Expression of GRP78 and its copartners in HEK293 and pancreatic cancer cell lines (BxPC-3/PANC-1) exposed to MRI and CT contrast agents.

Endoplasmic reticulum (ER) stress-associated chaperones trigger a defense mechanism called as unfolded protein response (UPR) which can manage apoptosis and be determinative in cell fate. Both anticancer drug effects and potential toxicity effects of magnetic resonance imaging (MRI) and computed tomography (CT) contrast agents were aimed to be evaluated. For this purpose, we investigated expression profiles of endoplasmic reticulum stress-associated chaperone molecules in human pancreatic tumor lines BxPC-3 and PANC-1 and control human embryonic kidney cells 293 (HEK293) induced with a variety of gadolinium and iohexol contrast agents. Protein expression levels of ER stress-associated chaperones (master regulator: GRP78/Bip and its copartners: Calnexin, Ero1, PDI, CHOP, IRE1α and PERK) were evaluated with Western blotting. Expression levels at mRNA level were also assessed for GRP78/Bip and CHOP with real-time PCR. Induction of cells was carried out with four different Gd-based contrast agents (GBCAs): (Dotarem, Optimark, Primovist and Gadovist) and two different iohexol agents (Omnipol, Omnipaque). CT contrast agents tested in the study did not result in significant ER stress in HEK293 cells. However, they do not seem to have theranostic potential in pancreas cancer through ER pathway. The potential efficiency of macrocyclic MRI contrast agents to provoke apoptosis via ER stress-associated chaperones in BxPC-3 cells lends credibility for their future theranostic use in pancreas cancer as long as undesired toxicity effects were carefully considered. ER stress markers and/or contrast agents seem to have promising potential to be translated into the clinical practice to manage pancreas cancer progression.

Elevated expression levels of COX-2, IL-8 and VEGF in colon adenocarcinoma.

There is growing evidence of a connection between inflammation and tumor development and NF-κB is an important transcription factor in the inflammation pathway. Genetic approaches have proven the role of NF-κB responsive genes in tumorigenesis. The NF-κB responsive genes products such as IL-8, VEGF and COX-2 are the key components of angiogenesis. MMP-2 and MMP-9 are playing important roles in the disruption of the extracellular matrix that may contribute to the metastasis of tumor cells. This study aimed to investigate gene expression levels of COX-2, IL-8, VEGF, MMP-2 and MMP-9 in colon tumors. A total of 34 fresh colon carcinoma specimens and paired normal adjacent tissues (NAT) were collected during the surgery and RNA isolations were carried out from specimens. Synthesis of cDNA was carried out from these RNAs with oligo dT18 primers. The transcribed cDNA was used for PCR amplification reactions for the investigated genes with ß-actin being the internal reference via the semi-quantitative RT-PCR method. A statistically significant difference was observed for COX-2, IL-8 and VEGF which were all upregulated in colon tumors compared with adjacent normal tissues (p<0.05). However, MMP-2 and MMP-9 expression levels did not change between tumor and normal tissues (p>0.05). Upregulated expression levels of COX-2, IL-8 and VEGF might occur in the early stages of tumorigenesis and detection of these mRNA levels may be beneficial for early diagnosis and management of colon tumors.

Single-strand conformation polymorphism-based genetic characterization of the Cyclospora cayetanensis strains collected from different provinces in Turkey.

INTRODUCTION AND OBJECTIVE: Cyclospora cayetanensis, a coccidian protozoan species, has been recently found to cause diarrhea in all age groups in immunocompetent and immunocompromised individuals in most regions of the world. This study aimed to conduct the molecular detection of C. cayetanensis and to determine the genetic diversity of the 18S ribosomal RNA (rRNA) gene sequence of C. cayetanensis isolated from individuals living in different provinces in Turkey by using PCR-single-strand conformation polymorphism (SSCP). MATERIAL AND METHODS: A total of 22 subjects were included in the study. Fourteen of the subjects were female and eight were male, with ages ranging between 7-65 years. Stool specimens were examined using wet mount and modified acid-fast staining methods, which revealed the presence of oocysts in the samples. The 18S rRNA ITS-1 Ccits37f-GCTTGCTATGTTTTAGCATGTGG and Ccits501r-GCACAATGAATGCACACACA gene regions were used as primers. The PCR products were analyzed by agarose gel electrophoresis and visualized on a UV transilluminator. For the SSCP, the PCR products were denatured with formamide, run for 16 h in 6% (49:1) polyacrylamide gel, and then imaged with silver staining. RESULTS: SSCP assay was performed given that the DNA strands demonstrated different folds; the DNA strands contain different nucleotides based on the PCR-SSCP results for the Cyclospora strains collected in 4 provinces. Moreover, 3 different band profiles were observed in the investigated samples. A slight mutation difference was observed among the strains collected. CONCLUSIONS: Further comprehensive studies involving more C. cayetanensis-positive specimens and utilizing different mutation screening methods are warranted to demonstrate mutation differences in Cyclopora strains in Turkey.

