Amplification of Tumor Transcripts from Limited Quantity of Esophageal Squamous Cell Carcinoma Tissue Samples

To examine the template-switching technology accompanied by in vitro transcription (the Switch Mechanism At the 5ʹ end of Reverse Transcript) to amplify enough amount of mRNA as input for gene expression experiments. We amplified limited quantity of esophageal squamous cell carcinoma (ESCC) transcripts samples using generated ds cDNA as template and in vitro transcription (IVT) reaction. In addition, the quality and quantity of amplified mRNA were assessed by comparative real-time PCR of genes such as stem cell markers CD44, OCT4 and SNAIL as well as MAGE-A4 as a cancer-testis antigens, and XRCC5 as an underexpressed gene in ESCC.The results obtained from this study demonstrated that optimal amounts of mRNA are generated by template-switching and IVT reaction. Integrity and purity of all RNA samples were assessed. By using this approach, over 10 micrograms of amplified mRNA were generated from 100 ng of starting total RNA. The results of comparative real-time PCR of five genes with different levels of expression illustrated that the expression level of amplified sense RNA was almost similar when compared with non-amplified RNA. Our results clearly showed the usefulness of the T7-based IVT technique for amplification of limited quantity of input total RNA

Lung-derived innate cytokines: new epigenetic targets of allergen-specific sublingual immunotherapy

Objective(s):Sublingual allergen-specific immunotherapy is a safe and effective method for treatment of IgE-mediated respiratory allergies; however, the underlying mechanisms are not fully understood. This study was planned to test whether sublingual immunotherapy (SLIT) can exert epigenetic mechanisms through which the airway allergic responses can be extinguished. Materials and Methods:BALB/c mice were sensitized intraperitoneally and challenged intranasally. Then, they received sublingual treatment with recombinant Che a 2 (rChe a 2), a major allergen of Chenopodium album. After SLIT, allergen-specific antibodies in sera, cytokine profiles of spleen cell cultures, mRNA and protein expression of lung-derived IL-33, IL-25, and TSLP (thymic stromal lymphopoietin), and histone modifications of these three genes were assessed. Results:Following Immunotherapy, systemic immune responses shifted from Th2 to Th1 profile as demonstrated by significant decrease in IgE and IL-4 and substantial increase in IgG2a and IFN-γ. At local site, mRNA and protein levels of lung-derived pro-inflammatory cytokines IL-33 and TSLP were markedly down-regulated following SLIT that was associated with marked enrichment of trimethylated lysine 27 of histone H3 at promoter regions of these two cytokines. Conclusion:In our study, sublingual immunotherapy with recombinant allergen effectively attenuated allergic immune responses, at least partly, by induction of distinct histone modifications at specific loci. Additionally, the lung-derived pro-allergic cytokines IL-33 and TSLP could be promising mucosal candidates for either monitoring allergic conditions or therapeutic approaches

Bee Venom Induces Unfolded Protein Response in A172 Glioblastoma Cell Line

Background: Glioblastoma is a type of brain tumor with poor response to available therapies, and shows high rate of mortality. Despite remarkable advancements in our knowledge about cytogenetic and pathophysiologic features of glioblastoma, current treatment strategies are mainly based on cytotoxic drugs; however, these therapeutic approaches are facing progressive failure because of the resistant nature of glioblastomas. In the recent years, however, promising results have emerged owing to targeted therapies toward molecular pathways within cancerous cells. Unfolded Protein Response (UPR) is a remarkable signaling pathway that triggers both apoptosis and survival pathways within cells, and therefore induces UPR-related apoptotic pathways in cancer cells by ER stress inducers. Objectives: Recently, the role of Bee venom (Bv), which contains powerful bioactive peptides, in inducing UPR-related apoptosis was revealed in cancer cell lines. Nevertheless, currently there are no reports of Bv potential ability in induction of UPR apoptotic routes in glioblastoma. The aim of current study was to evaluate possible role of Bee venome in inducing of UPR pathway within A172 glioblastoma cell line. Materials and Methods: We treated the A172 glioblastoma cell line with different Bv doses, and assessed UPR-related genes expression by real-time Polymerase Chain Reaction (PCR). Results: The IC50 of Bv for the studied cell line was 28 μg/mL. Furthermore, we observed that Bv can induce UPR target genes (Grp94 and Gadd153) over-expression through a dose-dependent mechanism. Conclusions: Our results suggest the potential role of Bv as a therapeutic agent for glioblastomas. Keywords: Glioblastoma; A172 Cell Line; Unfolded Protein Response; Bee Veno

Ectopic Expression of Human DPPA2 Gene in ESCC Cell Line Using Retroviral System

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Role of MAML1 in targeted therapy against the esophageal cancer stem cells

