Juglone-ascorbate treatment enhances reactive oxygen species mediated mitochondrial apoptosis in pancreatic cancer.

BACKGROUND: Treatment of Pancreatic Cancer (PC) is challenging due to its aggressiveness and acquired resistance to conventional chemotherapy and radiotherapy. Therefore, the discovery of new therapeutic agents and strategies is essential. Juglone, a naphthoquinone, is a secondary metabolite produced naturally in walnut-type trees having allelopathic features in its native environment. Juglone was shown to prevent cell proliferation and induce ROS-mediated mitochondrial apoptosis. Ascorbate with both antioxidant and oxidant features, shows selective cytotoxicity in cancer cells. METHODS AND RESULTS: In this study, we evaluated the anticancer effects of Juglone in combination with ascorbate in PANC-1 and BxPC-3 PC cells. The MTT assay was used to determine the IC50 dose of Juglone with 1 mM NaAscorbate (Jug-NaAsc). Subsequently, the cells were treated with 5, 10, 15 and 20 µM Jug-NaAsc for 24 h. Apoptotic effects were evaluated by analyzing the following genes using qPCR; proapoptotic Bax, antiapoptotic Bcl-2 related to the mitochondrial apoptotic pathway and apoptosis inhibitor Birc5 (Survivin). Immunofluorescence analysis was performed using Annexin V-FITC in PC cells. As an antioxidant enzyme, Trx2 protein levels were determined by a commercial ELISA test kit. Jug-NaAsc treatment decreased the expressions of antiapoptotic genes Bcl-2 and Birc5 while the apoptotic gene Bax expression increased at all doses. Additionally, a dose-dependently increase of apoptosis according to immunofluorescence analysis and the decreases of Trx2 enzyme levels at all treatments in both cell lines supported gene expression results. CONCLUSION: Our results suggest that Juglone is a potential anticancer agent especially when combined with ascorbate.

The effects of Juglone-Selenium combination on invasion and metastasis in pancreatic cancer cell lines.

Background: Pancreatic cancer does not show any symptoms in the early period and metastatic process is already passed when the diagnosis is done. Therefore, in the battle with pancreatic cancer, novel treatment strategies, particularly antiinvasive and antimetastatic strategies, are needed. The cytotoxic and anticancer effects of juglone and sodium selenite (NaSe) have been showed in various cancer cells. Objectives: In this study, it is aimed to investigate the synergistic effects of juglone and selenium on PANC-1 and BxPC-3 pancreatic cancer cells. Methods: Antimetastatic effects of juglone-NaSe were carried out by adhesion and invasion assays and the genes and protein expressions. Expression analysis of the CDH1, ITGB3 and COL4A3 genes and their proteins E-cadherin, ß3 integrin and tumstatin which play role in metastasis and angiogenesis processes, were done by qPCR and immunohistochemical analysis, respectively. Results: Study findings have provided evidences that the juglone-selenium has a cytotoxic and dose dependent suppressive effect on invasion and metastasis in PANC-1 and BxPC-3 cells. Conclusion: The juglone-NaSe has the potential to be a promising agent especially to inhibit invasion and metastasis in pancreatic cancer treatment. However, more in depth studies are needed to more clearly demonstrate the effects of juglone-selenium.

Type 2 diabetes is associated with the MTNR1B gene, a genetic bridge between circadian rhythm and glucose metabolism, in a Turkish population.

Type 2 diabetes (T2D) is a complicated public health problem in Turkey as well as worldwide. Genome-wide approaches have been guiding in very challenging situations, such as the elucidation of genetic variations underlying complex diseases such as T2D. Despite intensive studies worldwide, few studies have determined the genetic susceptibility to T2D in Turkish populations. In this study, we investigated the effect of genes that are strongly associated with T2D in genome-wide association (GWA) studies, including MTNR1B, CDKAL1, THADA, ADAMTS9 and ENPP1, on T2D and its characteristic traits in a Turkish population. In 824 nonobese individuals (454 T2D patients and 370 healthy individuals), prominent variants of these GWA genes were genotyped by real-time PCR using the LightSNiP Genotyping Assay System. The SNP rs1387153 C/T, which is located 28 kb upstream of the MTNR1B gene, was significantly associated with T2D and fasting blood glucose levels (P < 0.05). The intronic SNP rs10830963 C/G in the MTNR1B gene was not associated with T2D, but it was associated with fasting blood glucose, HbA1C and LDL levels (P < 0.05). The other important GWA loci investigated in our study were not found to be associated with T2D or its traits. Only the SNP rs1044498 (A/C variation) in the ENPP1 gene was determined to be related to fasting blood glucose (P < 0.05). Our study suggests, consistent with the literature, that the MTNR1B locus, which has a prominent role in glucose regulation, is associated with T2D development by affecting blood glucose levels in our population.