Determination the Subtypes of Blastocystis sp. and Evaluate the Effect of These Subtypes on Pathogenicity.

PURPOSE: The present study aimed to determine the frequency of subtypes of Blastocystis sp. in the immunosuppressed individuals, in patients with chronic urticaria, and in patients with GIS complaints to investigate the difference of Blastocystis sp. subtype distribution between patient and control groups. METHODS: A total of 345 stool samples were collected from the patients and samples were studied by native-Lugol, trichrome staining, and Jones medium culture method. Positively detected samples were subjected to PCR to determine the subtypes. RESULTS: This is the first study of nine subtypes of Blastocystis sp. investigated in our country and the most frequently found subtype was ST3, and then, the other subtypes were ST1, ST2, ST5, and ST6, respectively. Mix subtype was detected in the 11.6% and no subtype was detected in the 17.4% of the samples. The ST5 was detected first time in the control group and ST6, which is reported limitedly in our country, was found in patients with GIS complaints. ST1 and ST2 were found higher in the patient group. CONCLUSION: This study confirmed that the subtype (ST) differences are an important factor affecting the pathogenesis of Blastocystis sp.

Gene expression of ING4 gene in pancreatic cancers and splice forms of ING4 gene.

Pancreatic cancer is characterized by rapid metastasis and resistant to medical treatments. As the other cancers, mutations of tumor suppressor genes that involved in suppression of cell growth are observed in pancreatic cancers. ING4 protein is one of the proteins involved in the regulation of p53 tumor suppressor gene functions. ING4 involved in suppression of cell proliferation, chromosome rearrangement, cell migration, and angiogenesis. In this study, gene expressions and splicing variants of ING4 gene were investigated. Fresh tumor and normal specimens of the same pancreatic cancer patients were used. Gene expression study carried out by calculating the brightness of the bands on agarose gel and splicing variants were detected by direct sequencing.  According to the results, three splice forms of ING4 and a decrease in gene expression of ING4 were determined. Splicing type of ING4 affects the translocation of ING4 proteins into the nucleus. To determine the gene expression of each splicing variant, will further clarify the role of ING4 in pancreatic cancers.

Vitamin D can be used as a supplement against cancer stem cells.

Cancer is standing like a bottomless pit or a black hole in front of mankind. Scientists are trying all possible ways to find a solution against to cancer. As known, cancer is a phenomenon fed from internal dynamics. One of internal dynamic is cancer stem cells that are involved in the formation and development of cancer. Because of these dynamics, scientists began to search solution inside of the body. Another internal dynamic is vitamin D and it is not only important in calcium homeostasis but also it is important for cell proliferation, differentiation, and apoptosis. In this study, we investigated the effect of vitamin D on cancer stem cells that sorted from MCF-7 cell line and on HEK293 cell line as control. Our results showed that calcitriol treatment reduced the number of CSC (Cancer Stem Cell) in the MCF-7 cell while increased in HEK293 cell population. Gene expression analyses showed that effect of calcitriol on apoptosis plays an important role in this reduction. Deficiency or unavailability of vitamin D may take a role in the pathogenesis of breast cancer.

Effects of melatonin on apoptosis and cell differentiation in MCF-7 derived cancer stem cells.

Melatonin is a hormone of the pineal gland that has a wide range of biological effects such as antioxidant, anti-inflammatory, and anti-tumor activity. Previous studies have shown that melatonin also affects survival, proliferation, and apoptosis of the cells. In this study, we investigated the effect of melatonin on apoptosis, self-renewal, and differentiation. For this purpose, MCF-7 and HEK293 cells were subjected to melatonin treatment. Expression of genes related to apoptosis (Bax and Bcl2) and self-renewal and differentiation (Oct4, Sox2, and Nanog) analyzed after the sorting of cancer stem cells from MCF-7 cells. Results showed that the effect of melatonin is dependent on the melatonin concentration and treatment periods. Melatonin treatment decreased the cell proliferation rate of MCF-7 in contrast to HEK293. Also, this treatment increased apoptosis in MCF-7 cells and decreased in HEK293 cells. Gene expression of Nanog was decreased and Sox2 was increased in both cell groups after the melatonin treatment. Expression of Oct4 was decreased in MCF-7 cells and increased in HEK293 cells. We determined that melatonin decreases apoptosis and differentiation of stem cells in normal HEK293 stem cells, but increases apoptosis and differentiation in the MCF-7 cancer stem cells.

