Role of cardiac drugs and flavonoids on the IRE1-JNK pathway as revealed by re-ranked molecular docking scores, MM/PBSA and umbrella sampling.

One of the important causes of cardiac dysfunction is the triggering of apoptosis through the IRE1-JNK signaling pathway due to excessive ER stress (endoplasmic reticulum stress). Although there are various studies on beneficial or harmful side effects of cardiac drugs, knowledge about the molecular mechanism of their interactions on this pathway is very limited. In this study, we investigated interactions of statins, ace inhibitors, antiarrhythmic drugs and flavonoids in IRE1, ASK1(apoptosis signal-regulating kinase 1) and JNK1 at an atomic level in comparison with their well-known inhibitors. The rank of scores obtained from four different docking algorithms (Autodock 4, Autodock Vina, iGEMDOCK and GOLD) were combined so that they could be compared with each other and evaluated together. According to combined results, the most potent compound for each compound group was selected for molecular dynamics simulations, MM/PBSA (molecular mechanics/Poisson-Boltzmann surface area) and umbrella sampling calculations. We observed that the statin group drugs had the best affinity by interacting with ASK1 and JNK1 by having a similar effect with their inhibitors, and atorvastatin and pitavastatin came to the fore. Norizalpinine from the flavonoid group had a strong binding interaction with IRE1, and amiodarone from the antiarrhythmic drug group had high binding affinities with IRE1, ASK1 and JNK1. Our study has shown that atorvastatin, pitavastatin, norizalpinine and amiodarone may have a role in preventing cardiac dysfunctions caused by ER stress and may shed light on further in vitro and in vivo research.Communicated by Ramaswamy H. Sarma.

Effect of SIRT1 activators and inhibitors on CD44+/CD133+­enriched non­small cell lung cancer cells.

Lung cancer is one of the most commonly diagnosed cancers and it is associated with high rates of morbidity and mortality. Metastasis and relapse of the tumor depend on the survival and proliferation of lung cancer stem cells (LCSCs). The ability to identify CSCs may prevent recurrence and lead to more effective treatments. Sirtuins are a group of deacetylases that include seven variants (SIRT1­7), with sirtuin 1 (SIRT1) being the most intensively investigated. Evidence suggests that SIRT1 is both a tumor­suppressor gene and an oncogene. SIRT1 can deacetylate the tumor­suppressor protein p53 to decrease its activity. SIRT1 activators increase the deacetylation of p53, whereas SIRT1 inhibitors can stimulate p53 by inhibiting deacetylation. In the present study, CD44+ and CD133+­enriched A549 (non­small cell lung cancer) cells collected using the CD44 and CD133 CSC surface markers by fluorescence­activated cell sorting method were treated with SIRT1 inhibitors (tenovin­6 and sirtinol) and SIRT1 activators (resveratrol and SRT1720), and their effects on apoptosis, as well as the mRNA and protein expression of SIRT1 and p53 were investigated. Of these agents, it was found that resveratrol increased p53 expression by 4.1­fold, decreased SIRT1 expression by 0.2­fold, and it was the most potent inducer of apoptosis.

The relationship of the endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) gene polymorphism in Turkish type 2 diabetic patients with and without diabetic foot ulcers.

OBJECTIVE: This study aims to evaluate the influence of eNOS G894T and VEGF C936T gene polymorphism in diabetic foot ulcers. METHOD: We studied 50 patients with diabetic foot ulcers and 57 diabetic patients without diabetic foot ulcer and a control group of 75 healthy individuals. RESULTS: The genotype eNOS distribution did not differ between Type 2 Diabetic Patients group and Diabetic Foot Ulcer group (P>0.05). The frequency of the polymorphic T allele in Type 2 Diabetic Patients were significantly higher than the control group (42.3% and 24.5%, respectively)(p<0.01). The frequency of the polymorphic T allele between the Type 2 Diabetic Patients and Diabetic Foot Ulcer group was similar (p>0.05). The genotype VEGF distribution did not differ between Type 2 Diabetic Patients group and Diabetic Foot Ulcer group (P>0.05). The frequency of the polymorphic T allele between the Type 2 Diabetic Patients and Diabetic Foot Ulcer group was similar for both groups (p>0.05). CONCLUSION: Polymorphism of eNOS G894T is not a risk factor for diabetic foot ulcer formation. T allele is a risk factor for diabetes, but T allele is not a risk factor for diabetic foot ulcer formation. Polymorphism of VEGF C936T and T allele are not risk factors for diabetes occurence and diabetic foot formation.

