An evaluation the effect of glycyrrhetinic and glycyrrhizic acids derived from licorice extract on gastric cancer cell lines

BACKGROUND AND OBJECTIVE: Gastric cancer is the second most prevalent carcinogenic disease and surgery, chemotherapy and radiation are its principal treatment modalities. However, in most cases, poor response to treatment and adverse side effects are observed regarding these modalities. Given the lack of response to treatment and growing rates of gastric cancer, researchers are trying to come up with more efficient treatments with fewer side effects. In the traditional medicine, licorice has been suggested as a cancer treatment considering its high antioxidant properties and few side effects. This study aimed to evaluate the effect of licorice extract on gastric cancer cell lines. METHODS: In this experimental study, adenocarcinoma gastric cell lines were prepared from cell bank and were cultured. After passage, the cells were transferred into a 96-well plate. In each well, approximately 2,000 cells in RPMI-1640 culture medium with FBS (10%) were placed. The cells were repeatedly exposed to different concentrations of Glycyrrhetinic acid (0, 1, 10 and100.1 μM) and Glycyrrhizic acid (10, 1, 100 and 0.1 μM) for 24 and 48 hours. Finally, the obtained results of the experimental and control groups were compared with each other. FINDINGS: According to our results, the toxic effect of Glycyrrhetinic and Glycyrrhizic acids is dose and time dependent. In 24 hours, the mean optical density (MOD) in 100 μM concentration of Glycyrrhetinic acid was 0.41±0.02 and 0.79±0.04 in the experimental and control groups, respectively (p=0.0002). After 48 hours, MOD was 0.16±0.004 and 1.749±0.24 in the experimental and control groups, respectively (p=0.0003). Moreover, the MOD of 100 μM concentration of Glycyrrhizic acid was 0.78±0.53 and 2.09±0.49 in the experimental and control groups in 48 hours. There was a significant difference between the experimental and control groups (p=0.035). CONCLUSION: The results of this study demonstrated that the licorice compounds have a toxic effect on carcinogenic cells. Therefore, it is recommended to perform more study on both Glycyrrhizic and Glycyrrhizic acids as effective compounds on gastric cancer treatmen

Flow Cytometric Analysis of Inflammatory Cells in Experimental Acute Pancreatitis

 Background: Accumulating evidence indicates that inflammatory cells migrate into the pancreas tissue and play an important role in the pathogenesis of acute pancreatitis (AP). The aim of this study was to establish a flow cytometric method to enumerate these infiltrating cells in the pancreas of an experimental AP.Materials and Methods: Twelve hours after inducing of AP, mice pancreatic tissues were cut into small fragments and single cells were prepared by mechanical dissociation. The isolated cells were stained with either anti-mouse CD45-PerCP or isotype antibody and analyzed by flow cytometry. Using side scatter (SSC)/CD45 gating we were able to identify inflammatory cells from non-inflammatory cells.Results: The mean percentage of leukocytes was 5.9±1.6 in the control group whereas, it was 26.7±8.1 in the AP. Moreover, we found that the percentage of lymphocytes, monocytes and granulocytes were 1.1±0.2, 0.9±.04 and 2.9±1.8 of total pancreatic cells, respectively, in the control mice. In contrast to lymphocytes, the percentage of monocytes and granulocytes were significantly increased in the AP group and it was 3±1.3 and 18.2±3.2 for monocytes and granulocytes, respectively.Conclusion: Quantitative flow cytometric analysis is feasible and provides a reliable and rapid assay to determine the number and percentage of inflammatory cells in experimental AP

Hydrogen Peroxide Preconditioning Promotes Protective Effects of Umbilical Cord Vein Mesenchymal Stem Cells in Experimental Pulmonary Fibrosis

Purpose Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder with few available treatments. Mesenchymal stem cell therapy (MSCT), an innovative approach, has high therapeutic potential when used to treat IPF. According to recent data, preconditioning of MSCs can improve their therapeutic effects. Our research focuses on investigating the anti-inflammatory and antifibrotic effects of H2O2-preconditioned MSCs (p-MSCs) on mice with bleomycin-induced pulmonary fibrosis (PF). Methods Eight-week-old male C57BL/6 mice were induced with PF by intratracheal (IT) instillation of bleomycin (4 U/kg). Human umbilical cord vein-derived MSCs (hUCV-MSCs) were isolated and exposed to a sub-lethal concentration (15 pM for 24 h) of H2O2 in vitro. One week following the injection of bleomycin, MSCs or p-MSCs were injected (IT) into the experimental PF. The survival rate and weight of mice were recorded, and 14 days after MSCs injection, all mice were sacrificed. Lung tissue was removed from these mice to examine the myeloperoxidase (MPO) activity, histopathological changes (hematoxylin-eosin and Masson\u27s trichrome) and expression of transforming growth factor beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) through immunohistochemistry (IHC) staining. Results Compared to the PF+MSC group, p-MSCs transplantation results in significantly decreased connective tissue () and collagen deposition. Additionally, it is determined that lung tissue in the PF+pMSC group has increased alveolar space () and diminished expression of TGF-β1 and α-SMA. Conclusion The results demonstrate that MSCT using p-MSCs decreases inflammatory and fibrotic factors in bleomycin-induced PF, while also able to increase the therapeutic potency of MSCT in IPF

