Effects of thiostrepton alone or in combination with selumetinib on triple-negative breast cancer metastasis.

OBJECTIVE: FoxM1 transcription factor contributes to tumor metastasis and poor prognosis in many cancers including triple-negative breast cancer (TNBC). In this study, we examined the effects of FoxM1 inhibitor Thiostrepton (THIO) alone or in combination with MEK inhibitor Selumetinib (SEL) on metastatic parameters in vitro and in vivo. METHODS: Cell viability was determined by MTT assay. Immunoblotting and immunohistochemistry was used to assess metastasis-related protein expressions in 4T1 cells and its allograft tumor model in BALB/c mice. In vivo uPA activity was determined by enzymatic methods. RESULTS: Both inhibitors were effective on the expressions of FoxM1, ERK, p-ERK, Twist, E-cadherin, and Vimentin alone or in combination in vitro. THIO significantly decreased 4T1 cell migration and changed the cell morphology from mesenchymal-like to epithelial-like structure. THIO was more effective than in combination with SEL in terms of metastatic protein expressions in vivo. THIO alone significantly inhibited mean tumor growth, decreased lung metastasis rate and tumor foci, however, no significant changes in these parameters were observed in the combined group. Immunohistochemically, FoxM1 expression intensity was decreased with THIO and its combination with SEL in the tumors. CONCLUSIONS: This study suggests that inhibiting FoxM1 as a single target is more effective than combined treatment with MEK in theTNBC allograft model. The therapeutic efficacy of THIO should be investigated with further studies on appropriate drug delivery systems.

Flavopiridol's effects on metastasis in KRAS mutant lung adenocarcinoma cells.

BACKGROUND: There is still no clinically approved agent for mutant KRAS, which is the most common alteration in non-small-cell lung cancer (NSCLC). Flavopiridol is a semisynthetic flavonoid that inhibits cell growth through cyclin-dependent kinases in G1/S or G2/M of the cell cycle and induces apoptosis. In this study, we evaluated its effect on cellular apoptosis, survival, and metastasis mechanisms on KRAS mutant A549, Calu-1, and H2009 cell lines. METHODS: The cytotoxic effects of flavopiridol on NSCLC cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability test. The cells were treated with 200 and 400 nM flavopiridol, and, then, apoptosis, survival, and metastasis-related protein expressions were determined by Western blot analysis. The antimetastatic effects of flavopiridol were assessed by wound healing and Galectin-3 activity assay. RESULTS: Flavopiridol drastically affected toxicity in all KRAS mutant NSCLC cells at nanomolar concentrations. Also, it could efficiently inhibit wound healing and Galectin-3 activity in all the cells tested. However, the metastasis-related protein expressions did not reflect these obvious effects on blotting. p-Erk was activated as a cellular survival mechanism to escape apoptosis in all the cells tested. CONCLUSION: Although there are many mechanisms that still need to be elucidated, flavopiridol can be used as a metastasis inhibitor and an apoptosis inducer in KRAS mutant NSCLC.

Flavopiridol's antiproliferative effects in glioblastoma multiforme.

AIM OF STUDY: Glioblastoma multiforme (GBM) is largely refractory to surgical operation, radiotherapy, and chemotherapy in use today. Remaining lifetime accounting for the GBM-affected patients varies between 12 and 16 months generally. The most frequently altered genes in GBM are p53, epidermal growth factor receptor, PTEN, and cyclin-dependent kinase inhibitor 2A. Our aim is to investigate the antiproliferative and apoptotic effects of flavopiridol, a cyclin-dependent kinases and specific phosphokinase inhibitor, on glioblastoma cell lines having different genetic profiles: U87MG, U118MG, and T98G. MATERIALS AND METHODS: Cell viability and IC50 values were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, protein expressions were determined by Western blot and caspase activities were analyzed by activity kit. RESULTS: Western blot analysis showed down-regulation of the cyclin D1, c-Myc, and p53 protein activities, and up-regulation of p27KIP1 activity after flavopiridol treatment. Additionally, flavopiridol diminished p-Akt protein levels generally which induces inhibition of proliferation. CONCLUSION: The present study demonstrated that flavopiridol did not induce caspase-3/7 activation, BIM, and BAX pro-apoptotic proteins but it leads to the expression changes of various proteins that inhibit proliferation and eternity in glioblastoma cell lines which have different genetic alterations.