Investigation of TGFBI (transforming growth factor beta-induced) Gene Mutations in Families with Granular Corneal Dystrophy Type 1 in the Konya Region.

Objectives: Granular corneal dystrophies (GCD) are characterized by small, discrete, sharp-edged, grayish-white opacities in the corneal stroma. Among the genes responsible for the development of GCD, the most strongly related gene is transforming growth factor beta-induced (TGFBI), located in the 5q31.1 locus. Studies show that R124H in exon 4 and R555W in exon 12 are hot-spot mutations in the TGFBI gene that lead to GCD development. In this study, we aimed to investigate these two hot-spot mutations in exons 4 and 12 of the TGFBI gene and other possible mutations in the same regions, which code important functional regions of the protein, in Turkish families with GCD and to determine the relationship between the mutations and disease and related phenotypes. Materials and Methods: The study included 16 individuals diagnosed with GCD type 1 (GCD1), 11 of these patients' healthy relatives, and 28 unrelated healthy individuals. DNA was obtained from peripheral blood samples taken from each individual and polymerase chain reaction was used to amplify target gene regions. Genotyping studies were done by sequence analysis. Results: The R124S mutation in exon 4 of TGFBI was not detected in the patients or healthy individuals in our study. However, all individuals diagnosed as having GCD1 were found to be heterozygous carriers of the R555W mutation in exon 12 of TGFBI. This mutation was not detected in healthy family members or control individuals unrelated to these families. In addition, we detected the silent mutation F540F in exon 12 and c.32924 G>A substitution in an intronic region of the gene in a few patients and healthy individuals. Conclusion: Our study strongly supports the association of GCD1 with R555W mutation in exon 12 region of the TGFBI gene, as reported in the literature.

PIK3R1 gene polymorphisms are associated with type 2 diabetes and related features in the Turkish population.

BACKGROUND: The phosphatidylinositol 3-kinase p85 alpha regulatory subunit 1 gene (PIK3R1) encodes the PIK3R1 protein, which plays a direct role in insulin signaling. PIK3R1 (p85 regulatory subunit) connects firmly with the p110 catalytic subunit, and together these proteins form the phosphatidylinositol 3-kinase (PI3K) protein. PI3K is a key protein in the Akt signaling pathway, which regulates cell survival, growth, differentiation, glucose trafficking, and utilization. Defects in the insulin signaling cascade play an important role in the development of insulin resistance, which shares a common genetic basis for metabolic diseases such as type 2 diabetes (T2D), obesity and cardiovascular diseases. OBJECTIVES: In our study, we investigated the effect of single nucleotide polymorphisms (SNPs) rs3756668 in 3'UTR region, rs706713 and rs3730089 in exons 1 and 6, respectively, rs7713645 and rs7709243 in intron 1, and rs1550805 in intron 6 of PIK3R1 gene on T2D. MATERIAL AND METHODS: This study enrolled a total of 840 individuals, including 427 diabetic individuals (206 obese and 221 non-obese) and 413 nondiabetic individuals (138 obese and 275 non-obese). The target SNPs were analyzed using real-time polymerase chain reaction (RT-PCR). Statistical analysis was performed using SPSS18.0 (IBM Corp., Armonk, USA). The p-values ≤0.05 were considered statistically significant. RESULTS: The SNPs rs706713 (Tyr73Tyr) and rs3730089 (Met326Ile) located in exons, and rs7713645, rs7709243 and rs1550805 located in introns were determined to be significantly associated with T2D and phenotypic features such as obesity, insulin resistance and the lipid parameters. The association with SNP rs3756668, which is located in the 3'UTR, was not significant. CONCLUSIONS: Our study supports the role of PIK3R1, an important candidate gene due to its critical role in insulin signal transduction, in T2D development.

Transcription factor 7-like 2 (TCF7L2) gene polymorphisms are strong predictorsof type 2 diabetes among nonobese diabetics in the Turkish population.

