Hacer Ilke Onen

Evaluation of in vitro fertilization parameters and estrogen receptor alpha gene polymorphisms for women with unexplained infertility

PurposeAssociation of ESR1 gene PvuII, XbaI and (TA)n microsatellite polymorphisms and woman infertility was evaluated.MethodsInfertile(n = 104) and fertile(n = 107) women were included in this study. We performed polymerase chain reaction-restriction fragment-length polymorphism analysis for detecting ESR1 polymorphisms.Result(s)PvuII and XbaI polymorphisms confered risk for infertility in a simple dominant manner in which a significant relationship was observed between infertile and control women. Infertile women had fewer(<18) short repeat alleles in promotor region. ESR1 genotypes were compared concerning maturation, fertilization, pregnancy rates and embryo quality. Although no difference was found in terms of pregnancy rates, maturation and fertilization rates were significantly smaller in pp and xx genotypes. Also, pp genotypes had significantly lower number of good quality embryos. Long TA repeat in promotor was found to be associated with low fertilization rate.Conclusion(s)Polymorphisms at the ESR1 gene are associated with infertility in this Turkish infertile women population.

Genetic Variations in the Hypoxia-Inducible Factor-1αGene and Lung Cancer

Hypoxia-inducible factor-1 (HIF-1), an important genetic component of angiogenesis, becomes stable as a response to tumor hypoxia and facilitates tumor survival. The polymorphisms of the HIF-1αgene may cause changes in the activity of this protein, which serves as a transcription factor for many genes in tumorigenesis. In this study, we have investigated the relationship between seven HIF-1αpolymorphisms [C > T substitution in intron 8 (rs10873142), T418I (rs41508050) in exon 10, P564P (rs41492849), L580L (rs34005929), P582S (rs11549465), A588T (rs11549467) in exon 12 and dinucleotide GT repeats in intron 13 (rs10645014)] among lung cancer patients in the Turkish population. Genomic DNA was isolated from 141 lung cancer cases and 156 controls and subjected to PCR for amplification. Genotyping was carried out with RFLP and DNA sequencing methods. There was no significant difference between the lung cancer cases and controls in terms of the distribution of genotyping frequencies of seven HIF-1αpolymorphisms (P > 0.05). No significant relationship was found between the C > T substitution in intron 8 and P582S haplotypes and development of lung cancer. In addition, there were no significant associations between the genotypes and clinopathological characteristics of the cases examined. These findings show that polymorphisms in the HIF-1αgene do not confer susceptibility to lung cancer. Exp Biol Med 234:1109–1116, 2009

Endometrial mRNA expression of matrix metalloproteinases, their tissue inhibitors and cell adhesion molecules in unexplained infertility and implantation failure patients

The aim of this study was to analyse whether some cases of unexplained infertility and implantation failure after IVF could be explained by different expression levels of the matrix metalloproteinases (MMP-2, 9), their tissue inhibitors (TIMP-2, 3) and intercellular (ICAM-1) and vascular (VCAM-1) adhesion molecules in endothelial cells. Total RNA was extracted from the endometrial tissues of 41 women (unexplained infertile, group 1, n = 15; fertile volunteers, group 2, n = 15 and patients with implantation failure after IVF, group 3, n = 11). MMP-2, MMP-9, TIMP-2, TIMP-3, ICAM-1 and VCAM-1 mRNA expression levels were measured quantitatively using real-time polymerase chain reaction. In the endometrium from women with unexplained infertility and implantation failure after IVF, MMP-2 and TIMP-3 expression were significantly decreased when compared with the fertile group (P < 0.05 and P </= 0.001 respectively). In addition, a marked decrease was observed in the expression of VCAM-1 in women with unexplained infertility. These results suggest that the expression of gelatinase A (MMP-2), TIMP-3 and VCAM-1, at least at the transcriptional level, might be regulated by common factors and signalling pathways. The present study adds new and important data in this field and highlights the complex preparation of the endometrium for implantation at the molecular level.

NLF2 gene expression in the endometrium of patients with implantation failure after IVF treatment.

The aim of this study was to analyze the expression of microfibril-associated protein 2 (MFAP2), microfibril-associated protein 5 (MFAP5) and nuclear localized factor 2 (NLF2) genes in patients with repeated IVF failure and compare with fertile population. Total RNA was isolated from 38 patients (repeated implantation failure, group 1, n=22; fertile patients, group 2, n=16). mRNA expression levels were measured quantitatively using real-time polymerase chain reaction. Our results showed that mRNA expression of NLF2 significantly decreased in the infertility group as compared to control group (P=0.023). In addition a marked decrease was observed in the expression of MFAP2 in women with repeated implantation failure. In conclusion, NLF2 gene expression levels and differences in MFAP2 and MFAP5 gene expressions (albeit being insignificant) between infertile group and control group draw attention to a genetic basis under implantation failure.

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.