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.

Flavopiridol Induces Apoptosis via Mitochondrial Pathway in B16F10 Murine Melanoma Cells and a Subcutaneous Melanoma Tumor Model.

Flavopiridol is a cyclin-dependent kinase (CDK) inhibitor that promotes cell cycle arrest. We aimed to examine the anti-proliferative effects of the flavopiridol and oxaliplatin combination on p16INK4A deficient melanoma cells B16F10 and also its apoptotic effects on a subcutaneously injected B16F10 allograft melanoma tumor model. Flavopiridol and oxaliplatin treated B16F10 cell viability was determined by MTT assay. C57BL6 mice were injected with B16F10 cells and treated with flavopiridol after tumor implantation. BRAF and BCL2L1 mRNA expression levels were measured using reverse transcription-polymerase chain reaction (RT-PCR). Caspase 9 and caspase 3/7 activity were determined by activity assay kits. Proliferating cell nuclear antigen (PCNA) and B-cell lymphoma 2 (BCL-2) protein expression levels were analyzed immunohistochemically. Flavopiridol and oxaliplatin decreased cell death. Flavopiridol enhanced caspase 3/7 and caspase 9 activities in vitro and in vivo in a dose dependent manner via the mitochondrial apoptotic pathway. Even though there was a significant increase in Bcl-2 staining, PCNA staining was decreased in flavopiridol-administered mice. Decreased PCNA expression showed antiproliferative effects of flavopiridol which might be the result of cell-cycle arrest. Flavopiridol can be used as a cell cycle inhibitor.

The comparison between dual inhibition of mTOR with MAPK and PI3K signaling pathways in KRAS mutant NSCLC cell lines.

KRAS mutations are found in 15-25 % of patients with lung adenocarcinoma, and they lead to constitutive activation of KRAS signaling pathway that results in sustained cell proliferation. Currently, there are no direct anti-KRAS therapies available. Therefore, it is rational to target the downstream molecules of KRAS signaling pathway, which are mitogen-activated protein kinase (MAPK) signaling pathway (RAF-MEK-ERK) and PI3K pathway (PI3K-AKT-mTOR). Here, we examined the inhibition of both these pathways alone and in combination and analyzed the anti-proliferative and apoptotic events in KRAS mutant NSCLC cell lines, A549 and Calu-1. Cytotoxicity was determined by MTT assay after the cells were treated with LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), and RAD001 (mTOR inhibitor) for 24 and 48 h. The expression levels of p-ERK, ERK, AKT, p-AKT, p53, cyclinD1, c-myc, p27(kip1), BAX, BIM, and GAPDH were detected by western blot after 6 and 24 h treatment. Although PI3K/mTOR inhibition is more effective in cytotoxicity in A549 and Calu-1 cells, MEK/mTOR inhibition markedly decreases cell proliferation protein marker expressions. Our data show that combined targeting of MEK and PI3K-AKT with mTOR is a better option than single agents alone for KRAS mutant NSCLC, thus opening the possibility of a beneficial treatment strategy in the future.

SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines.