Case-control study on PCSK9 R496W (rs374603772) and D374Y (rs137852912) mutations in Turkish patients with primary dyslipidemia.

OBJECTIVE: The aim of this study was to investigate the relationships between F216L (rs28942112), R496W (rs374603772), S127R (rs28942111), and D374Y (rs137852912) PCSK9 gain-of-function (GOF) mutations and primary dyslipidemia and serum lipid levels in patients with primary dyslipidemia. METHODS: In this case-control study, DNA was isolated from blood samples collected from patients diagnosed with primary dyslipidemia in cardiology outpatient clinic of Ege University (n=200) and healthy individuals (n=201). F216L, R496W, S127R, and D374Y GOF mutations in the PCSK9 gene were evaluated and genotyped according to the results of melting curve analysis performed in a real-time polymerase chain reaction (PCR) 480 instrument using specific primers for each mutation. RESULTS: There were statistically significant differences between the patient and individuals in control groups in the R496W and D374Y mutations (x2=10.742 p=0.005; x2=6.078 p=0.048, respectively). In addition, triglyceride levels in patients with primary dyslipidemia heterozygous for R496W and D374Y mutations were 12.8-fold (p=0.015) and 3.4-fold (p=0.03) higher than that in mutant and wild-type genotype, respectively. Additionally, in the entire study group (n=401), PCSK9 R496W and D374Y mutation carriers had increased total cholesterol (p=0.021), triglycerides (p=0.0001), HDL cholesterol (p=0.028), and low-density lipoproteins (LDL) cholesterol (p=0.028) levels. However, F216L (rs28942112) and S127R (rs28942111) mutations were not detected in patients with primary dyslipidemia and healthy controls. CONCLUSION: We conclude that the PCSK9 R496W (rs374603772) and D374Y (rs137852912) GOF mutations may be significant risk factors in the development of primary dyslipidemia and may have significant impact on lipid parameters in general population.

Matrine induced G0/G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia (T-ALL) cells.

Matrine, a natural product extracted from the root of Sophora flavescens, is a promising alternative drug in different types of cancer. Here, we aimed to investigate the therapeutic effects and underlying molecular mechanisms of matrine on human acute lymphoblastic leukemia (ALL) cell line, CCRF-CEM. Cell viability and IC50 values were determined by WST-1 cell cytotoxicity assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Expression patterns of 44 selected miRNAs and 44 RNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the Applied Biosystems 7500 Fast Real-Time PCR System. Matrine inhibited cell viability and induced apoptosis of CCRF-CEM cells in a dose-dependent manner. Cell cycle analysis demonstrated that matrine-treated CCRF-CEM cells significantly accumulated in the G0/G1 phase compared with the untreated control cells. hsa-miR-376b-3p (-37.09 fold, p = 0.008) and hsa-miR-106b-3p (-16.67 fold, p = 0.028) expressions were decreased, whereas IL6 (95.47 fold, p = 0.000011) and CDKN1A (140.03 fold, p = 0.000159) expressions were increased after matrine treatment. Our results suggest that the downregulation of hsa-miR-106b-3p leads to the upregulation of target p21 gene, CDKN1A, and plays a critical role in the cell cycle progression by arresting matrine-treated cells in the G0/G1 phase.

PCSK 9 gain-of-function mutations (R496W and D374Y) and clinical cardiovascular characteristics in a cohort of Turkish patients with familial hypercholesterolemia.

