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.

Roles of Cytokines in Pathological and Physiological Gastroesophageal Reflux Exposure.

Background/Aims: Gastroesophageal reflux disease is frequently observed and has no definitive treatment. There are 2 main views on the pathogenesis of gastroesophageal reflux disease. The first is that epithelial damage starts from the mucosa by acidic-peptic damage and the inflammatory response of granulocytes. The other view is that T-lymphocytes attract chemoattractants from the basal layer to the mucosa, and granulocytes do not migrate until damage occurs. We aim to investigate the inflammatory processes occurring in the esophageal epithelium of the phenotypes at the molecular level. We also examined the effects of these changes on tissue integrity. Methods: Patients with mild and severe erosive reflux, nonerosive reflux, reflux hypersensitivity, and functional heartburn were included. Inflammatory gene expressions (JAK/STAT Signaling and NFKappaB Primer Libraries), chemokine protein levels, and tissue integrity were examined in the esophageal biopsies. Results: There was chronic inflammation in the severe erosion group, the acute response was also triggered. In the mild erosion group, these 2 processes worked together, but homeostatic cytokines were also secreted. In nonerosive groups, T-lymphocytes were more dominant. In addition, the inflammatory response was highly triggered in the reflux hypersensitivity and functional heartburn groups, and it was associated with physiological reflux exposure and sensitivity. Conclusions: "Microinflammation" in physiological acid exposure groups indicate that even a mild trigger is sufficient for the initiation and progression of inflammatory activity. Additionally, the anti-inflammatory cytokines were highly increased. The results may have a potential role in the treatment of heartburn symptoms and healing of the mucosa.

Revisiting the Role of Esophageal Mucosal Dilated Intercellular Spaces in the Diagnosis and Pathophysiology of Heartburn.

Background/Aims: Dilated intercellular spaces (DISs) facilitate the diffusion of noxious agents into the deep layers of the esophageal epithelium. The role of DIS in heartburn pathogenesis is still controversial. Therefore, we aim to reinvestigate DIS in an extensively evaluated group of patients and healthy controls (HCs). Methods: We classified 149 subjects into the following groups: 15 HC, 58 mild erosive reflux disease (ERD), 17 severe ERD, 25 nonerosive reflux disease (NERD), 15 reflux hypersensitivity (RH), and 19 functional heartburn (FH). A total of 100 length measurements were performed for each patient's biopsy. Results: The overall intercellular spaces (ISs) value of gastroesophageal reflux disease (GERD) patients was higher than that of HC (P = 0.020). In phenotypes, mild ERD (vs HC [P = 0.036], NERD [P = 0.004], RH [P = 0.014]) and severe ERD (vs HC [P = 0.002], NERD [P < 0.001], RH [P = 0.001], FH [P = 0.004]) showed significantly higher IS. There was no significant difference between the HC, NERD, RH, and FH groups. The 1.12 µm DIS cutoff value had 63.5% sensitivity and 66.7% specificity in the diagnosis of GERD. There was a weak correlation (r = 0.302) between the IS value and acid exposure time, and a weak correlation (r = -0.359) between the IS value and baseline impedance. A strong correlation was shown between acid exposure time and baseline impedance (r = -0.783). Conclusions: Since the IS length measurement had better discrimination power only in erosive groups, it is not feasible to use in daily routine to discriminate other nonerosive phenotypes and FH. The role of DIS in heartburn in nonerosive patients should be reconsidered.

Epigenetic Alterations from Barrett's Esophagus to Esophageal Adenocarcinoma.

Barrett's esophagus (BE) is a disease entity that is a sequela of chronic gastroesophageal reflux disease that may result in esophageal adenocarcinoma (EAC) due to columnar epithelial dysplasia. The histological degree of dysplasia is the sole biomarker frequently utilized by clinicians. However, the cost of endoscopy and the fact that the degree of dysplasia does not progress in many patients with BE diminish the effectiveness of histological grading as a perfect biomarker. Multiple or more quantitative biomarkers are required by clinicians since early diagnosis is crucial in esophageal adenocancers, which have a high mortality rate. The presence of epigenetic factors in the early stages of this neoplastic transformation holds promise as a predictive biomarker. In this review, current studies on DNA methylations, histone modifications, and noncoding RNAs (miRNAs) that have been discovered during the progression from BE dysplasia to EAC were collated.

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.

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.

In Vitro and In Vivo Effects of Nonsteroidal Anti-inflammatory Drugs and Aspirin on Rabbit Esophageal Epithelium.