OBJECTIVES: Primary and acquired resistance to EGFR TKIs in EGFR mutant lung cancer occurs primarily through secondary mutations in EGFR or Met amplification. Drug resistance can also be mediated by expression of pluripotency transcription factors, such as OCT4, SOX2 and NANOG that decrease terminal differentiation. In this study, we investigated the expression and role of SOX2 in model systems of EGFR mutant tumors. MATERIALS AND METHODS: Immunoblotting or immunohistochemistry was used to assess expression of pluripotency transcription factors in lungs of transgenic mice or in human NSCLC cell lines. Expression of SOX2 was reduced by shRNA knockdown, and response to erlotinib and cellular proliferation were assessed. RESULTS AND CONCLUSION: Induction of mutant EGFR in transgenic CCSP-rtTA/TetO-EGFR(L858R/T790M) mice correlated with increased OCT4 and SOX2 expression in lung tissue prior to tumor development. Established lung tumors retained SOX2 expression. To assess a role for SOX2 in tumorigenesis, a panel of NSCLC cell lines with activating EGFR mutations was assessed for SOX2 expression. Two of six cell lines with mutant EGFR showed detectable SOX2 levels, suggesting SOX2 expression did not correlate with EGFR mutation status. To assess the role of SOX2 in these cell lines, HCC827 and H1975 cells were infected with lentivirus containing SOX2 shRNA. Knockdown of SOX2 decreased proliferation in both cell lines and increased sensitivity to erlotinib in HCC827 cells. Because constitutive activation of the PI3K/Akt pathway is associated with EGFR TKI resistance, cells were treated with PI3K/AKT inhibitors and expression of SOX2 was examined. PI3K/Akt inhibitors decreased SOX2 expression in a time-dependent manner. These data suggest targeting SOX2 may provide therapeutic benefit in the subset of EGFR-mutant tumors with high constitutive levels of SOX2, and that until more direct means of inhibiting SOX2 are developed, PI3K/Akt inhibitors might be useful to inhibit SOX2 in EGFR TKI resistant tumors.

Investigation of ocular neovascularization-related genes and oxidative stress in diabetic rat eye tissues after resveratrol treatment.

Changes in vascular endothelial growth factor (VEGF), angiotensin-converting enzyme (ACE), matrix metalloproteinase (MMP)-9, and endothelial nitric oxide synthase (eNOS) mRNA expression profiles and oxidative stress in the eye tissue microenviroment may have important roles in ocular neovascularization and permeability in proliferative diabetic retinopathy. The present study investigated the effects of resveratrol (RSV) treatment on the mRNA expression profile of VEGF, ACE, MMP-9, and eNOS, which are associated with vascular neovascularization, and glutathione, protein carbonyl, and nitrite-nitrate levels, which are markers of oxidative stress in eyes of diabetic rats. Twenty-four Wistar albino male rats were divided into four groups. After diabetes induction with streptozotocin (10 mg/kg/day) RSV was administered to the RSV and diabetes mellitus (DM) + RSV groups for 4 weeks. The mRNA levels were measured by quantitative real-time polymerase chain reaction assay, and biochemical estimations were determined with spectrophotometric assays in eye homogenates. The mRNA expression levels of VEGF, ACE, and MMP-9 were increased in the DM group compared with the control group, and RSV treatment decreased their mRNA levels. Expression of eNOS mRNA was increased in the RSV and DM groups and decreased in the DM + RSV group. Nitrite-nitrate levels and protein carbonyl content were increased and glutathione levels were decreased in the DM group compared with controls. Consequently, these data suggest that RSV suppressed the expression of eNOS, which is actively involved in the inflammation and healing process in chronic diabetes. Although oxidative stress was increased in eye tissue from diabetic rats, mRNA levels of VEGF, MMP-9, and ACE genes associated with vascular remodeling did not change in diabetic eyes.

Inhibition of ErbB2 by herceptin reduces viability and survival, induces apoptosis and oxidative stress in Calu-3 cell line.

Human epidermal growth factor receptor 2 (ErbB2) amplification and overexpression has been seen in many cancer types including non-small cell lung cancer (NSCLC). Thus, ErbB2 is an important target for cancer therapies. Increased ErbB2 expression has been associated with drug resistance in cancer cells. Herceptin is a humanized monoclonal antibody that targets the extracellular domain of ErbB2. In this study, we aimed to block ErbB2 signaling with Herceptin and assess cytotoxicity and effects on apoptosis, oxidative stress, nuclear factor kappa-B (NF-kB), and Survivin expression in Calu-3 cell line. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay were used to assess cell viability as a marker of proliferation. Acridine orange/ethidium bromide (AO/EB) staining and caspase 3/7 activity were measured as the markers of apoptosis. The relative expressions of NF-kB-p50 and Survivin mRNAs were evaluated. Activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and the levels of glutathione (GSH) and reactive oxygen species (ROS) were determined in a time- and dose-dependent manner. Our results show that Herceptin treatment inhibits cell proliferation and activates apoptosis but without effects on Survivin and NF-kB expression in Calu-3 cell line. Intracellular glutathione levels and SOD and CAT activities were decreased in a time- and dose-dependent manner associated with oxidative stress. Also, ROS were increased at 24 h. These results provide evidence that Herceptin can be used as a cytotoxic and apoptotic agent in NSCLC.

