Inflammatory responses in esophageal mucosa before and after laparoscopic antireflux surgery.

BACKGROUND: Currently, the primary treatment for gastroesophageal reflux is acid suppression with proton pump inhibitors, but they are not a cure, and some patients don't respond well or refuse long-term use. Therefore, alternative therapies are needed to understand the disease and develop better treatments. Laparoscopic anti-reflux surgery (LARS) can resolve symptoms of these patients and plays a significant role in evaluating esophageal healing after preventing harmful effects. Successful LARS improves typical gastroesophageal reflux symptoms in most patients, mainly by reducing the exposure time to gastric contents in the esophagus. Amelioration of the inflammatory response and a recovery response in the esophageal epithelium is expected following the cessation of the noxious attack. AIM: To explore the role of inflammatory biomolecules in LARS and assess the time required for esophageal epithelial recovery. METHODS: Of 22 patients with LARS (pre- and post/5.8 ± 3.8 months after LARS) and 25 healthy controls (HCs) were included. All subjects underwent 24-h multichannel intraluminal impedance-pH monitoring and upper gastrointestinal endoscopy, during which esophageal biopsy samples were collected using endoscopic techniques. Inflammatory molecules in esophageal biopsies were investigated by reverse transcription-polymerase chain reaction and multiplex-enzyme-linked immunosorbent assay. RESULTS: Post-LARS samples showed significant increases in proinflammatory cytokines [interleukin (IL)-1ß, interferon-γ, C-X-C chemokine ligand 2 (CXCL2)], anti-inflammatory cytokines [CC chemokine ligand (CCL) 11, CCL13, CCL17, CCL26, CCL1, CCL7, CCL8, CCL24, IL-4, IL-10], and homeostatic cytokines (CCL27, CCL20, CCL19, CCL23, CCL25, CXCL12, migration inhibitory factor) compared to both HCs and pre-LARS samples. CCL17 and CCL21 levels were higher in pre-LARS than in HCs (P < 0.05). The mRNA expression levels of AKT1, fibroblast growth factor 2, HRAS, and mitogen-activated protein kinase 4 were significantly decreased post-LARS vs pre-LARS. CCL2 and epidermal growth factor gene levels were significantly increased in the pre-LARS compared to the HCs (P < 0.05). CONCLUSION: The presence of proinflammatory proteins post-LARS suggests ongoing inflammation in the epithelium. Elevated homeostatic cytokine levels indicate cell balance is maintained for about 6 months after LARS. The anti-inflammatory response post-LARS shows suppression of inflammatory damage and ongoing postoperative recovery.

Investigating the potential therapeutic role of targeting STAT3 for overcoming drug resistance by regulating energy metabolism in chronic myeloid leukemia cells.

Objectives: STATs are one of the initial targets of emerging anti-cancer agents due to their regulatory roles in survival, apoptosis, drug response, and cellular metabolism in CML. Aberrant STAT3 activity promotes malignancy, and acts as a metabolic switcher in cancer cell metabolism, contributing to resistance to TKI nilotinib. To investigate the possible therapeutic effects of targeting STAT3 to overcome nilotinib resistance by evaluating various cellular responses in both sensitive and nilotinib resistant CML cells and to test the hypothesis that energy metabolism modulation could be a mechanism for re-sensitization to nilotinib in resistant cells. Materials and Methods: By using RNAi-mediated STAT3 gene silencing, cell viability and proliferation assays, apoptotic analysis, expressional regulations of STAT mRNA transcripts, STAT3 total, pTyr705, pSer727 protein expression levels, and metabolic activity as energy metabolism was determined in CML model K562 cells, in vitro. Results: Targeting STAT3 sensitized both parental and especially nilotinib resistant cells by decreasing leukemic cell survival; inducing leukemic cell apoptosis, and decreasing STAT3 mRNA and protein expression levels. Besides, cell energy phenotype was modulated by switching energy metabolism from aerobic glycolysis to mitochondrial respiration in resistant cells. RNAi-mediated STAT3 silencing accelerated the sensitization of leukemia cells to nilotinib treatment, and STAT3-dependent energy metabolism regulation could be another underlying mechanism for regaining nilotinib response. Conclusion: Targeting STAT3 is an efficient strategy for improving the development of novel CML therapeutics for regaining nilotinib response, and re-sensitization of resistant cells could be mediated by induced apoptosis and regulation in energy metabolism.