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Glycyrrhetinic Acid Inhibits Cell Growth and Induces Apoptosis in Ovarian Cancer A2780 Cells

Purpose: Accumulating evidence indicates that glycyrrhizin (GZ) and its hydrolyzed metabolite 18-β glycyrrhetinic acid (GA) exhibit anti-inflammatory and anticancer activities. The objective of this study was to examine the in vitro cytotoxic activity of GA on human ovarian cancer A2780 cells. Methods: A2780 cells were cultured in RPMI1640 containing 10% fetal bovine serum. Cells were treated with different doses of GA and cell viability and proliferation were detected by dye exclusion and 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays. Apoptosis induction and expression of Fas and Fas ligand (FasL) were analyzed by flow cytometry. Results: We observed that GA decreases cell viability and suppressed cells proliferation in a dose-dependent manner as detected by dye-exclusion and XTT assayes. In addition, our flow cytometry data show that GA not only induces apoptosis in A2780 cells but also upregulates both Fas and FasL on these cells in a dose-dependent manner. Conclusion: we demonstrate that GA causes cell death in A2780 cells by inducing apoptosis

Impaired immunomodulatory ability of type 2 diabetic adipose-derived mesenchymal stem cells in regulation of inflammatory condition in mixed leukocyte reaction

The immunomodulatory properties of type 2 diabetic patients’ adipose-derived mesenchymal stem cells (D-ASCs) has not been extensively studied. In this study, we compared the immunomodulatory properties of D-ASCs and non-diabetic subjects mesenchymal stem cells (ND-ASCs) in co-culture with mixed leukocyte reaction (MLR). ASCs were isolated from adipose tissue samples of type 2 diabetic and non-diabetic subjects (age: 40-55). D-ASCs and ND-ASCs were co-cultured with two-way MLR. Peri pheral blood mononuclear ce lls (PBMCs) proliferation ratio, protein levels of IFN- γ and IL-10, mRNA expression of COX-2 , TNF- α , TGF- β 1 and IL-6 genes in MLR, D-ASCs and ND-ASCs co-cultures were assessed using XTT, ELISA and Real-time qRT-PCR, respectively. PBMCs proliferation on days 2 and 4 of D-ASCs co-culture was higher than ND-ASCs co-culture of the same days ( p < 0.001). IFN- γ level decreased on day 4 compared to day 2 of ND-ASCs co-culture, but its level had not changed in D-ASCs co-culture. COX-2 expression on days 2 and 4 of D-ASCs co-culture was lower than ND- ASCs co-culture of the same days ( p < 0.05). The expression of TNF- α and IL-6 on days 2 and 4 of D-ASCs co- culture were higher than ND-ASCs co-culture of the same days ( p < 0.001). TGF- β 1 on day 4 of ND-ASCs co- culture showed a slightly higher expression than D-ASCs co-culture of the same day. Lower suppression of PBMCs proliferation, declined expre ssion of anti-inflammatory and upregulated expression of pro-inflammatory factors in D-ASCs co-culture, indicated an impairment of these cells in modulation of the inflammatory condition

The chemokine receptor CXCR4 is associated with the staging of gastric cancer

Background: CXCR4 is a cognitive receptor for stromal-derived factor-1 (SDF-1) and has been previously shown to be associated with tumor growth and invasion of many cancers. However, its expression and function in gastric cancer has not been well clarified. Materials and Methods: Herein, we studied the expression of CXCR4 on gastric samples from patients with gastric adenocarcinoma in comparison with precancerous lesions by employing qRT-PCR. Results: Our qRT-PCR data show that CXCR4 is highly expressed in tissue samples from patients with gastric cancer than precancerous lesions (2.4 times higher, P value < 0.05). When we correlated the level of CXCR4 with clinicopathological findings, we observed that CXCR4 level is associated with staging of the disease and lymphatic invasion. In conclusion: We present evidence that CXCR4 level is significantly elevated in later stages of gastric cancer. Thus, CXCR4 may play a crucial role in gastric cancer progression

Nuclear Pattern of CXCR4 Expression Is Associated with a Better Overall Survival in Patients with Gastric Cancer

Introduction. Previous studies have shown that stromal-derived factor-1 (CXCL12) and its receptor, CXCR4, play a crucial role in metastasis of various tumors. Similarly, it has been cleared that CXCR4 is expressed on the cell surface of gastric cancers. However, nuclear expression of CXCR4 and its clinical importance have not been yet studied. Materials and Methods. Herein, we studied the expression of CXCR4 in gastric samples from patients with gastric adenocarcinoma as well as human gastric carcinoma cell line, AGS, by employing RT-PCR, immunohistochemistry, and flow cytometry techniques. Results. RT-PCR data showed that CXCR4 is highly expressed on AGS cells. This was confirmed by IHC and FACS as CXCR4 was detected on cell membrane, in cytoplasm, and in nucleus of AGS cells. Moreover, we found that both cytoplasmic and nuclear CXCR4 are strongly expressed in primary gastric cancer and the cytoplasmic pattern of CXCR4 tends to be associated with a shorter overall survival than nuclear staining. In conclusion, we present evidence for the first time that both cytoplasmic and nuclear expression of CXCR4 are detectable in gastric cancer tissues. However, the role of both cytoplasmic and nuclear CXCR4 needs to be further elucidated

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

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