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

PURPOSE: Association of ESR1 gene PvuII, XbaI and (TA)n microsatellite polymorphisms and woman infertility was evaluated. METHODS: Infertile(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.

Polymorphisms in the vitamin D receptor gene and risk of lung cancer.

BACKGROUND: Differences between the individual variations in DNA may modulate lung cancer process. Many studies reported that Vitamin D Receptor (VDR) gene polymorphisms may influence the cancer risk due to their antiproliferative, antiangiogenic, antimetastatic and apoptotic effects. MATERIAL/METHODS: The genotype and haplotype frequencies of three polymorphisms of VDR, i.e. TaqI (rs731236), BsmI (rs1544410), and ApaI (rs7975232), were studied using PCR-RFLP in 137 patients with lung cancer and 156 controls. RESULTS: Differences were observed in genotype (P=0.024) and allele (P=0.011) frequencies of TaqI polymorphism due to the "T" allele. Furthermore, compared with the "tt" genotype, the odds ratio for the "TT" genotype increased 2.24 times (95%CI=1.05-4.77, P=0.037). Comparing cases and controls, smoking habit (P=0.012) and gender distribution (P=0.005) were found to increase the risk of lung cancer in patients with "TT" homozygotes, demonstrating the role of gene-environment interaction in lung cancer. In addition, when age and gender within the case group only were evaluated in relation to genotype, the adjusted odds ratios for the "TT" genotype increased 2.20 times (95%CI=1.01-4.78, P=0.047) for age and 2.24 times (95%CI=1.05-4.80, P=0.037) for gender. However, no differences were observed for the distribution of variant genotypes of the BsmI and ApaI polymorphisms (P>0.05). To evaluate the joint effects of these polymorphisms, haplotype analysis was performed which showed that the haplotype baT was associated with higher lung cancer risk compared with the most common haplotype BAt (P=0.026). CONCLUSIONS: This is perhaps the first study suggesting that TaqI polymorphism of the VDR gene might be a risk factor for lung cancer and that age, gender, and smoking habit could have an impact on lung cancer risk.

Association of genetic polymorphisms in vitamin D receptor gene and susceptibility to sporadic prostate cancer.

Genetic and environmental factors are involved in prostate cancer (PCa) etiology. Single nucleotide polymorphisms (SNPs) may contribute to the PCa pathogenesis. The goal of this study is to determine the role of vitamin D receptor (VDR) gene polymorphisms and haplotypes in the development and progression of sporadic PCa. One hundred and thirty-three PCa patients and 157 age-matched healthy controls were genotyped for the Apa I (rs7975232), Bsm I (rs1544410) and Taq I (rs731236) polymorphisms in VDR gene by using polymerase chain reaction-restriction fragment length polymorphism. An association was observed between the Apa I polymorphism and PCa predisposition (P = 0.03). When compared with AA genotype, there was a highly notable difference in the frequencies of the Aa (P = 0.02), aa (P = 0.026) and Apa I ''a'' allele carriers (Aa + aa) (P = 0.009) genotypes. Furthermore, we found a statistical difference in the allele frequencies of the Apa I polymorphism between the sporadic PCa patients and control subjects (P = 0.013). The genotype distribution for the Bsm I and Taq I polymorphisms were similar between cases and controls (P > 0.05). No clinically significant relationship was found between the three-locus haplotypes and development of sporadic PCa. The genotype frequencies for the three polymorphisms of the VDR gene within subgroups of PCa (defined by tumor stage, Gleason score, PSA levels) were also analyzed, but no statistically noteworthy difference was observed (P > 0.05). As far as we know, this is the first study which investigates the relationship between VDR genotypes and sporadic PCa in the Turkish population. Our findings suggest that the VDR ApaI (rs7975232) polymorphism may play a role in the development of sporadic PCa.

GJB2 and mitochondrial A1555G gene mutations in nonsyndromic profound hearing loss and carrier frequencies in healthy individuals.