The Risk Factors and Clinical Features of Acanthamoeba Keratitis: First Time Detection of Acanthamoeba T5 Genotype from Keratitis Patients in Turkey.

PURPOSE: The primary aim of this study is to investigate Acanthamoeba in clinical samples of keratitis cases (n = 60), in contact lens (CL) and lens care solutions of asymptomatic CL users (n = 41), and to identify the genotypes in positive samples. The secondary aim is to assess the risk factors and clinical features of Acanthamoeba keratitis (AK) patients. METHODS: All samples from patients and asymptomatic CL users were examined by microscopy and inoculated in non-nutrient agar plates. PCR was performed using the DNA isolated from corneal scrapings, CL and lens care solution samples. Positive DNA samples were sequenced to determine the genotype of Acanthamoeba. RESULTS: In none of the samples, Acanthamoeba was identified by microscopy, while Acanthamoeba was detected in a patient with keratitis by culture method. However, Acanthamoeba was detected in 11.66% (7/60) of the keratitis patients by PCR. The genotypes of these isolates detected by sequencing were T4 (4), and T5 (3). Acanthamoeba was detected in none of the samples of asymptomatic CL users by any of the three methods. CONCLUSION: To best of our knowledge, this is the first study to detect T5 genotype in AK patients from Turkey. In addition, the CL use was found to be an important risk factor for AK.

Genotyping and Molecular Identification of Acanthamoeba Genotype T4 and Naegleria fowleri from Cerebrospinal Fluid Samples of Patients in Turkey: Is it the Pathogens of Unknown Causes of Death?

PURPOSE: This study was aimed to investigate the presence of pathogenic free-living amoebae (FLA) in suspected cases of meningoencephalitis with unknown causes of death in Turkey. METHOD: A total of 92 patients, who were diagnosed as meningoencephalitis, were enrolled. All cerebrospinal fluid (CSF) samples were directly microscopically examined and cultured. Acanthamoeba, N. fowleri and B. mandrillaris were further investigated using molecular diagnostic tools including real-time PCR, sequencing, and phylogenetic analyses. RESULTS: The examined CSF samples were not found positive for the presence of FLA by microscopic examination and culture method. However, two CSF samples were detected positive by real-time PCR assay. Of the positive CSF samples, one was identified as Acanthamoeba genotype T4 and the second positive sample was identified as N. fowleri belonging to genotype II. Furthermore, the pathogens diagnoses was verified through Sanger sequencing. CONCLUSION: This study was significant to report the presence of Acanthamoeba genotype T4 and N. fowleri genotype II in CSF samples by real-time PCR assay. The present study shows the significance of primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE) as one of the differential diagnoses to be considered by clinicians during the evaluation of suspected meningoencephalitis or cases of unknown cause in Turkey. Using real-time PCR, this has made the rapid detection, in a short time-frame, of Acanthamoeba and N. fowleri in CSF samples from patients. The problems with qPCR is that it is not available in every laboratory, reagents are expensive, and it requires skilled and expert personnel to set up these assays.

Targeting UPR signaling pathway by dasatinib as a promising therapeutic approach in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease that mediated by BCR/ABL oncogenic signaling. CML can be targeted with the imatinib, dasatinib, and nilotinib TKI inhibitors, the latter two of them have been approved for imatinib-resistant or -intolerant CML patients. The TKIs resistance occurs by different molecular mechanisms, including overexpression of BCR-ABL, mutations in the TKI binding site of BCR/ABL, and ER-stress. Unfolded protein responses (UPR) is a cytoprotective mechanism which is activated by ER-stress. The IRE1, PERK, and ATF6 are three main arms of the UPR mechanism and are activated by a common mechanism involving the dissociation of the ER-chaperone BiP/GP78. There is a correlation between ER-stress, CML progression, and response to TKI treatment. In the present study, we aimed to determine alterations of the expression levels of genes related to UPR pathway signaling after treatment with dasatinib in K562 chronic myeloid leukemia cell line by quantitative RT-PCR relatively. The array-data revealed that treatment with dasatinib significantly decreased the UPR mechanism-related genes (including HSPA1B, HSPA2, HSPA4L, ATF6, ATF6B, CEBPB, PERK, TRIB3, DNAJB, ERN1, and UHRF1) in K562 cells. In conclusion, the results showed that dasatinib regulates the UPR mechanism that plays a significant role in cancer progression and therapy resistance in CML. Thus, dasatinib-induced dysfunction of the UPR mechanism may promise encouraging therapy for CML.