BACKGROUND/AIM: Type 2 diabetes (T2D) is1397645907a multifactorial disease, determined by environmental and genetic factors. Currently, the transcription factor 7-like 2 (TCF7L2) gene shows the strongest association with T2D. In this study, we investigated whether TCF7L2 gene polymorphisms are associated with T2D in a Turkish population. MATERIALS AND METHODS: Using PCR-RFLP and PCR-SSCP, we genotyped six intronic polymorphisms in the TCF7L2 gene, commonly associated with T2D, in 169 individuals with diabetes and 119 healthy controls. RESULTS: We found that rs7903146 C → T substitution in intron 3 (OR: 1.9, P = 0.005) and rs12255372 G → T substitution in intron 4 (OR: 2.1, P = 0.002) were significantly associated with T2D while other SNPs were not associated (P > 0.05). We determined no association between TCF7L2 gene polymorphisms and fasting glucose, fasting insulin, HbA1c, or HOMA-IR levels (P > 0.05), except for rs7903146 C → T substitution, which was significantly associated with the fasting glucose level (P = 0.003). CONCLUSION: Our results indicate that, in the Turkish population, the T allele of the rs7903146 (C → T) and rs12255372 (G → T) polymorphisms in the TCF7L2 gene is an independent risk factor for the development of T2D.

Investigation of juglone effects on metastasis and angiogenesis in pancreatic cancer cells.

Juglone, a natural component, is shown to have cytotoxic and apoptotic effects in several cancer cell lines. However, little is known about its effects on invasion and metastasis. In this study, we aimed to determine the antimetastatic effect of juglone in the BxPC-3 and PANC-1 pancreatic cancer cell lines. Cytotoxic effect of juglone was evaluated by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) test. The cells were treated with juglone at

The Adiponectin variants contribute to the genetic background of type 2 diabetes in Turkish population.

Adiponectin, an adipose tissue specific protein encoded by the Adiponectin gene, modulates insulin sensitivity and plays an important role in regulating energy homeostasis. Many studies have shown that single nucleotide polymorphisms (SNPs) in the Adiponectin gene are associated with low plasma Adiponectin levels, insulin resistance and an increased risk of type 2 diabetes mellitus. The aim of the present study was to evaluate the contribution of the Adiponectin gene polymorphisms in genetic background of type 2 diabetes in a Turkish population. In total, 169 unrelated and non-obese diabetic patients and 119 age- and BMI-matched nondiabetic individuals with no family history of diabetes were enrolled in this study. We detected a significant association between type 2 diabetes and two SNPs: SNP −11391G N A, which is located in the promoter region of the Adiponectin gene, and SNP +276G N T, which is found in intron 2 of the gene (P b 0.05). The silence SNP G15G (+45TN G) in exon 1 and SNP+349ANG in intron 2 also showed a weak association with type 2 diabetes (P=0.06 and P=0.07, respectively),while SNPs−3971ANG in intron 1 and Y111H, R112C and H241P in exon 3 showed no association (P N 0.05). In conclusion, these findings suggest that Adiponectin gene polymorphisms might be effective on susceptibility for type 2 diabetes development which emerged from the interactions between multiple genes, variants and environmental factors.

IRS1 gene polymorphisms Gly972Arg and Ala513Pro are not associated with insulin resistance and type 2 diabetes risk in non-obese Turkish population.

Insulin receptor substrate 1 (IRS1), plays a critical role in insulin signaling and its control has an important place in the development of insulin resistance. The tyrosine phosphorylation of IRS1 serves as docking molecules for downstream effectors such as Phosphatidylinositol 3-kinase and phosphotyrosine phosphatase-2. We focused on the Gly972Arg and Ala513Pro variants of the IRS1 gene, since these specific allelic variants are located near the Tyr-Met-X-Met (YMXM) motifs around Tyr987 and Tyr612. Thus, we aimed to investigate the effects of Gly972Arg/Ala513Pro polymorphisms in IRS1 gene on development of insulin resistance and the risk of type 2 diabetes in a non-obese Turkish population. This work included 306 individuals comprising 178 subjects with type 2 diabetes and 128 healthy subjects matched for body mass index. Gly972Arg/Ala513Pro polymorphisms had no effect on type 2 diabetes risk and its phenotypes (P > 0.05). Although IRS1 gene and its variants are associated with type 2 diabetes and insulin resistance in several studies worldwide, our data showed that there is no association between Gly972Arg and Ala513Pro variants in IRS1 and disease in Turkish population.

Effects of single nucleotide polymorphisms in K(ATP) channel genes on type 2 diabetes in a Turkish population.