OBJECTIVE: The molecular basis of the mutations in the PCSK9 gene that produces familial hypercholesterolemia (FH) in the Turkish population is unknown. This study was conducted to determine the presence of four different PCSK9 gain-of-function (GOF) mutations (F216L, R496W, S127R, and D374Y) in a group of patients with FH. METHODS: A total of 80 consecutive patients with FH (mean age: 56±11 years; mean maximum LDL cholesterol: 251±76 mg/dL) were included in the study. Patients with FH were diagnosed according to the Dutch Lipid Clinic Network criteria based on serum cholesterol levels, personal and family histories of cardiovascular disease, tendon xanthomas, and genetic analysis. To identify F216L, R496W, S127R, and D374Y mutations of the PCSK9 gene, high-resolution melting analysis was performed on isolated DNAs. RESULTS: Of the 80 patients, there were 11 patients (13.8%) with PCSK9 GOF mutations. Detected mutations were D374Y mutation in four (5.0%) patients and R496W in seven patients (8.7%). Only one patient was homozygous for R496W mutation. The other two GOF mutations (S127R and F216 variants) were not detected. There was no significant difference with regard to demographic characteristics and CV disease risk factors and clinical course of the disease between the PCSK9 mutation-positive and PCSK9 mutation-negative groups. CONCLUSION: This is the first study from a Turkish FH cohort, revealing a higher frequency (approximately 14%) of two PCSK9 GOF mutations (D374Y and R496W) and a different disease course compared to the world literature.

Polymorphisms of lipid metabolism enzyme-coding genes in patients with diabetic dyslipidemia.

OBJECTIVE: The polymorphisms/mutations of genes encoding proteins and enzymes involved in lipoprotein metabolism play important roles in the development of diabetic dyslipidemia. The aim of our study was to investigate the effects of LPL (rs320), LIPC (rs2070895), SCARB1 (rs5888), LCAT (rs2292318), CETP (rs708272), ADIPOQ (rs1501299), RETN (rs3745367), PON1 (rs662), and MNSOD (rs4880) gene polymorphisms on lipid metabolism and diabetic dyslipidemia. METHODS: This case-control study included 217 patients with diabetic dyslipidemia and 212 healthy age- and gender-matched individuals. Genomic DNA isolation was performed from blood samples, and genotype analysis was performed using melting curve analysis on a LightCycler® 480 Instrument. The chi-square test was used to compare genotype distribution and allele frequencies between the groups. RESULTS: Significant associations were observed between LPL (rs320) (p<0.001), LIPC (rs2070895) (p<0.001), SCARB1 (rs5888) (p<0.001), LCAT (rs2292318) (p<0.001), CETP (rs708272) (p<0.001), ADIPOQ (rs1501299) (p=0.01), RETN (rs3745367) (p<0.001), and MNSOD (rs4880) (p<0.001) polymorphisms and diabetic dyslipidemia. However, no association was observed between PON1 (rs662) polymorphisms and diabetic dyslipidemia (p=0.611). CONCLUSION: LPL (rs320), LIPC (rs2070895), SCARB1 (rs5888), LCAT (rs2292318), CETP (rs708272), ADIPOQ (rs1501299), RETN (rs3745367), and MNSOD (rs4880) polymorphisms play an important role in basic molecular metabolism in diabetic dyslipidemia. Therefore, these polymorphisms may be used as a predictive marker for diabetic dyslipidemia in high-risk patients.

LDLR C1725T Gene Polymorphism Frequency in Type 2 Diabetes Mellitus Patients With Dyslipidemia.

BACKGROUND: Dyslipidemia has a substantial role in the development of cardiovascular diseases in patients with type 2 diabetes mellitus (T2DM). Determining the genetic profile of T2DM patients with dyslipidemia is important in order to reduce the risk of microvascular and macrovascular complications. Low-density lipoprotein receptor (LDLR) plays a critical role in plasma lipoprotein hemostasis. LDLR mutations/polymorphisms cause changes at the lipoprotein level. The objective of this study is to determine the frequency of LDLR (rs179989) polymorphisms in Turkish T2DM patients with dyslipidemia. METHODS: The study group consisted of 217 T2DM patients with dyslipidemia including 28 cases with myocardial infarction and 212 healthy controls. Genomic DNA was isolated from venous blood samples and genotype analysis was carried out on the LightCycler® 480 instrument. The χ2 test was used to compare genotype distributions. RESULTS: There were no significant differences in the frequency or allelic distribution of the LDLR C1725T (rs1799898) genotype between the type 2 diabetic dyslipidemia patients and the control group (P > 0.05). CONCLUSION: LDLR C1725T polymorphism was not associated with lipid parameters, and dyslipidemia in T2DM patients.

miR-15a enhances the anticancer effects of cisplatin in the resistant non-small cell lung cancer cells.