EF24 and RAD001 potentiates the anticancer effect of platinum-based agents in human malignant pleural mesothelioma (MSTO-211H) cells and protects nonmalignant mesothelial (MET-5A) cells:

The most widespread neoplasm of the pleura is malignant pleural mesothelioma (MPM) with low prevalence rate. The mechanistic target of rapamycin signaling pathway, inhibited by RAD001, was shown to be deregulated in MPM development and considered a novel target for the MPM therapy. The EF24, a curcumin analog, also affects several signaling pathways and kills cancer cells as a single agent or in combination with classical drugs. We aimed to evaluate possible effects of RAD001, EF24, cisplatin, and oxaliplatin treatments on both malignant pleural mesothelioma (MSTO-211H) and nonmalignant mesothelial (Met-5A) cell lines. The effects of the agents on MSTO-211H and Met-5A cells were evaluated in terms of cell viability, cytotoxicity, DNA synthesis rate, quantitation of apoptotic DNA fragmentation, and cleaved caspase 3 levels. Moreover, quantitative messenger RNA (mRNA) analysis of apoptotic (CASP9) and antiapoptotic (BCL2L1 and BCL2) genes were also performed. We found that both EF24 and RAD001 alone treatments decreased only MSTO-211H cell viability, but cisplatin and oxaliplatin affected both cell lines. Pretreatment with EF24 or RAD001 followed by cisplatin increased the effects of cisplatin alone application. EF24 and RAD001 pretreatment decreased DNA fragmentation rate when compared with cisplatin alone treatment in Met-5A cells. Sequential treatments resulted in a significant increase of CASP9 mRNA expression in MSTO-211H cells but not in Met-5A cells. Our preliminary results suggest that pretreatment with EF24 or RAD001 may reduce cytotoxic effect of cisplatin on nonmalignant mesothelial cells and increase cell death response of MPM cells. Further analyses using animal models are needed to confirm these findings in vivo.

Effects of cisplatin and panobinostat on human mesothelial (Met-5A) and malignant pleural mesothelioma (MSTO-211H) cells.

New therapeutic approaches are still needed for effective malignant pleural mesothelioma treatment. The use of classical chemotherapy agents in combination with newly developed molecules may shed light on new therapeutic approaches. We aimed to determine the efficacy of panobinostat, alone and in combination with cisplatin, on cell survival and mRNA expression of FOXO3A, CCND1, and CASP9 genes in both mesothelioma and healthy mesothelial cell lines. Cells were treated with 1-100 µM cisplatin and 25-1000 nM panobinostat. Methylthiazol tetrazolium assays were performed to determine cell viability. mRNA expression levels of genes were analyzed with quantitative real-time polymerase chain reaction. Cisplatin and panobinostat exposure of the cells for 24 h resulted in decreased cell survival. The combined treatment was found to be more effective. No significant changes were observed with respect to CCND1 expression after exposure to agents alone or in combination. However, agents in combination resulted in upregulation of FOXO3A and CASP9 in MSTO-211H cells. Gene expression levels were not affected by any agents in healthy cells. Use of cisplatin in combination with new chemotherapeutic agents may reduce the toxic effects of cisplatin in normal cells and result in more effective removal of tumor cells.

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.

Upregulated mrna expression of mtor in surgically resected early stage non-small cell lung carcinoma: a potential molecular targeted therapy

Background: Non-small cell lung cancer (NSCLC) comprises about 85% of all lung cancers. Although many attempts for early detection and treatment, prognosis of NSCLC is still poor. In recent years the pathways and the genes that play role in lung cancer development were researched widely. PI3K/AKT/MTOR which is thought to be efficacious in the development of many cancer which controls the expression of many genes playing an important role in cell proliferation, metastasis, resistance to apoptosis and angiogenesis. Also the increase in CCND1 expression was shown in several cancer types. The aim of this study is to search the mRNA expression profile of AKT, MTOR and CCND1 genes which has been thought to play role in NSCLC development, and their expression in different pathological stages and histological types of the disease. Materials and Methods: Forty-four NSCLC patients who didn’t get neoadjuvant therapy were included in this study. The samples from tumor and matched normal lung tissue were obtained from resection specimens (lobectomy or pneumonectomy). Total cellular RNA was isolated from the samples. The mRNA expression levels of AKT1, mTOR and CCND1 genes were measured by quantitative real-time polymerase chain reaction (qPCR). Results: Our findings revealed a statistically significant increase of mTOR expression on mRNA levels (P < 0.05). Although AKT and CCND1 expression slightly increased in malign tissues, these changes in the expression were not significant (P > 0.05). The mRNA expression of mTOR was also upregulated and it was statistically significant for early stage disease and adenocarcinoma subtype (P < 0.05). Conclusions: Inhibition of mTOR gene expression at mRNA level might be potential target for future treatment strategies of NSCLC.

Different surgical techniques and L-carnitine supplementation in an experimental varicocele model.

We aimed to investigate the impact of various varicocelectomy techniques and/or L‐carnitine as an adjunct treatment, following the emergence of oxidative stress, on the expression levels of SCF/c‐kit signalling pathways in spermatogenesis. Forty‐two rats were divided into seven groups: group 1 (G1) control; group 2 (G2) sham; group 3 (G3) varicocele; group 4 (G4) varicocele + varicocelectomy with testicular nonartery sparing; group 5 (G5) same as G4 but with artery sparing; group 6 (G6) same as G4 but with L‐carnitine and group 7 (G7) same as G5 with L‐carnitine. mRNA expression levels of SCF and c‐kit were measured quantitatively using real‐time polymerase chain reaction. CASP‐3 activity at protein level was determined, and histological evaluation was performed. mRNA expression level of SCF increased in G6 as compared to control group (3.52‐folds change; P = 0.035), whereas mRNA expression level of c‐kit gene remained the same. We found that in the left testis of G6 group, mRNA expression level of SCF increased 2.2‐folds in comparison with the right testis (P < 0.05). There were no statistically significant differences in the CASP‐3 protein expression levels between the control and other groups. When Cosentino Score analyses of immunostaining were conducted, we observed no significant differences among groups. Spermatogenic failure could be primarily due to a sertoli cell dysfunction. Although surgical treatment has been the best option for management of varicocele, auxiliary agents like L‐carnitine may be considered as supportive treatment regimes in addition to conventional surgical treatments.