A reliable mouse model of liver and lung metastasis by injecting esophageal cancer stem cells (CSCs) through tail-vein injection.

BACKGROUND: Esophageal Squamous Cell Carcinoma (ESCC) is a highly aggressive tumor with increased metastatic potential. Recent evidence suggests that esophageal CSCs have a crucial role in tumor initiation, progression, and resistance to conventional anti-cancer therapies. The study aimed to develop mouse model to mimic the late steps of the metastasis process using a tail-vein injection of esophageal CSCs. METHODS AND RESULTS: The sphere formation assay was used to enrich CSCs. For analysis of tumorigenicity, YM-1 adherent cells and enriched CSCs were injected subcutaneously into dorsal flank of nude mice. The expression of SLUG, E-cad, and CTHRC1 genes was examined by Real-Time qRT-PCR and immunohistochemistry (IHC) methods. To assess the metastatic potential of adherent YM-1 cells and their enriched CSCs, we injected the cells into the tail vein of nude mice. Our findings showed the up-regulation of SLUG and down-regulation of E-cad in the esophageal CSC-derived tumors (ECSCTs) compared to adherent cells-derived tumors. There was no statistically significant difference between CTHRC1 gene expressions in both groups of tumors. IHC staining confirmed the higher expression of SLUG protein in ECSCTs compared to adherent cell-derived tumors. Enriched CSCs were able to metastasize to the lungs and livers after three months, but, metastasis of adherent cells wasn't observed. CONCLUSION: Our study showed esophageal CSCs injected through the tail-vein injection can migrate and metastasize to the lung and liver after three months. The developed metastatic mouse model can be a valuable and relevant model to investigate the molecular and cellular mechanisms of metastasis and develop successful targeted therapies against ESCC. The present study is one of the few studies that investigate the metastasis of esophageal cancer stem cells (ESCC type) through injection into the tail vein of nude mice.

Upregulation of Fucosyltransferase 3, 8 and protein O-Fucosyltransferase 1, 2 genes in esophageal cancer stem-like cells (CSLCs).

Recently, studies have shown that Fucosylation plays an important role in the invasion and metastatic process of CSLCs. Understanding the expression pattern of fucosyltransferase (FUT) genes may help to suggest better-targeted therapy strategies for esophageal squamous cell carcinoma (ESCC). The study aimed to address the expression pattern of FUT gene variants in esophageal CSLCs and parental adherent cells. Sphere formation method was used to enrich CSLCs. Expression of FUT genes was examined in tumor sphere and parental adherent cells using the RT-PCR method and then relative expression of detected variants was performed by the Real-Time PCR method in both groups. The detected FUTs, also, were assessed in fresh ESCC tumors and the matched healthy controls. Analysis of The cell surface carbohydrate Lewis x (LeX, CD15) was performed by flow cytometry. Molecular analysis showed that the expression of FUT 3, 8 and POFUT1, 2 genes in tumorsphere were significantly higher than parental adherent cells. Analysis of fresh ESCC tumor tissues and the matched healthy controls showed that FUT8 and POFUT1, 2 genes in contrast to FUT 3 have higher expression in tumor tissues than controls. Flow cytometric analyses revealed that tumorsphere and their parent cells do not differ significantly in Lewis x surface marker. The present study showed that FUT 3, 8 and POFUT1, 2 genes upregulated in esophageal CSLCs in comparison to adherent cells. Understanding the expression pattern of FUT gene variants may help to suggest better-targeted therapy strategies for ESCC.

Beta-catenin Forms Protein Aggregation at High Concentrations in HEK293TCells.

BACKGROUND: The canonical Wnt signal transduction (or the Wnt/ß-catenin pathway) plays a crucial role in the development of animals and in carcinogenesis. Beta-catenin is the central component of this signaling pathway. The activation of Wnt/ß-catenin signaling results in the cytoplasmic and nuclear accumulation of ß-catenin. In the nucleus, ß-catenin interacts with the TCF/LEF transcription factors and, therefore, participates in the upregulation or downregulation of some important genes involved in diverse cellular activities. In addition, ß-catenin is a critical component of the cadherin-mediated cell adherens junction. We had previously noticed that very high cellular concentrations of ß-catenin had a negative effect on the transcriptional activity of this protein and, therefore, the aim of this study was to find a mechanism for this negative interaction. METHODS: Cell fractionation, western blotting, and immunofluorescence microscopy experiments were performed to measure ß-catenin protein levels and ß-catenin cellular localization in HEK293Tcells transfected with various amounts of a ß-catenin-encoding plasmid. Also, total RNA was extracted from the cells and used for reverse transcriptase-PCR experiments to measure the expression of the ß-catenin target genes. SPSS, version 16, was used to analyze the results statistically. RESULTS: We demonstrated that overexpression of ß-catenin led to the formation of rod-shaped protein aggregates. The aggregate structures were mainly formed in the cell nucleus and were heavy enough to be isolated by centrifugation. Beta-catenin aggregate formation was accompanied by a decrease in the expression of the ß-catenin target genes used in this study. CONCLUSION: Since deregulation of ß-catenin function occurs in several human diseases, including cancer and neurological disorders, the results of this paper further support the possible biological and clinical significance of ß-catenin aggregate formation.