This study aimed to assess mutations in GJB2 gene (connexin 26), as well as A1555G mitochondrial mutation in both the patients with profound genetic nonsyndromic hearing loss and healthy controls. Ninety-five patients with profound hearing loss (>90 dB) and 67 healthy controls were included. All patients had genetic nonsyndromic hearing loss. Molecular analyses were performed for connexin 26 (35delG, M34T, L90P, R184P, delE120, 167delT, 235delC and IVS1+1 A-->G) mutations, and for mitochondrial A1555G mutation. Twenty-two connexin 26 mutations were found in 14.7% of the patients, which were 35delG, R184P, del120E and IVS1+1 A-->G. Mitochondrial A1555G mutation was not encountered. The most common GJB2 gene mutation was 35delG, which was followed by del120E, IVS1+1 A-->G and R184P, and 14.3% of the patients segregated with DFNB1. In consanguineous marriages, the most common mutation was 35delG. The carrier frequency for 35delG mutation was 1.4% in the controls. 35delG and del120E populations, seems the most common connexin 26 mutations that cause genetic nonsyndromic hearing loss in this country. Nonsyndromic hearing loss mostly shows DFNB1 form of segregation.

Polymorphisms in the aurora-A gene is not associated with lung cancer in the Turkish population.

Lung cancer, a complex neoplasm of lung tissue, is influenced by several environmental and genetic factors which could be changed in each individual. Aurora-A gene is related to mitotic events such as: chromosome instability, cell cycle regulation, spindle formation, and kinetechore-microtubule connections. This centrosomic serine/threonine kinase provides a strong connection between mitotic errors and carcinogenesis. The genomic alterations such as single nucleotide polymorphisms (SNPs) can exist in molecular pathways of lung cancer. Therefore, we evaluated the role of genetic polymorphisms of Aurora-A gene in the lung cancer in the Turkish population. Genotypes of five Aurora-A polymorphisms (F31I, V57I, 6328G/A, P50L, and S104L) were determined in 102 healty controls and 102 new diagnosed lung cancer cases. All samples were genotyped with DNA sequence technique. There were not any genotype variations in P50L, S104L, and 6328G/A polymorphisms. The frequencies of both genotypes F31I and V57I in lung cancer patients were not significantly different from those in controls (p > 0.05). A multivariable logistic regression analysis including patient characteristics, such as age and gender, did not change the results.

Single nucleotide polymorphisms in the hypoxia-inducible factor-1alpha (HIF-1alpha) gene in human sporadic breast cancer.

BACKGROUND: DNA sequence variations in hypoxia-inducible factor-1alpha (HIF-1alpha) gene, which have been demonstrated to be correlated with tumor angiogenesis, may yield changes both in the production outcomes and in the activities of the gene. In this study, we investigated the relationship between three single nucleotide polymorphisms (SNPs) [C1772T and G1790A in exon 12 and C111A in exon 2 of the HIF-1alpha gene] in the HIF-1alpha gene coding regions and development of sporadic breast cancer in the Turkish population. These three polymorphisms result in an amino acid change from proline 582 to serine, from alanine 588 to threonine and from serine 28 to tyrosine, respectively. METHODS: Genomic DNA was isolated from 102 sporadic breast cancer patients and 102 healthy female controls. All three HIF-1alpha gene regions were amplified by PCR, and genotypes were determined by RFLP and DNA sequencing. RESULTS: There were no significant differences between patients and controls in terms of the distribution of C1772T and G1790A polymorphisms of HIF-1 gene (p >0.05). As for HIF-1alpha C111A polymorphism, we did not find CA and AA variants of the gene in either controls or patients. Multivariable logistic regression analysis was performed between CC and CT + TT genotypes of C1772T polymorphism. No significant differences were found between these two genotypes in terms of clinicopathological characteristics of the patients including age at enrollment, age at menarche and first delivery, number of full-term pregnancies, body mass index, use of oral contraceptives and postmenopausal hormones, family history of breast and ovarian cancers, menopausal status, histopathological features, oophorectomy, smoking habits, and alcohol consumption (p >0.05). CONCLUSIONS: Our results suggest that none of the polymorphisms studied in the HIF-1alpha gene influence susceptibility to sporadic breast cancer. The present study is the first case-control study that investigates the association of HIF-1alpha polymorphisms with sporadic breast cancer in the Turkish population.