BACKGROUND: Gastroesophageal reflux disease has a high incidence of 23%, with 29% of those with gastroesophageal reflux disease consuming nonsteroidal anti-inflammatory drugs. There are insufficient data concerning the effects of nonsteroidal anti-inflammatory drugs on the esophageal tissue. We aimed to examine the effects of well-known nonsteroidal anti-inflammatory drugs using electrophysiologic criteria on the rabbit esophageal epithelium. METHODS: Esophageal epithelium mounted on Ussing chambers enabled in vitro investigation of the electrophysiological properties. Doses of 1 mg/mL, 2.5 mg/mL, 5 mg/mL ibuprofen, naproxen, and aspirin were dissolved in dimethyl sulfoxide and added to the luminal side. Esophagi were cannulated from both sides for the administration of high-dose ibuprofen in vivo, and the potential difference was monitored. RESULTS: Ibuprofen and aspirin inhibited tissue transport functions in a dose-dependent manner. pH 4 acid and 0.1 mg/mL ibuprofen alone were not harmful; however, the combination of these agents had an additive and significance effect: 78% decrease in the potential difference and 85% decrease in the short-circuited current (Isc). The change in the potential difference in the in vivo experiments (5 mg/mL ibuprofen) was similar (52 ± 7% decrease) with in vitro experiments in the first 30 minutes. CONCLUSION: Nonsteroidal anti-inflammatory drugs were harmful to the rabbit esophageal epithelium in both the in vitro and in vivo experiments. Even though aspirin and ibuprofen affected the transport mechanisms of the esophageal epithelium, the dose-dependent decrease of tissue potential difference and Isc with ibuprofen was more pronounced than those with aspirin. The combination of harmless doses of ibuprofen and acid demonstrated that even low acidic conditions can create a disruptive environment.

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.

Pepsin and pH of Gastric Juice in Patients With Gastrointestinal Reflux Disease and Subgroups.

GOAL: The aim of this study was to investigate the pepsin values and pH results of gastric juice among the subtypes of gastroesophageal reflux disease (GERD) and functional heartburn. BACKGROUND: The major destructive agents of GERD on the esophageal epithelium are gastric acid and pepsin. No precise information about pepsin concentration in gastric juice exists. STUDY: Ninety patients with GERD, 39 erosive reflux disease (ERD) Los Angeles (LA) grade A/B, 13 ERD LA grade C/D, 19 nonerosive reflux disease (NERD), 8 esophageal hypersensitivity, 11 functional heartburn, and 24 healthy controls were included in the study. During endoscopy gastric juices from the patients were aspirated and their pH readings immediately recorded. Gastric juice samples were analyzed using Peptest, a lateral flow device containing 2 unique human monoclonal antibodies to detect any pepsin present in the gastric juice sample. RESULTS: The highest mean gastric pepsin concentration (0.865 mg/mL) and the lowest median gastric pH (1.4) was observed in the LA grade C/D group compared with the lowest mean gastric pepsin concentration (0.576 mg/mL) and the highest median gastric pH (2.5) seen in the NERD group. Comparing pH, the NERD patient group was significantly higher (P=0.0018 to P=0.0233) when compared with all other GERD patient groups. CONCLUSIONS: The basal gastric pepsin level in the healthy control group was comparable to literature values. There was good correlation and a significant linear relationship between the gastric pepsin level and gastric pH within the patient groups. The severity of the GERD disease is related to the lowest pH and the highest pepsin concentration in gastric juice.

The Evaluation of Effect of Aurora Kinase Inhibitor CCT137690 in Melanoma and Melanoma Cancer Stem Cell.

