pathway is activated in most cancer types when Wnt antagonist genes are inactivated. Glycogen synthase kinase 3 (GSK3ß) is an important regulator of the Wnt/ß-catenin signaling pathway. The mechanisms underlying GSK3ß regulation of neoplastic transformation and tumor development are unclear. Studies have raised the possibility that the Wnt signaling pathway may be implicated in renal cell carcinoma (RCC). Therefore, in the present study, we hypothesize that the expression and methylation status of the secreted frizzled-related protein 2 (sFRP2) gene, one of the secreted antagonists that bind Wnt protein, and re-expression of this gene with the demethylation agent (5-aza-2'-deoxycytidine; DAC) may induce apoptosis in RCC cells. To test this hypothesis, we investigated the relationship among epigenetic inactivation of sFRP2 and p-GSK3ß (Ser9) and other Wnt antagonists (sFRP1, DKK3, WIF-1) and apoptotic factors (Bax and Caspase3) as well as the anti-apoptotic factor BCL2. Our results indicate that DAC-mediated inhibition of DNA methylation led to a re-activation of sFRP2 expression and increased expression levels of the Wnt antagonists and apoptotic factors. In contrast, the level of ß-catenin (CTNNB1) expression decreased. The p-GSK3ß (Ser9) protein level in Caki-2 cells was significantly down-regulated, while the DNA fragmentation rate increased after treatment with 5 µM DAC at 96 h. Our data show that sFRP2 functions as a tumor suppressor gene in RCC and that its restoration may offer a new therapeutic approach for the treatment of RCC. Moreover, our study draws attention to the regulatory features of epigenetic molecules and analyses their underlying molecular mechanisms of action and their potential use in clinical practice.

Expression of cyclooxygenase-2, 12-lipoxygenase, and inducible nitric oxide synthase in head and neck squamous cell carcinoma.

AIM: The objective of this study was to investigate whether cyclooxygenase-2 (COX-2), 12-lipoxygenase (12-LOX), and inducible nitric oxide synthase (iNOS) have a role in carcinogenesis of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: Twenty-two patients with HNSCC were included in the study. Cancer tissues and adjacent normal mucosa were obtained from each patient. Real-time PCR was used to assess the expression of COX-2, 12-LOX, and iNOS. RESULTS: COX-2 and 12-LOX mRNA expressions are significantly increased in HNSCC compared with adjacent normal mucosa. Expression of iNOS was not significantly elevated in overall head and neck cancer tissues compared with normal mucosa. However, iNOS expression was found to be significantly elevated in patients with laryngeal cancer. CONCLUSION: These data suggest that COX-2 and 12-LOX may play a role in carcinogenesis of head and neck cancer. iNOS as well as COX-2 and 12-LOX may play a role in carcinogenesis of laryngeal cancer.

Effects of the adverse life events and Disrupted in Schizophrenia-1 (DISC1) gene polymorphisms on acute symptoms of schizophrenia.

The aim of this study was to evaluate the effects of traumatic childhood events and recent adverse life events, as well as the Disrupted in Schizophrenia-1 (DISC1) gene polymorphisms on types of last acute symptoms of patients with schizophrenia. Hundred patients with schizophrenia were given the Childhood Trauma Questionnaire, the Social Readjustment Rating Scale, Scale for Assessment of Positive Symptoms (SAPS), Scale for Assessment of Negative Symptoms (SANS), Brief Psychiatric Rating Scale (BPRS), and Calgary Depression Scale for Schizophrenia (CDSS). The patients' and healthy controls' DISC1 gene was evaluated for the -274G>C, c.791G>A, and c.2110A>T polymorphisms. There was no statistically significant difference with regard to the DISC1 gene polymorphisms between patient and healthy control groups. No significant relationship was found between the -274G>C, c.791G>A, and c.2110A>T haplotypes and development of different acute symptoms of schizophrenia. Having a recent stressful life event significantly affected SAPS (95% confidence interval [CI]=-67.547, -21.473; p=0.00) and BPRS-1 scores (95% CI=-51.405, -6.885; p=0.01), whereas emotional abuse at childhood significantly affected SANS scores (95% CI=-37.300, -10.401; p=0.00). This study shows that features of acute symptoms in schizophrenia are not influenced by the polymorphisms on the DISC1 gene, but are influenced by recent adverse life events and emotional abuse at childhood.

Fractalkine gene receptor polymorphism in patients with multiple sclerosis.

BACKGROUND: Although the immunopathogenesis of multiple sclerosis (MS) has been intensely investigated in recent years, some associated molecules still have not been examined. For instance, no study has been conducted to investigate a possible polymorphism in the fractalkine receptor gene. METHODS: In order to examine fractalkine gene receptor polymorphisms, 3 mL of serum from 92 MS patients and 91 controls were stored at -20°C. DNA was extracted from the serum samples that were purified, and the gene regions in CX3CR1 (i.e., the fractalkine regions) containing the T280M and V294I fractalkine receptor haplotypes were amplified via the polymerase chain reaction (PCR) technique. The obtained fragments were then cut using restriction enzymes, and agarose gel electrophoresis was performed. RESULTS: In a comparison of the patients and controls, we found that the median values of the Expanded Disability Status Scale (EDSS) scores among genotypes of the V294I polymorphism in the fractalkine gene receptor were statistically higher in genotype II than genotype VI. Also, relapsing/remitting MS (RRMS) was statistically higher in genotype VI than in genotype II, whereas the frequency of secondary progressive MS (SPMS) was statistically higher in genotype VV than in the genotype VI for the same polymorphism. CONCLUSIONS: Although many polymorphism studies have focused on patients with MS, there is no polymorphism study about the fractalkine receptor which is a chemokine and plays an important role in neuroinflammation and neurodegeneration. Our results provide information about disease progression and may also be beneficial in developing new strategies for the treatment of the disease.