No association between polymorphism in the vascular endothelial growth factor gene at position -460 and sporadic prostate cancer in the Turkish population.

The development and progression of prostate cancer (PCa) has biologically and genetically remained a mystery. A man's risk of developing PCa is influenced by both genetic and environmental factors. Angiogenic cytokines like vascular endothelial growth factor (VEGF) play a pivotal role in tumor angiogenesis. Single nucleotide polymorphisms in angiogenesis-dependent genes affect the sensibility of cancer development and progression. Therefore, we hypothesized a potential association between DNA sequence variations in VEGF -460 gene region and sporadic PCa patients in the Turkish population. 133 sporadic PCa patients and 157 healthy controls were studied. Genotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. The distribution of genotype and allele frequencies of the polymorphism did not yield a statistically significant difference between patients and controls (P>0.05). Furthermore, classification of patients by tumor-lymph nodes-metastasis (TNM), Gleason Scores (GS) and serum prostate-specific antigen (PSA) levels did not show significant differences among the VEGF -460 C>T genotypes (P>0.05). This is the first demonstration showing that the VEGF -460 C>T polymorphism in men is not associated with sporadic PCa in the Turkish population.

Lack of association between -460 C/T and 936 C/T of the vascular endothelial growth factor and angiopoietin-2 exon 4 G/A polymorphisms and ovarian, cervical, and endometrial cancers.

Tumor growth, which employs a number of regulators, requires the formation of new blood vessels. The most important regulators are vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANGPT-2). DNA sequence variations in VEGF and ANGPT-2 genes may lead to altered productions and/or activities of these genes. In this study, we aimed to determine the polymorphic effects of the changes in the VEGF -460 C/T, VEGF 936 C/T, and ANGPT-2 exon 4 G/A, which we perceive as risk factors in the progress and metastasis of cancer, on the gynecologic cancer patients in the Turkish population. Forty-seven ovarian, 32 cervical, and 21 endometrial cancer patients and 106 healthy controls were studied. The genomic DNA was extracted from the whole blood by using DNA extraction techniques. DNA samples were analyzed by polymerase chain reaction and restriction fragment length polymorphism. There were no significant differences between any of the three types of gynecologic cancer patients and controls in terms of the distribution of VEGF -460, VEGF 936, and ANGPT-2 genotypes and alleles (p > 0.05). Odds ratios (ORs) were calculated by logistic regression analysis in comparison with the most common homozygote genotype observed in the studied population. No evidence of a relationship that would constitute a risk factor (p > 0.05) was found between genotype and allele frequencies of patients and controls for VEGF -460, VEGF 936, and ANGPT-2 genes. A multivariable logistic regression analysis with the involvement of covariant factors, such as the history of gynecologic cancer and/or other cancer types in the family, stages of tumor, smoking habits, and existence of other diseases, did not change the results. The present study is the first case-control study of VEGF and ANGPT-2 polymorphisms in relation to ovarian, cervical, and endometrial cancers.

An investigation of relationships between hypoxia-inducible factor-1 alpha gene polymorphisms and ovarian, cervical and endometrial cancers.