Platinum-based chemotherapies have long been used as a standard treatment in non-small cell lung cancer. However, cisplatin resistance is a major problem that restricts the use of cisplatin. Deregulated cell death mechanisms including apoptosis and autophagy could be responsible for the development of cisplatin resistance and miRNAs are the key regulators of these mechanisms. We aimed to analyse the effects of selected miRNAs in the development of cisplatin resistance and found that hsa-miR-15a-3p was one of the most significantly downregulated miRNAs conferring resistance to cisplatin in Calu1 epidermoid lung carcinoma cells. Only hsa-miR-15a-3p mimic transfection did not affect cell proliferation or cell death, though decreased cell viability was found when combined with cisplatin. We found that induced expression of hsa-miR-15a-3p via mimic transfection sensitised cisplatin-resistant cells to apoptosis and autophagy. Our results demonstrated that the apoptosis- and autophagy-inducing effects of hsa-miR-15a-3p might be due to suppression of BCL2, which exhibits a major connection with cell death mechanisms. This study provides new insights into the mechanism of cisplatin resistance due to silencing of the tumour suppressor hsa-miR-15a-3p and its possible contribution to apoptosis, autophagy and cisplatin resistance, which are the devil's triangle in determining cancer cell fate.

Role of mTOR in glioblastoma.

Mammalian target of rapamycin (mTOR), which is a member of the serine/threonine protein kinase family, is a protein complex that has a central role of cell growth and proliferation. mTOR emerges as a critical cell growth checkpoint on phosphoinositide 3-kinase (PI3K) signaling pathway. In this case mTOR has become an important therapeutic target for glioblastoma (GBM) that is one of the most deadly types of cancer. Various combination treatments including inhibition of mTOR may provide more significant results in the treatment of GBM. In addition to new mTOR targets, which may have a plant origin form, more potent mTOR inhibitors by utilizing the computational methodology may emerge as a hope for GBM therapy. In the future, a better understanding of the functional properties of mTORC2 with its potent effective inhibitors may help design more efficiently GBM treatment modalities.

Sequencing of mutations in the serine/threonine kinase domain of the bone morphogenetic protein receptor type 2 gene causing pulmonary arterial hypertension.

OBJECTIVE: Germline mutations in the bone morphogenetic protein receptor type-2 (BMPR2) gene are considered to be a major risk factor for pulmonary arterial hypertension (PAH). BMPR2 mutations have been reported in 10%-20% of idiopathic PAH and in 80% of familial PAH cases. The aim of this study was to evaluate the frequency of mutations in the serine/threonine kinase domain of the BMPR2 gene in a group of patients from a single PAH referral center in Turkey. METHODS: This cross-sectional study used a DNA-sequencing method to investigate BMPR2 mutations in the serine-threonine-kinase domain in 43 patients diagnosed with PAH [8 with idiopathic PAH and 35 with congenital heart disease (CHD)] from a single PAH referral center. Patients were included if they had a hemodynamically measured mean pulmonary arterial pressure of >25 mm Hg with a mean pulmonary capillary wedge pressure of ≤15 mm Hg. Patients with severe left heart disease and/or pulmonary disease that could cause pulmonary hypertension were excluded. Associations between categoric variables were determined using the chi-square test. Differences between idiopathic and CHD-associated PAH groups were compared with the unpaired Student's t-test for continuous variables. RESULTS: We detected a missense mutation, [p.C347Y (c.1040G>A)], in one patient with idiopathic PAH in exon 8 of the BMPR2 gene. The mutation was detected in a 27-year-old female with a remarkable family history for PAH. She had a favorable response to endothelin receptor antagonists. No mutations were detected in the exons 5-11 of the BMPR2 gene in the PAH-CHD group. CONCLUSION: A missense mutation was detected in only one of the eight patients with idiopathic PAH. The BMPR2 missense mutation rate of 12.5% in this cohort of Turkish patients with idiopathic PAH was similar to that seen in European registries. The index patient was a young female with a family history remarkable for PAH; she had a good long-term response to PAH-specific treatment, probably due to the early initiation of the treatment. Genetic screening of families affected by PAH might have great value in identifying the disease at an early stage.