The effects of Epigallocatechin-3-gallate and Dabrafenib combination on apoptosis and the genes involved in epigenetic events in anaplastic thyroid cancer cells.

Anaplastic thyroid cancer cases with poor prognosis are associated with epigenetic modifications such as abnormal DNA methylation. Epigallocathesin-3-gallate (EGCG) is a polyphenol compound of green tea that is still under investigation on its role in cancer prevention. EGCG is known as an epigenetic diet in DNA methyltransferase inhibitor. The cytotoxic effects of Dabrafenib, EGCG, and dabrafenib in combination with EGCG were assessed by using WST-8 assay; and also, Flow cytometry was utilized to identify cells undergoing apoptosis after treatments of the SW-1736 cells. We investigated the mRNA expression of genes involved in epigenetic events in SW-1736 cells by real-time qRT-PCR following the treatments. We demonstrated for the first time that the Dabrafenib-EGCG combination reduced cell viability significantly depending on concentration and induced apoptosis by 8.49-fold through investigating the additive effect together on SW-1736 cells. The IC50 doses of Dabrafenib and EGCG for 48 h were determined as 6.7 µM and 22.5 µM, respectively. The results of qRT-PCR demonstrated that the Dabrafenib-EGCG combination significantly caused the down-regulation of genes involved in epigenetic regulation. We suggest that the combination of Dabrafenib and EGCG following in vivo phase studies will contribute as an alternative treatment option for the treatment of ATC.

Functional roles of miR-625-5p and miR-874-3p in the progression of castration resistant prostate cancer.

AIMS: Androgen receptor (AR) signaling is important in normal prostate and prostate tumor tissues. Thus, the new therapeutic strategies targeting ARs may also be important for treatment of prostate cancer (PC) and its biology. The studies have shown that miRNAs to be dysregulated in PC progression. Therefore, in the present study, differentially expressed miRNAs that predictively target the ARs were identified and investigated by in silico analysis. MAIN METHODS: Cellular proliferation, qPCR, western blot and apoptosis assays were performed to investigate the molecular mechanism of the selected miRNAs in the PC cells. KEY FINDINGS: In our miRNA qPCR study, several miRNAs were found to be differentially regulated in castration resistant prostate cancer (CRPC) cells (LNCaP-Abl and LNCaP-104R2) compared with androgen dependent (AD) cells (LNCaP). The expression levels of miR-625-5p and miR-874-3p were significantly increased in LNCaP-Abl (2.62-fold, p = 0.0002; 4.00-fold, p = 0.00002, respectively) and LNCaP-104R2 (2.44-fold, p = 0.0455; 3.77-fold, p = 0.0383, respectively) compared with AD cells. The expression levels of AR and prostate specific antigen were increased in PC cells compared with AD cells. Furthermore, transfection of PC cells with anti-miRs suppressed their proliferation and AR protein levels (p < 0.05). SIGNIFICANCE: Several differentially regulated miRNAs were identified in CRPC cells, including miR-625-5p and miR-874-3p that are potentially involved in PC progression. These results may provide novel insights into the molecular mechanism underlying CRPC cells and miRNA applications may constitute a new and alternative method to prevent development of CRPC cells in the future.

Combination of dasatinib and okadaic acid induces apoptosis and cell cycle arrest by targeting protein phosphatase PP2A in chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is a cancer type of the white blood cells and because of BCR-ABL translocation it results in increased tyrosine kinase activity. For this purpose, dasatinib is the second-generation tyrosine kinase inhibitor that is used for inhibition of BCR-ABL. Effectively and safetly, dasatinib has been used for imatinib-intolerant/resistant CML patients. Protein phosphatase 2A (PP2A) is the major serine/threonine phosphatase ensuring cellular homeostasis in cells and is associated with many cancer types including leukemias. In this study, we aimed to investigate the effects of dasatinib and okadaic acid (OA), either alone or in combination, on apoptosis and cell cycle arrest and dasatinib effect on enzyme activity and protein-level changes of PP2A in K562 cell line. The cytotoxic effects of dasatinib were evaluated by WST-1 analysis. Apoptosis was determined by Annexin V and Apo-Direct assays by flow cytometry. Cell cycle arrest analysis was performed for the investigation of the cytostatic effect. We also used OA as a PP2A inhibitor to assess apoptosis and cell cycle arrest changes in case of reducing the level of PP2A. PP2A enyzme activity and protein levels of PP2A were examined by serine/threonine phosphatase assay and Western blot analysis, respectively. Apoptosis was increased with dasatinib and OA combination. Cell cycle arrest was determined especially after OA treatment. The enzyme activity was decreased depending on time after dasatinib application. PP2A regulatory and catalytic subunit protein levels were decreased compared to control. Targeting the PP2A by dasatinib and OA has potential for CML treatment.