BACKGROUND: DNA sequence variations in HIF-1 alpha gene might yield changes both in the production outcomes and in the activities of the gene. Overexpression of the HIF-1 alpha subunit, resulting from intratumoral hypoxia and genetic alterations, has been demonstrated in common human cancers and is correlated with tumor angiogenesis and patient mortality. In this study, we aimed to determine how the three single nucleotide polymorphisms (SNPs, C1772T and G1790A exon 12, C111A exon 2) in the HIF-1 alpha gene coding regions affect the ovarian, cervical and endometrial cancer patients in the Turkish population. A study on this relationship has not been conducted to date. METHOD: 102 gynecologic cancer patients and 107 healthy controls were studied. Genotypes of the three polymorphisms were analyzed by PCR-RFLP. RESULTS: There was no significant difference between ovarian cancer patients and controls in terms of the distribution of C1772T genotypes and alleles (P>0.05). However, there was a highly significant increase in the frequency of both CT 1772 and TT 1772 genotypes in patients with cervical and endometrial cancers compared with healthy controls. In fact, 1772T allele-carriers (CT+TT genotypes) showed an association with the risk of cervical and endometrial cancers compared to the wild type (OR=3.84, 95% CI: 1.65-8.93; OR=7.41, 95% CI: 2.33-23.59, respectively). C1772T polymorphism was not associated with family history concerning gynecologic and/or other cancer types, stages (I-IV) and grades of tumor, smoking habits and existence of other diseases that generate a hypoxic microenvironment even after multivariable logistic regression analysis. As for HIF-1 alpha G1790A genotypes, the frequencies of G alleles were 98% in ovarian patients and 100% in the control group. We found no significant difference in the genotype distribution and allele frequencies between the ovarian patients and healthy control subjects. There were no GA and AA genotypes among the cervical and endometrial cancer patients. As for HIF-1 alpha C111A polymorphism, we did not find CA and AA variants of the gene in controls or in any of the three types of patients. CONCLUSION: Our results suggest that the C1772T polymorphism of the HIF-1 alpha may be associated with cervical and endometrial cancers.

Apolipoprotein E gene polymorphism in nonalcoholic fatty liver disease.

The aim of this study was to evaluate the relationship between apolipoprotein E gene polymorphism and nonalcoholic fatty liver disease. The study group consisted of 237 nonalcoholic fatty liver disease patients who were detected by ultrasonography and 201 controls with ultrasonographically normal livers. DNA amplifications were performed by polymerase chain reaction technique and apolipoprotein E genotypes were evaluated after digestion with CfoI restriction enzyme. Serum levels of glucose, lipids, lipoproteins, and apolipoproteins were measured in all subjects. Additionally, viral hepatitis markers, liver enzymes, and body mass index were assessed. Patients were found to have significantly higher triglyceride, glucose, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyltransferase levels and lower high-density lipoprotein cholesterol and apolipoprotein (a) levels than controls (P<0.05). There were no statistically significant differences in genotypes and allele frequencies between all patients and controls. Comparing nonobese patients with controls, the frequencies of allele epsilon2 and genotype epsilon2epsilon3 were statistically significantly different in the controls (P=0.04 and P=0.01, respectively). In conclusion, occurrence of the epsilon2 allele and epsilon2epsilon3 genotype may be protective against development of nonalcoholic fatty liver disease.

The association of 5alpha-reductase II (SRD5A2) and 17 hydroxylase (CYP17) gene polymorphisms with prostate cancer patients in the Turkish population.

To date, research has led to the invention of multiple genes and their single nucleotide polymorphisms (SNPs) and environmental factors that influence the prostate cancer (PCa) pathogenesis. Therefore, the genes involved in these pathways are candidates for PCa predisposition. It is thought that polymorphisms of 5alpha-reductase II (SRD5A2) and 17 hydroxylase (CYP17) genes are likely to increase susceptibility. The aim of this study was to investigate the risk association of SRD5A2 and CYP17 gene polymorphisms in the development and progression of PCa in the Turkish population. In this study, 100 PCa patients and 105 healthy controls were studied. SRD5A2 and CYP17 gene polymorphisms were determined by real-time PCR and polymerase chain reaction-restriction length polymorphisms (PCR-RFLP) techniques. First, the AT and TT genotypes of SRD5A2 gene at codon 49 were not observed. Second, there was no significant association between the polymorphisms at codon 89 and the risk of PCa. Third, in the CYP17 gene, the A1A1 genotype is more common (46%) in cases than controls (32.4%). The odds ratios (ORs) of the A1A1 genotype was found at 1.69 (95% confidence interval [CI], 0.77-3.74) compare with the A2A2 genotype. Genotyping results of the SRD5A2 and CYP17 genes were also analyzed in relation to prostate-specific antigen (PSA) levels, Gleason score (GS), and tumor stage, but no statistically significant difference was observed (P > 0.05). Finally, we conclude that there was no evidence of an association between CYP17 (P = 0.134) and SRD5A2 (P = 0.784) polymorphism and PCa risk in the Turkish population.