An Association Study Between Gene Polymorphisms of Folic Acid Metabolism Enzymes and Biochemical and Hormonal Parameters in Acromegaly.

AIM: Folate metabolism is fundamental to several biological functions and required for cell replication, division, and survival. The mammalian folic acid cycle is highly complex and the enzymes, methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR), have crucial roles in this metabolic pathway. The common polymorphisms of the MTHFR (C677T and A1298C), MTRR (A66G), and MTR (A2756G) enzymes are well documented as folate deficiency-related disorders, but their roles have not been examined in acromegalic patients. The aim of this study was to compare the genotypic distribution of these gene polymorphisms between patients with acromegaly and controls and explore whether these polymorphisms were associated with biochemical and hormonal parameters in acromegaly. We examined 91 acromegaly patients and 112 healthy subjects who were compared in terms of age and gender. Blood specimens of the subjects were collected in tubes containing ethylenediaminetetraacetic acid. Genomic DNA was isolated from peripheral blood leukocytes and genotyping of the MTHFR (C677T and A1298C) gene polymorphisms was assessed by melting temperature analyses after real-time polymerase chain reaction (PCR), whereas MTRR A66G and MTR A2756G gene polymorphism analyses were performed by PCR/restriction fragment length polymorphism from the isolated DNA of the subjects. RESULTS: MTHFR-677TT genotype frequency was significantly higher in the acromegaly group than the control group (p=0.017), and a significant increase was found in fibrinogen (p=0.032) levels in 677TT-carrying acromegaly patients. MTRR-66AA genotype was significantly higher in the control group than the acromegaly group (p=0.004). Total cholesterol (p=0.048) and C-reactive protein (p=0.046) levels decreased significantly in 66AA genotypes. Although MTR-2756AG genotype frequency was not different between the control and acromegaly groups, 2756AG genotype-carrying individuals have higher left carotid intima-media thickness levels within the patient group. CONCLUSION: Our results suggest that polymorphisms of the genes encoding the folic acid metabolism enzymes affect biochemical parameters in acromegaly and this may result in predispositions to some complications associated with folate metabolism and acromegaly.

Suppression of STAT3 by chemically modified siRNAs increases the chemotherapeutic sensitivity of parental and cisplatin-resistant non-small cell lung cancer cells.

PURPOSE: Increased activation of the JAK-STAT signaling pathway is frequently observed in several primary cancers as well as cancer cell lines. Thus, targeting JAK-STAT pathway components by different molecular-biologic approaches in the search for new anticancer therapies has become widespread and resulted in encouraging outcomes. In this study, the effects of chemically modified anti-STAT3 small interfering (si)RNAs on cell viability, proliferation and apoptosis of parental and cisplatin resistant non-small cell lung cancer (NSCLC) cells were investigated with the aim to provide a new therapeutic strategy for overcoming cisplatin resistance in lung cancer. METHODS: The parental NSCLC cell line Calu1 and its cisplatin- resistant subline CR-Calu1 were used to study the effects of STAT3 suppression with chemically modified anti-STAT3 siRNAs. STAT3 gene and protein expressions were analyzed by real-time (RT) quantitative (q) PCR and Western blot, respectively. Apoptosis was evaluated by Caspase-3 activity and cell death assays. RESULTS: STAT3 messenger (m)RNA and protein expression were significantly increased in CR-Calu1 cells and suppressing its expression with specific siRNAs increased the rate of apoptosis through Caspase-3 activation. STAT3 suppression also significantly increased cisplatin sensitivity of Calu1 and CR-Calu1 cells after transfection with STAT3 siRNAs. CONCLUSIONS: NSCLC cells could be sensitized to cisplatin by targeting STAT3 with chemically modified siRNAs together, a fact which was accompanied with increased apoptosis.