TLR4 Asp299Gly and Thr399Ile and TLR2 intron 2 microsatellite gene polymorphism in patients with acute biliary pancreatitis: Does it cause the disease?

OBJECTIVES: There has been coverage of Toll-like receptor 4 and Toll-like receptor 2 gene polymorphisms in inflammatory episodes in a number of studies. In view of the inflammatory nature of acute pancreatitis, we aimed to determine the predictive value of mutations in Asp299Gly and Thr399Ile of the Toll-like receptor 4 gene, and the intron 2 microsatellite polymorphism of the Toll-like receptor 2 gene on the occurrence of acute biliary pancreatitis. MATERIAL AND METHODS: The study included 86 patients for the Toll-like receptor 4 Thr399Ile polymorphism, 100 patients for the Toll-like receptor 4 Asp299Gly polymorphism with acute biliary pancreatitis, and 101 healthy volunteers. At the same time, 93 patients and 92 healthy volunteers were included in the study to research the Toll-like receptor 2 intron 2 microsatellite polymorphism. Genotypes were determined using the restriction fragment length polymorphism analysis of PCR products and by an allele-specific PCR. RESULTS: The Toll-like receptor 4 Thr399Ile homozygotes mutant variants (p=0.005) and Toll-like receptor 2 MM genotype (p<0.001) were detected with a significantly higher frequency in patients with acute biliary pancreatitis than in the healthy blood donors. CONCLUSION: The Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms and the Toll-like receptor 2 intron 2 microsatellite polymorphism are statistically associated with ABP.

Studying the C1772T polymorphism of Hif-1α and TGF-ß3 IVS5+104 A/G polymorphism in children with congenital non-syndromic neural tube defects and their mothers.

Prevalence of neural tube defect (NTD) has reduced after folic acid intake. However; which mechanisms are effective in NTD are not known exactly. In this study; due to the possible effects on hypoxic pathway and embryonic development, particularly on extracellular matrix components, Hif-1α Pro582Ser and TGF-ß3 IVS5+104 A/G SfaN1 polymorphisms were studied by PCR-RFLP method both on children with NTDs and mothers. Statistical differences were seen for Hif-1α and TGF-ß3 IVS5+104 A/G SfaN1 polymorphisms in children with NTDs but no difference was seen in mothers. Both genes are effective on many pathways and our results suggest that regulation of extracellular matrix components of children during fetal life is important in neural tube defects formation. The results of this study show that Hif-1α Pro582Ser and TGF-ß3 IVS5+104 A/G SfaN1 polymorphisms may play a role in NTDs.

Fentanyl Inhibits Tumorigenesis from Human Breast Stem Cells by Inducing Apoptosis

Fentanyl is an opioid analgesic that it is widely used in cancer patients. Since there have been reports of effects of analgesic medications on the recurrence and development of resistance to treatment, influences of of fentanyl on MCF-7 and HEK293 cells were evaluated. Cell viability and apoptosis were assessed by MTT assay and flow cytometry, respectively. Gene expression analysis was performed by quantitative real-time PCR assay for the Oct4, Sox2 and Nanog genes as stem cell markers and Bax, Bcl2, and p53 genes as apoptosis markers. MTT assay results showed that fentanyl significantly inhibited the growth of MCF-7 cells in a dose-and time-dependent manner while significantly increasing apoptosis. In contrast, decrease was noted in HEK-293 cells. In MCF-7 derived cancer stem cells, fentanyl treatment decreased the expression of Bax, Bcl2, Oct4, Sox2, Nanog genes when compared to untreated cells. In HEK-293 stem cells, decrease was noted for Sox2, Nanog and Bax, but increase for Oct4. Our study supports an antitumor role of fentanyl by inducing apoptosis and reducing numbers of cancer stem cells in the MCF-7 human breast adenocarcinoma line.

IL-8 gene polymorphism in acute biliary and non biliary pancreatitis: probable cause of high level parameters?