Abstract Background Esophageal cancer is the sixth-leading cause of cancer-related deaths worldwide. Cancer stem cells (CSCs) are the main reason for tumor relapse in esophageal squamous cell carcinoma (ESCC). The NOTCH pathway is important in preservation of CSCs, therefore it is possible to target such cells by targeting MAML1 as the main component of the NOTCH transcription machinery. Methods In present study we isolated the CD44+ ESCC CSCs and designed a MAML1-targeted therapy to inhibit the NOTCH signaling pathway. CSCs were isolated using magnetic cell sorting utilizing the CD44 cell surface marker. Several stem cell markers were analyzed in the levels of protein and mRNA expression. The isolated CSCs were characterized in vivo in NUDE mice. Biological role of MAML1 was assessed in isolated CD44+ CSCs. A drug resistance assay was also performed to assess the role of MAML1 in CD44+ CSCs with 5FU resistance. Results The CD44+ CSCs had ability to form tumors in NUDE mice. MAML1 silencing caused a significant decrease (p = 0.019) and ectopic expression caused a significant increase in migration of CD44+ CSCs (p = 0.012). Moreover, MAML1 silencing and ectopic expression significantly increased and decreased 5FU resistance, respectively (p < 0.05). MAML1 silencing significantly increased the number of cells in G1 phase (p = 0.008), and its ectopic expression significantly increased the number of CD44+ CSCS in S phase (p = 0.037). Conclusions MAML1 may be utilized for targeted therapy with a low side effect to eliminate the CD44+ CSCs through inhibition of canonical NOTCH pathway in ESCC patients

Effects of Vitamin-E treatment on CatSper genes expression and sperm quality in the testis of the aging mouse

Background: CatSper genes are a novel family of four sperm-specific calcium channels, which indicate testis-specific expression patterns. Despite the crucial role of CatSper genes in the male reproduction, very little is known about the factors that regulate their expression. Objective: The objective of this study was to investigate the effects of vitamin E treatment on the expression of CatSper 1 and CatSper 2 genes as well as sperm quality in the aged male mice. Materials and Methods: Twenty four 11-12 months old aged male mice and twenty four 2-3-months old young male mice were randomly divided into four groups. Control groups received no injection. The experimental groups of male mice were received intraperitoneal injection of 106 mg/kg vitamin E daily for 35 days. Left testis and cauda epididymides from each mouse were collected on the days 21, 28 and 35 following vitamin E treatment and were used for Real-Time PCR and immunohistochemistry. Also, sperm analysis was performed according to the WHO guidelines given for human sperm examination. Data were analyzed using SPSS software. Results: Administration of vitamin E improved sperm parameters in the aged as well as young adult male mice. In addition, the expression of CatSper genes increased following vitamin E treatment. Also, intensity of signal for CatSper1 and CatSper2 increased in the head and middle piece of sperm in experimental group as compared to those of control ones. Conclusion: The vitamin E treatment significantly improved the sperm quality, especially in terms of sperm motility, count and morphology rate. Furthermore, CatSper genes expression could be up-regulated by the vitamin E treatment

Expression analysis of CD44 isoforms S and V3, in patients with esophageal squamous cell carcinoma

Objective(s):  CD44 is a member of the cell adhesion molecules family. Naturally, CD44S, along with CD44V3 influence the cell motility, migration, and adhesion, while in tumor cells they lead to tumor invasion, progression, and metastasis. The purpose of this research is to evaluate the CD44S and CD44V3 expression in Esophageal Squamous Cell Carcinoma (ESCC) and to reveal their correlations with clinicopathological features of patients. Materials and Methods:Fresh tumoral and distant tumor-free esophageal tissues were obtained from 50 patients with ESCC. Using quantitative real-time PCR, the expression levels of CD44S and CD44V3 were quantified and compared in both groups of cells. The patients had not received any therapeutic interference, such as chemotherapy or radiation, prior to sampling. Results:Significant overexpression of CD44S and CD44V3 mRNA was observed in 13 (26.0%, P=0.03) and 11 (22.0%, P=0.007) tumor specimens, respectively. The expression of the genes were significantly correlated not only with each other (P=0.0001), but also with differentiation grade of tumor (P=0.033), stage of tumor progression (P=0.003), and depth of tumor invasion         (P=0.00). In addition, low level of CD44V3 mRNA expression was attended to be associated with tumor invasion. Conclusion:There is no correlation between CD44S expression with clinicopathological features of patients; however, simultaneous expression of these genes has an important effect on tumorigenesis

Loss of heterozygosity and microsatellite instability as predictive markers among Iranian esophageal cancer patients

Objective(s): Variation in microsatellite sequences that are dispersed in the genome has been linked to a deficiency in cellular mismatch repair system and defects in several genes of this system are involved in carcinogenesis. Our aim in this study was to illustrate microsatellite DNA alteration in esophageal cancer. Materials and Methods: DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues from surgical and matched margin-normal samples. Microsatellite instability (MSI) and loss of heterozygosity (LOH) were studied in 50 cases of esophageal squamous cell carcinoma (ESCC) by amplifying six microsatellite markers: D13S260 (13q12.3), D13S267 (13q12.3), D9S171 (9p21), D2S123 (2p), D5S2501 (5q21) and TP53 (17p13.1) analyzed on 6% denaturing polyacrylamide gel electrophoresis. Results: Statistical analysis indicated a near significant reverse correlation between grade and LOH (P= 0.068, correlation coefficient= -0.272). Specifically, increased LOH in tumor DNA has a significant correlation with increased differentiation from poorly differentiated to well differentiated tumors (P= 0.002 and P= 0.016 respectively). In addition, higher number of chromosomal loci with LOH showed a reverse correlation with lymph node metastasis (P= 0.026, correlation coefficient= -0.485). Furthermore, there was a positive correlation between addiction and MSI (P= 0.026, correlation coefficient= 0.465). Conclusion: Microsatellite DNA alterations may be a prognostic tool for detection and the evolution of prognosis in patients with SCC of esophagus. It can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci and addiction with any of opium, cigarette, water pipe or alcohol can be a susceptibility factor(s) for MSI