BACKGROUND AND AIMS: ATP-sensitive potassium (K(ATP)) channels of pancreatic ß-cells play a key role in glucose-stimulated insulin secretion mechanism. The Kir6.2 protein, forming the K(ATP) channel pore inwardly, and the SUR1 protein that surrounds it forming the outside part of the channel were encoded by ABCC8 and KCNJ11 genes, respectively. Recent studies reported that the single nucleotide polymorphisms (SNPs) established in these genes are associated with defects in insulin secretion and type 2 diabetes mellitus (T2DM). We aimed to investigate the allele profiles and the risk alleles of the ABCC8 and KCNJ11 genes and to highlight the associations with the disease in patients in Konya region of Turkey where T2DM is common. METHODS: In this study, 169 patients with T2DM and 119 healthy controls were included. A total of 29 SNPs in ABCC8 and KCNJ11 genes were screened by PCR-SSCP technique and sequenced. Biochemical parameters and genotype-phenotype relationships were analyzed using variance analysis. RESULTS: R1273R silent substitution in exon 31 and 16/-3t→c substitution in noncoding region of exon 16 of ABCC8 gene showed a significant association (OR 4.8 [95% CI 2.41-9.77], p <0.001 and OR 3.5 [95% CI 1.64-7.40], p <0.001 under dominant and recessive models, respectively). We detected a significant association between E/K heterozygote genotype and reduced plasma insulin level in patients with T2DM (p <0.05). CONCLUSIONS: ABCC8 exons 16 and 31 variants increase susceptibility to T2DM and KCNJ11 E23K decreases insulin secretion in a Turkish population.

Association between the T-593A and C6982T polymorphisms of the osteopontin gene and risk of developing nephrolithiasis.

BACKGROUND AND AIMS: Increased synthesis of several urinary proteins including osteopontin (OPN) has been shown to be associated with stone formation within the urinary tract. The objective of this study was to analyze the genotype distributions and allele frequencies for OPN gene promoter T-593A and C6982T (in exon 7) polymorphisms among patients with kidney stones. METHODS: In this case-control study, the study group consisted of 121 patients with radiologically confirmed nephrolithiasis. Genomic DNA from patients and control cases (n = 100) was analyzed by single-strand conformation polymorphism method and nucleotide sequence analysis. RESULTS: Homozygous carriers of the T-593T genotype were more frequent, but carriers of the A-593A genotype were less frequent in patients than in controls. There was also an increase in -593T allele (88% in patients vs. 79% in controls) and decrease in -593A allele frequencies (21% in control vs. 12% in patients) in the nephrolithiasis groups (p = 0.013). The carriers of C6982C genotype were less frequent, but marked increases in T6982T genotype (25.6% in patients vs. 7% in controls, p = 0.001) and 6982T allele frequency (53.3% in patients vs. 37.5% in controls, p = 0.001) were noted in patients of Turkish ancestry. CONCLUSIONS: These results are the first to demonstrate the existence of T-593A promoter polymorphism of the OPN gene and significant association with risk of developing nephrolithiasis. Our results showed marked associations between polymorphisms (C6982T and T-593A) of the OPN gene and the stone-forming phenotypes in the Turkish population.

Expression profile and polymorphisms of actin genes in protoscoleces of Echinococcus granulosus from sheep in central Turkey.

The expression levels and polymorphisms of two actin genes, EgactI and EgactII, were investigated in the protoscoleces of Echinococcus granulosus isolated from sheep in the central region of Turkey. Only the EgactII gene was found to be expressed at detectable levels by protoscoleces in the present study. PCR-RFLP analysis and following sequence analysis revealed that a partial EgactI gene is more polymorphic than a partial EgactII gene. And, three alleles for EgactI gene were identified. In all samples, including three alleles, there were nucleotide substitutions at six positions of the partial EgactI gene, and were distinct from the reference sequence from GenBank(TM) database. These differences are referred to as the Anatolian haplotype.

Micronucleus frequency in acquired middle ear cholesteatoma.

OBJECTIVE: To determine the micronucleus (MN) frequency of acquired cholesteatoma tissue using an MN assay. MATERIAL AND METHODS: Eighteen patients were diagnosed as having chronic otitis media with acquired cholesteatoma and were divided into primary and secondary acquired cholesteatoma groups. Cholesteatoma tissue and normal tissue specimens from the external ear canal skin were taken from the patients during surgical operations. MN frequencies of cholesteatoma and control samples were determined according to standard criteria. RESULTS: The MN frequencies of the cholesteatoma and control tissues were 0.54%+/-0.31% and 0.24%+/-0.11%, respectively (p<0.01). MN frequencies for the primary and secondary acquired cholesteatoma groups were 0.63%+/-0.36% and 0.46%+/-0.26%, respectively (p>0.05). MN frequencies in cholesteatoma patients without and with complications were 0.42%+/-0.19% and 0.85%+/-0.37%, respectively (p<0.05). CONCLUSION: MN frequencies were found to be increased in cholesteatoma tissues when compared with external ear canal skin. The MN frequency in five cases with complications was higher than in cases without complications. These results indicate that there could be associations between MN frequency and acquired cholesteatoma and between MN frequency and complications.