Beta-myosin heavy-chain mutations R403QLW, V606M, K615N and R663H in patients with hypertrophic cardiomyopathy.

OBJECTIVE: Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium with an autosomal-dominant pattern of inheritance mainly caused by single heterozygous mutations in sarcomere genes. In this study we aimed to detect the presence of R403QLW, V606M, K615N, and R663H mutations in beta-myosin heavy-chain gene (MYH7) and figure out the genotype-phenotype correlations in Turkish patients with HCM. METHODS: This case-control study based on genotype-phenotype correlation included 69 patients (mean age, years: 50±13.16) diagnosed with HCM constituting the study group and 50 healthy individuals (mean age, years: 52±1.4) constituting the control group. DNA was extracted from peripheral blood and the genotyping of mutations was performed by real-time PCR technique and high resolution melting analysis. Associations between categoric variables were determined using chi-square tests. Differences between two groups were compared with unpaired Student's t-test for continuous variables. RESULTS: None of the patients in the HCM group were carrying the index mutations. One healthy individual was found to be heterozygous for the R663H mutation with mildly abnormal IVS and LVPW thickness. The allele frequency for R663H (G>A) mutation was found to be 0.01% in control group. CONCLUSION: We performed a mutational screening of 6 HCM-associated mutations in 69 Turkish HCM patients (not previously studied except R403Q). There was no significant difference in the prevalence of the mutations between the patients with HCM and the healthy controls (p>0.05).

Is there any association between the Ser326Cys polymorphism of the 8-oxoguanine glycosylase 1 (OGG1) gene and risk of colon polyp and abnormal glucose tolerance in acromegaly patients?

AIM: Evidence arising from experimental studies indicates an association between increased levels of the growth hormone/insulin-like growth factor 1 and oxidative stress. The association of the Ser326Cys polymorphism in the 8-oxoguanine glycosylase (OGG1) gene with a colon carcinoma and diabetes mellitus has been examined. The aim of the study was to compare the genotypic distribution of OGG1 Ser326Cys between acromegaly patients and nonacromegalic subjects and to explore whether this polymorphism is associated with a colon polyp risk and abnormal glucose tolerance. METHODS: We examined 98 acromegaly patients, and 99 healthy subjects who can be compared in terms of age and gender. All participants were evaluated by anthropometric and biochemical measurements. Also, a 75-g oral glucose test and colonoscopy was applied to the patients. Genomic DNA was isolated from peripheral blood leucocytes and the genotype was assessed by melting temperature analyses after using a real-time polymerase chain reaction protocol. RESULTS: Colon polyps were detected in 13 (30.2%) of 43 patients who underwent the colonoscopy. Except for diastolic blood pressure, clinical and biochemical characteristics were similar between the patients diagnosed with and without a colon polyp. A higher proportion of acromegaly patients had the Ser326Ser genotype when compared to the control group (p=0.007). Genotypes were similar between the patients with a normal glucose tolerance and an abnormal glucose tolerance (p=0.774). The frequency of the Cys allele was significantly higher in patients with polyps than those without a polyp (38.5% vs. 18.3%) (p=0.029). CONCLUSION: Our results suggest that the Cys allele may influence the colon polyp risk in acromegaly patients. Large-scale studies with acromegaly patients are required to show whether being a carrier of the Cys allele is associated with the risk of a colorectal polyp.

Role of 14-3-3σ in resistance to cisplatin in non-small cell lung cancer cells.

The efficacies of chemotherapeutic agents are often limited by side effects and acquired drug resistance. We have investigated whether the differential expression pattern of 14-3-3σ affects cisplatin response in non-small cell lung cancer cell lines. Two pairs of parental/cisplatin resistant cell lines (A549/CRA549 and Calu1/CR-Calu1) and clinical lung cancer biopsy samples were analysed for 14-3-3σ expression. Cell viability was assessed by WST assay; and 14-3-3σ expression was suppressed by siRNA transfection. 14-3-3σ mRNA expression increased in CR-A549 and CR-Calu1 compared with their cisplatin-sensitive parental A549 and Calu1 cell lines. But when 14-3-3σ expression was suppressed, elevated cisplatin response was seen in A549 and CR-Calu1 cell lines. Increased 14-3-3σ expression might also account for reduced cisplatin response in vivo, since, 14-3-3σ expression in clinical biopsy samples obtained from lung cancer patients undergoing cisplatin-based chemotherapy significantly higher in the non-responder compared with the responder group. We therefore propose that increased 14-3-3σ expression is correlated with cisplatin response in non-small cell lung cancer cells; monitoring its expression might become useful in the future in predicting poor outcome to cisplatin treatment and/or the verification of acquired cisplatin resistance in lung cancer patients.