Lower serum zinc level is associated with higher fasting insulin in type 2 diabetes mellitus (T2DM) and relates with disturbed glucagon suppression response in male patients.

AIMS: Zinc ion can play critical role in glycemic control in diabetes mellitus (DM), contributing to both insulin synthesis and secretion. In this study, we aimed to investigate the level of zinc in diabetic patients and its association with glycemic parameters, insulin, and glucagon level. METHODS: 112 individuals (59 cases of type 2DM and 53 non-diabetic controls) were included in this study. Biochemical parameters (FBG, 2hpp, HbA1C), and zinc level in the serum were measured using colorimetric assays. Insulin and glucagon were measured by ELISA method. HOMA-IR, HOMA-B, reciprocal HOMA-B, and Quicki indices were calculated using appropriate formula. For further analysis, patients were divided into two groups: high (>135.5 µg/dl) and low (<135.5 µg/dl) zinc. Glucagon suppression was considered yes if 2hpp glucagon < fasting glucagon. RESULTS: Our results showed that serum Zn level in type 2 DM patients was lower than control (P value=0.02). Patients with lower Zn had higher fasting insulin (P value=0.006) and higher ß-cell activity index (HOMA-B, p value=0.02), however fasting glucagon and parameters of hyperglycemia (FBG, 2hpp, Hba1C) were not different. Moreover, insulin sensitivity and resistance indices (Quicki, HOMA-IR,1/HOMA-IR) showed non-significantly improved status in high Zn group. We found non-significant association between glucagon suppression and Zn level in both genders (N = 39, p value = 0.07), however, it was significant in males (N = 14, p value = 0.02). CONCLUSION: Altogether, our results indicated reduced serum Zn in type 2DM can exacerbate hyperinsulinemia and glucagon suppression (only significant in the male), highlighting its importance in type 2DM control.

A1 adenosine receptor antagonist induces cell apoptosis in KYSE-30 and YM-1 esophageal cancer cell lines.

Background and aim: Adenosine A1 receptor (AA1R) has been shown to have an inhibitory effect on cell growth in several cancers; however, its function in esophageal cancer is still unclear. In this study, we examined the effect of AA1R on cell growth and apoptosis in esophageal cancer cells. Materials and methods: In this study, YM-1 and KYSE-30 esophageal cancer cell lines were cultured. AA1R gene expression was determined by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). As well, the AA1R antagonist (DPCPX) effect on cell viability was evaluated by the MTT assay. Moreover, apoptosis was assessed by annexin-V and propidium iodide staining, and the caspase-3/7 activity assay kit. Result: qRT-PCR results indicated that the AA1R was expressed in YM-1 and KYSE-30 cells. In addition, DPCPX significantly decreased cell proliferation in both cell lines. Furthermore, the A1AR antagonist induced apoptosis in KYSE-30 and YM-1 cells. After treatment of both cell lines with DPCPX, the caspase 3/7 activity was increased. Conclusion: Our finding indicates the AA1R antagonist induces apoptosis through caspase 3/7 activation and can be considered a potential target in esophageal cancer therapy.

Association of interleukin-17A gene promoter polymorphism with the susceptibility to generalized chronic periodontitis in an Iranian population.

Background: Chronic periodontitis (CP) is characterized by an immune response, leading to the destruction of periodontal supporting tissue. The effect of inflammatory and genetic factors on periodontitis has been evaluated previously. The interleukin (IL-17) as an inflammation regulator seems to play a critical role in periodontitis pathogenesis. Here, in the current study, we aimed to investigate the association of -197 G > A (rs2275913) IL-17 gene promoter polymorphism with generalized severe CP in an Iranian population. Materials and Methods: In this case-control study, a total of 54 patients with periodontitis and 118 normals were enrolled. The polymerase chain reaction-restriction fragment length polymorphism technique was applied to detect IL-17 promoter rs2275913 genotypes in association with the susceptibility to severe CP. Chi-square test or Fisher's exact test was employed to compare genotype frequencies between groups. P < 0.05 were considered statistically significant. Results: The distribution of genotypes and alleles was in Hardy-Weinberg equilibrium. Although no significant association was observed between the risk of periodontitis and genotype frequencies under any of the inheritance models, the GG genotype was higher in healthy controls, while the AG genotype was more frequently observed in patients under the codominant model ([odd ratio [OR] = 2.14, 95% confidence interval (CI) (1.01-4.53), P = 0.13]). The frequency of AG-AA genotype was higher in patients under dominant inheritance model ([OR = 1.92, 95% CI (0.94-3.93), P = 0.068]), while GG-AA and AG genotypes were higher in healthy controls under over dominant model (OR = 0.1.95, 95% CI [0.98-3.86], P = 0.055). Conclusion: The results of this study showed that the presence of allele A and AG genotypes could be considered possible factors in increasing the risk of developing CP, although the differences of allele and genotype frequencies were remarkable but not statistically significant between the two groups.