Preparation, characterization and in vitro evaluation of cisplatin-bound triblock polymeric micelle solution for ovarian cancer treatment.

The main objective of this study was to prepare cisplatin (CDDP) bound triblock polymeric micelle solution which will have a hydrophilic shell not being phagocytosed by mononuclear phagocyte system, and evaluate in vitro behavior for the treatment of ovarian cancer. For this aim, CDDP was bound to polyglutamic acid (PGA) and the triblock polymer was prepared using polyethylene glycol)-polylactide-co-glycolide (PEG-PLGA). CDDP-bound triblock copolymer conjugation was characterized, in vitro release and permeability studies were performed using USP II method and Caco-2 cell lines, respectively. The release of CDDP from CDDP-bound triblock polymeric micelle solution was found 87.3 ± 3.56% at the end of the 24th hour. CDDP bound triblock polymeric micelle solution was detected as biocompatible, and permeable according to in vitro studies. According to the MTT results, the measured cytotoxicity was found to be maximum in CDDP-bound triblock polymeric micelle solution when compared with CDDP solution and conjugate in SKOV-3 and OVCAR-3 cells, whereas annexin V-FITC apoptosis results were found to be maximum in A2780 cells.

Brain-targeted, drug-loaded solid lipid nanoparticles against glioblastoma cells in culture.

The aim of this study was the preparation of solid lipid nanoparticles (SLN) formed from cetyl palmitate with having targeting molecules for monocarboxylate transporter-1 (MCT-1): ß-hydroxybutyric acid and anticancer agents: carmustine (BCNU) and temozolomide (TMZ) for enhanced anti-proliferation against glioblastoma multiforme (GBM). Properties including size, morphology, chemical structure, zeta potential, drug encapsulation efficacy, drug release, biocompatibility, stability were determined, and in vitro studies were done. BCNU and TMZ loaded SLNs had a hydrodynamic size of 227 nm ± 46 a zeta potential of -25 mV ± 4 with biocompatible features. The data showed rapid drug release at first and then continuous release. Nanoparticles could be stored for nine months. BCNU and TMZ loaded SLNs exhibited a remarkable increment in the antitumor activity compared to the free-drugs and induced apoptosis on U87MG cells. In addition, targeted nanoparticles were more uptaken by MCT-1 expressing brain cells. This study indicated that BCNU and TMZ loaded SLNs could act as a useful anticancer system for targeted GBM therapy.

The investigation of T-cell receptor subtypes in ovarian cancer: effects on survival and prognostic factors.

The aim of this study was to investigate T-cell receptor (TCR) changes in ovarian carcinoma (OC). The study included 24 malignant and 23 benign adnexal masses. DNA was isolated from ovarian samples. Multiplex PCR was used to determine T-cell gene clonality. PCR products were loaded onto polyacrylamide gel electrophoresis and imaged. The relationship between prognostic parameters and T-cell rearrangement was evaluated. In the study group (SG), TCRB-B positivity was higher than control group (CG) and TCRD receptor positivity was higher in CG. In SG, TCRG-B levels were higher in patients with stage I-II tumours compared to stage III. TCRG-B receptor was higher in patients with overall survival of 36 months and above. In our study, subgroups of TCRs were analysed in OC. According to our findings, significant differences in TCRB-B and TCRD subgroups may be applied to immune therapies. Understanding of TCR pathways will provide new treatment approaches in OC.IMPACT STATEMENTWhat is already known on this subject? Ovarian cancer is the most challenging kind of gynaecologic cancer. Although the main route of spread is direct invasion and peritoneal spread in the abdominal cavity, lymphatic invasion is also very important. Recent studies put forward that immunological mechanisms play crucial role in ovarian cancer. CD3 and CD8 positive lymphocyte infiltration in ovarian tumour is related with better prognosis where, FoxP3 positive lymphocyte infiltration is a predictor of poor survival. Also, immune checkpoints and inhibitors are important topics in ovarian cancer.What the results of this study add? Despite all the improvements and studies regarding immune system and ovarian cancer, the role T-cell receptor (TCR) subtypes is not clear and there are very few number of studies in this area. This is one of the first studies that describe the rearrangement of TCR subtypes between normal ovarian tissue and ovarian cancer tissue.What the implications are of these findings for clinical practice and/or further research? Understanding the role of TCR subtypes has a key role because studies about lymphocyte infiltration in ovarian cancer varies from region to region in the world and same type of lymphocytes has different effects in different studies. Further studies on TCR subtypes may elucidate us about the behaviour of lymphocytes. Additionally, these receptor may be targeted if their roles are better understood.