Identification of Y chromosome microdeletions in infertile Turkish men.

OBJECTIVE: The aim of this study was to determine the frequencies of Y chromosome microdeletions in infertile azoospermic and oligozoospermic Turkish men and in healthy control subjects. MATERIAL AND METHODS: Sixty-four azoospermic and 51 oligozoospermic patients infertile patients, and 70 healthy men who had a child without the aid of assisted reproductive technologies were included in this study. DNA was extracted from peripheral blood samples collected from the patients. Following multiplex PCR performed with 15 different primer sequences, Y chromosome AZFa, AZFb, AZFc and AZFd region microdeletions were determined by agarose gel electrophoresis. RESULTS: Y chromosome microdeletions were detected in 8 (12.5%) patients in the azoospermia group and 3 (5.9%) patients in the oligozoospermia group. The overall frequency of Y chromosome microdeletions in all infertile cases was 9.6%. Y chromosome microdeletions were not found in the healthy control group. Among the infertile cases, there were 4 (3.48%) AZFa, 2 (1.74%) AZFb, 3 (2.61%) AZFc and 7 (6.09%) AZFd region microdeletions. Y chromosome microdeletions were not found among healthy men in the control group. CONCLUSION: The presence of Y chromosome microdeletions among azoospermic and oligozoospermic infertile males suggests that routine genetic testing and genetic counseling prior to the use of assisted reproduction techniques are necessary.

Acquired activated protein C resistance in sarcoma patients.

Acquired activated protein C resistance (aAPCR) is seen more frequently in solid and hematological cancer patients. We aimed to investigate the presence of aAPCR and the frequency of clinically detectable thrombosis in sarcoma patients. Normalized activated protein C sensitivity ratio (nAPCSR), factor V Leiden (FVL) mutation, factor V (FV) levels and factor VIII (FVIII) levels were prospectively measured in 52 patients and in 52 healthy controls. Clinically detectable thrombosis was present in one patient (1.92%). Compared with healthy controls (106%), the sarcoma patients had significantly lower values of the nAPCSR at pre (87.25%) and post (94.35%) treatment period (P < 0.0001). aAPCR was found as 4.2, 13 and 0%, respectively. The post-treatment FV levels (178.1 U/dl) were significantly (P < 0.001) higher than the pretreatment levels (147.5 U/dl). Inverse correlation was found between post-treatment FV levels and nAPCSR values (r = -0.38, P < 0.02). We found out a slightly increased frequency of venous thromboembolism in sarcoma patients. As an original finding which has not been reported previously in the literature, we also found out a decrease in the nAPCSR, persisting even after treatment. Thirdly, we found out that the significantly higher rate of aAPCR at the time of diagnosis totally disappeared after treatment.

Cisplatin resistance induced by decreased apoptotic activity in non-small-cell lung cancer cell lines.

We have investigated defective steps in apoptosis that might account for the development of resistance. For this purpose, A549 and Calu1 NSCLC (non-small-cell lung cancer) cell lines were treated with cisplatin to obtain resistant sub-lines. Gene expression profiles and the phosphorylation status of the BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) protein were determined for each cell line. Cell death and cytochrome c release were analysed after treating cell lines with their appropriate cisplatin doses. Gene expression of BAD, Bid, caspases 4 and 6 were clearly decreased in the resistant cell lines, and the differential phosphorylation status of BAD also seemed to play a role in the development of cisplatin resistance. Since this is a new cisplatin-resistant Calu1 cell line, it is noteworthy that DNA fragmentation, apoptotic cell ratio and cytochrome c levels were most decreased in the CR-Calu1 cell line.