Regulation of GSK-3beta and beta-Catenin by Galphaq in HEK293T cells.

Recent studies have shown that heterotrimeric G proteins are involved in the regulation of the canonical Wnt/beta-Catenin pathway. However, the mechanism(s) behind this involvement is (are) poorly understood. Our previous results have shown that activation of Galphaq in Xenopus oocytes leads to inhibition of GSK-3beta and stabilization of the beta-Catenin protein, suggesting that Galphaq might stabilize beta-Catenin via inhibition of GSK-3beta. In this study, we have observed similar results in HEK293T cells. In these cells optimal activation of endogenous Galphaq by expressing M3-muscarinic acetylcholine receptor (with or without carbachol treatment), or exposing the cells to thrombin led to an increase of 2 to 3-fold in endogenous cytoplasmic beta-Catenin protein levels. In addition, expression of the activated mutant of Galphaq (GalphaqQL) dramatically enhanced accumulation of exogenous beta-Catenin with no effect on beta-catenin (CTNNB1) gene transcription. The Galphaq-mediated cellular accumulation of beta-Catenin was blocked by expression of a minigene encoding a Galphaq specific inhibitory peptide but not by a minigene encoding a Galphas blocking peptide. Also, expression of GalphaqQL led to a significant reduction in GSK-3beta kinase activity, supporting the idea that the positive role of Galphaq signaling in inducing cellular accumulation of beta-Catenin is mediated through inhibition of GSK-3beta.

Oral poliovirus vaccine-induced programmed cell death involves both intrinsic and extrinsic pathways in human colorectal cancer cells.

PURPOSE: Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. Replication-competent viruses, which are naturally able to infect and lyse tumor cells, seem to be promising in this field. The aim of this study was to evaluate the potential of oral poliovirus vaccine (OPV) on human CRC cells and elucidate the mechanism of apoptosis induction. MATERIALS AND METHODS: Protein and gene expression of poliovirus (PV) receptor (CD155) on four human CRC cell lines including HCT116, SW480, HT-29, and Caco-2 and normal fetal human colon (FHC) cell line as a control were examined by flow cytometry and SYBR Green Real-Time PCR, respectively. Cytotoxicity of OPV on indicated cell lines was tested using MTT assay. The ability of OPV on apoptosis induction for both intrinsic and extrinsic pathways was examined using caspase-8 and caspase-9 colorimetric assay kits. The PV propagation in mentioned cell lines was investigated, and the quantity of viral yields (cells associated and extracellular) was determined using TaqMan PCR. RESULTS: CD155 mRNA and protein were expressed significantly higher in studied CRC cell lines rather than the normal cell line (P=0). OPV induced cell death in a time- and dose-dependent manner in human CRC cells. Apoptosis through both extrinsic and intrinsic pathways was detected in CRC cells with the minimum level found in FHC. PV viral load was significantly correlated with apoptosis via extrinsic (R=0.945, P=0.0001) and intrinsic (R=0.756, P=0.001) pathways. CONCLUSION: This study suggests that OPV has potential for clinical treatment of CRC. However further studies in animal models (tumor xenografts) are needed to be certain that it is qualified enough for treatment of CRC.

Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells.

OBJECTIVES: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA) as a recently emerged anti-neoplastic histone deacetylase (HDAC) inhibitor and pioglitazone (PGZ) as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ) agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. MATERIALS AND METHODS: Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. RESULTS: Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after 24 hr; however, PGZ 400 µM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A) phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27) expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. CONCLUSION: These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies.

Long non-coding RNA SOX2OT: expression signature, splicing patterns, and emerging roles in pluripotency and tumorigenesis.

SOX2 overlapping transcript (SOX2OT) is a long non-coding RNA which harbors one of the major regulators of pluripotency, SOX2 gene, in its intronic region. SOX2OT gene is mapped to human chromosome 3q26.3 (Chr3q26.3) locus and is extended in a high conserved region of over 700 kb. Little is known about the exact role of SOX2OT; however, recent studies have demonstrated a positive role for it in transcription regulation of SOX2 gene. Similar to SOX2, SOX2OT is highly expressed in embryonic stem cells and down-regulated upon the induction of differentiation. SOX2OT is dynamically regulated during the embryogenesis of vertebrates, and delimited to the brain in adult mice and human. Recently, the disregulation of SOX2OT expression and its concomitant expression with SOX2 have become highlighted in some somatic cancers including esophageal squamous cell carcinoma, lung squamous cell carcinoma, and breast cancer. Interestingly, SOX2OT is differentially spliced into multiple mRNA-like transcripts in stem and cancer cells. In this review, we are describing the structural and functional features of SOX2OT, with an emphasis on its expression signature, its splicing patterns and its critical function in the regulation of SOX2 expression during development and tumorigenesis.