Assessing Anticancer Potential of Blueberry Flavonoids, Quercetin, Kaempferol, and Gentisic Acid, Through Oxidative Stress and Apoptosis Parameters on HCT-116 Cells.

In recent years, natural products gained popularity with their anti-inflammatory and antioxidant effects mediated by chemical compounds within their composition. Study results offering them as palliative therapy options in cancer or as anticancer agents with high levels of cytotoxicity brought a new approach to combine cancer treatment protocols with these products. From a different perspective, edible types of these products are suggested in daily diets due to their potential cancer preventive effects. Our preliminary work was on blueberry extracts (Vaccinium myrtillus) as a main representative of these natural products, and the contents of the extracts were analyzed with liquid chromatography tandem mass spectrometry (LC MS/MS) to reveal the composition and distribution of polyphenolic compounds within. The most abundant polyphenols detected in V. myrtillus extracts were quercetin, kaempferol, and a phenolic acid, gentisic acid (GA). The compounds were further evaluated on treated HCT-116 cells for their potential anticancer effects by measuring total antioxidant status, total oxidant status, and 8-hydroxydeoxyguanosine levels for evaluation of oxidative stress and through protein array analysis and flow cytometric analysis for evaluation of apoptosis. In analysis of oxidative stress parameters, reduced total oxidant levels and reduced oxidative stress index levels were found in cells treated with the compounds in comparison with untreated cells. In apoptosis-related protein profiles, at least twofold reduction in various apoptotic proteins was observed after quercetin and kaempferol treatment, whereas a different profile was observed for GA. Overall, results of this study showed that quercetin and kaempferol have strong cytotoxic, antioxidant, and apoptotic effects, although GA is mostly effective as an antioxidant polyphenol on HCT-116 cells.

Synergistic effect of ponatinib and epigallocatechin-3-gallate induces apoptosis in chronic myeloid leukemia cells through altering expressions of cell cycle regulatory genes.

PURPOSE: Ponatinib (P) has been used for the treatment of chronic myeloid leukemia (CML) and it is known that inhibition of BCR-ABL fusion protein by ponatinib induces apoptosis of CML cells. Epigallocatechin-3-gallate (EGCG), which is a polyphenol in green tea, induces apoptosis in different types of cancer cells. The purpose of this study was to determine the cytotoxic and apoptotic effects of ponatinib and EGCG combination in K562 CML cell line. This study also aimed to detect alterations of the expression levels of cell cycle-regulation related genes after ponatinib and EGCG combination in K562 CML cell line. METHODS: The cytotoxic effects of the compounds on K562 cells were determined in a time-and dose-dependent manner by using WST-1 analysis. The combination index (CI) isobologram was used to analyze the data. Apoptotic effects of P-EGCG were defined by flow cytometry and gene expressions were detected by RT-qPCR. RESULTS: IC50values of ponatinib and EGCG were 87.13 nM and 50µM, respectively. CI value of the P-EGCG was 0.658 and the combination showed synergistic effect (ED90 value: 28.39 nM ponatinib, 117.12 µg/ml EGCG). Ponatinib, EGCG and P-EGCG induced apoptosis compared to control cells. CyclinD1 and CDC25A were downregulated by P-EGCG by 2.49 and 2.63-fold, respectively. TGF-ß2 was upregulated by 4.57-fold. CONCLUSION: EGCG possesses cytotoxic and apoptotic properties and may cooperate with the growth inhibiting activity of ponatinib synergistically against CML cells. P-EGCG mediated apoptosis might be associated with upregulation of TGF-ß2 gene and downregulation of cyclinD1 and CDC25A genes.