BACKGROUND: Dysregulation of the cell cycle is one of the main causes of melanomagenesis. Genomewide studies showed that the expression of Aurora -A and -B significantly has been upregulated in melanoma. However, there is no FDA approved drug targeting aurora kinases in the treatment of melanoma. In addition, the development of resistance to chemotherapeutic agents in the treatment of melanoma and, as a result, the relapse due to heterogeneous cell groups in patients is a second phenomenon that causes treatment failure. Therefore, there is an urgent need for therapeutic alternatives targeting both melanoma and Melanoma Cancer Stem Cells (MCSCs) in treatments. At this stage, cell cycle regulators become promising targets. OBJECTIVE: In this study, we aimed to identify the effects of Aurora kinase inhibitor CCT137690 on the cytotoxicity, apoptosis, cell cycle, migration, and colony formation and expression changes of genes related to proliferation, cell death and cell cycle in melanoma and melanoma cancer stem cell. In addition, we investigated the apoptotic and cytostatic effects of CCT137690 in normal fibroblast cells. METHODS: We evaluated the cytotoxic effect of CCT137690 in MCSCs, NM2C5 referring as melanoma model cells and WI-38 cells by using the WST-1 test. The effect of CCT137690 on apoptosis was detected via Annexin V and JC-1 method; on cell cycle progression by cell cycle test; on gene expression by using RT-PCR, on migration activity by wound healing assay and clonal growth by clonogenic assay in NM2C5 cells and MCSCs. The effects of CCT137690 in WI-38, referring as healthy fibroblast cell, were assessed through Annexin V and cell cycle method. RESULTS: CCT137690 was determined to have a cytotoxic and apoptotic effect in MCSCs and melanoma. It caused polyploidy and cell cycle arrest at the G2/M phase in MCSCs and melanoma cells. The significant decrease in the expression of MMP2, MMP7, MMP10, CCNB1, IRAK1, PLK2 genes, and the increase in the expression of PTEN, CASP7, p53 genes were detected. CONCLUSION: Aurora kinases inhibitor CCT137690 displays promising anticancer activity in melanoma and especially melanoma cancer stem cells. The effect of CCT137690 on melanoma and MCSC may provide a new approach to treatment protocols.

Effect of Dasatinib on Genes Related to Mitotic Catastrophe Pathway in Chronic Myeloid Leukemia Cells.

BACKGROUND: Chronic Myeloid Leukemia (CML) is characterized by a reciprocal translocation t(9;22) and forms BCR/ABL1 fusion gene called the Philadelphia chromosome. The therapeutic targets for CML patients mediated with BCR/ABL1 oncogenic are tyrosine kinase inhibitors such as imatinib, dasatinib, and nilotinib. The latter two of which have been approved for the treatment of imatinib-resistant or intolerance CML patients. Mitotic Catastrophe (MC) is one of the non-apoptotic mechanisms initiated in types of cancer cells in response to anti-cancer therapies. Pharmacological inhibitors of G2 checkpoint members or genetic suppression of PLK1, PLK2, ATR, ATM, CHK1, and CHK2 can trigger DNA-damage-stimulated mitotic catastrophe. PLK1 and AURKA/B are anomalously expressed in CML cells, where phosphorylation and activation of PLK1 occur by AURKB at centromeres and kinetochores. OBJECTIVE: The purpose of this study is to investigate the effect of dasatinib on the expression of genes in MC and apoptosis pathways in K562 cells. METHODS: Total RNA was isolated from K-562 cells treated with the IC50 value of dasatinib and untreated cells as a control group. The expression of MC and apoptosis-related genes, was analyzed by the qRT-PCR system. RESULTS: The array-data demonstrated that dasatinib-treated K562 cells significantly caused the decrease of several genes (AURKA, AURKB, PLK, CHEK1, MYC, XPC, BCL2, and XRCC2). CONCLUSION: The evidence supplies a basis to support clinical researches for the suppression of oncogenes such as PLKs with AURKs in the treatment of types of cancer, especially chronic myeloid leukemia.

Revealing genome-wide mRNA and microRNA expression patterns in leukemic cells highlighted "hsa-miR-2278" as a tumor suppressor for regain of chemotherapeutic imatinib response due to targeting STAT5A.

BCR-ABL oncoprotein stimulates cell proliferation and inhibits apoptosis in chronic myeloid leukemia (CML). For cure, imatinib is a widely used tyrosine kinase inhibitor, but developing chemotherapeutic resistance has to be overcome. In this study, we aimed to determine differing genome-wide microRNA (miRNA) and messenger RNA (mRNA) expression profiles in imatinib resistant (K562/IMA-3 µM) and parental cells by targeting STAT5A via small interfering RNA (siRNA) applications. After determining possible therapeutic miRNAs, we aimed to check their effects upon cell viability and proliferation, apoptosis, and find a possible miRNA::mRNA interaction to discover the molecular basis of imatinib resistance. We detected that miR-2278 and miR-1245b-3p were most significantly regulated miRNAs according to miRNome array. Upregulating miR-2278 expression resulted in the inhibition of resistant leukemic cell proliferation and induced apoptosis, whereas miR-1245b-3p did not exhibit therapeutic results. Functional analyses indicated that AKT2, STAM2, and STAT5A mRNAs were functional targets for miR-2278 as mimic transfection decreased their expressions both at transcriptional and translational level, thus highlighting miR-2278 as a tumor suppressor. This study provides new insights in discovering the mechanism of imatinib resistance due to upregulating the tumor-suppressor hsa-miR-2278 which stands for a functional therapeutic approach, inhibited leukemic cell proliferation, induced apoptosis, and regain of chemotherapeutic drug response in CML therapy.