BACKGROUNDS/AIMS: Inflammatory mediators of the innate immune response play fundamental roles in the pathogenesis of acute pancreatitis. The correlation between interleukin-8 (IL-8) gene polymorphism with types of acute pancreatitis and severity of pancreatitis, was evaluated in this study. METHODS: According to the diagnostic criteria, 176 patients with acute pancreatitis were grouped into biliary (n=83) and nonbiliary pancreatitis (n=93). Healthy blood donors (n=100) served as controls. Serum alanine transaminase, aspartate transaminase, total and direct bilirubin, amylase, lypase, white blood cell count and c-reactive protein levels were evaluated to correlate with IL-8 rs4073 (-251T/A) polymorphism, which was analyzed using a real-time polymerase chain reaction method with melting point analysis. RESULTS: The IL-8 AA genotype was detected with a significantly higher frequency among the patients with acute biliary pancreatitis having higher alanine transaminase levels than the median range. Homozygote alleles were significantly higher among patients with acute biliary pancreatitis having amylase levels higher than the median range. CONCLUSIONS: Determination of the frequency of IL-8 polymorphism in acute pancreatitis is informative and provides further evidence concerning the role of IL-8 in laboratory tests.

Prevalence and antimicrobial susceptibility of Arcobacter species in cow milk, water buffalo milk and fresh village cheese.

In this study, the presence of Arcobacter spp. was examined in cow milk (n=50), water buffalo (WB) milk (n=50) and fresh village cheese (n=50) samples. The 16S rDNA-RFLP method was used for the identification of Arcobacter spp. The disc diffusion method was used to investigate the susceptibility of all strains identified to 18 different antimicrobial substances. The most commonly isolated Arcobacter species were found to be Arcobacter butzleri (38.89%), Arcobacter cryaerophilus (22.23%) and Arcobacter skirrowii (11.12%) in cow milk; A. cryaerophilus (33.33%), Arcobacter cibarius (20.83%) and A. butzleri (12.50%) in WB milk; and A. skirrowii (28.57%), A. butzleri (21.43%) and A. cryaerophilus (14.29%) in fresh village cheese. This is the first study to identify the presence of Arcobacter nitrofigilis, Arcobacter cloacae, Arcobacter halophilus, Arcobacter bivalviorum and A. cibarius species in analyzed samples. It was found that all of the A. cryaerophilus (n:16) isolates were resistant to cefoperazone, cloxacillin and penicillin G; all of the A. skirrowii (n:12) and A. butzleri (n:10) isolates were resistant to cefoperazone, tetracycline, ampicillin, erythromycin, cloxacillin and penicillin G. It was concluded that cow milk, WB milk and fresh village cheese samples are an important source of Arcobacter species and pose a risk to public health.

Increased micronucleus frequencies in peripheral blood lymphocytes in women with polycystic ovary syndrome.

OBJECTIVE: We aimed to assess possible genomic instability in women with polycystic ovary syndrome (PCOS). DESIGN: The frequency of micronuclei in cultured peripheral lymphocytes was used as a biomarker of genomic instability in somatic cells. METHODS: Nineteen women, diagnosed with PCOS and 19 healthy female volunteers of corresponding ages and body-mass index (BMI) were included in the study. Micronuclei frequencies were assessed in cytokinesis-blocked lymphocytes. RESULTS: The frequency of micronucleated cells (per thousand) was 9.00 (5.00) (interquartile range in parentheses) for patient group and 3.0 (3.0) for the control group (P < 0.0001, Mann-Whitney U-test). The serum levels of follicle-stimulating hormone (FSH), estradiol, prolactin, glucose and dehydroepiandrosterone sulfate (DHEAS) and the homeostasis model of assessment of insulin resistance (HOMA-IR) were not different between the two groups (P > 0.05). Serum total testosterone, luteinizing hormone (LH) and insulin levels and hirsutism score in the PCOS group were significantly (P = 0.007, P < 0.0001, P = 0.009 and P < 0.0001 respectively) higher than those of the control group (2.3 (2.1) nmol/l vs 1.7 (0.4) nmol/l; 8.5 (5.88) mU/ml vs 4.8 (4.4) mU/ml; 6.8 (5.1) microU/ml vs 9.7 (4.2) microU/ml; 19.5 (6.5) vs 4.0 (2.5) respectively). However, the mean level of sex hormone-binding globulin (SHBG) in PCOS group was significantly (P = 0.004) lower than in control group (36.4(22.6) nmol/l vs 48.6(25.2) nmol/l respectively). CONCLUSION: These findings suggest that women with PCOS have a high incidence of genomic instability, and this condition is positively correlated with the hirsutism score, BMI, LH and serum total testosterone and insulin levels, and is negatively correlated with SHBG.