Additive effect of recombinant Mycobacterium tuberculosis ESAT-6 protein and ESAT-6/CFP-10 fusion protein in adhesion of macrophages through fibronectin receptors

Background/purposeTuberculous granulomas are the sites of interaction between the T cells, macrophages, and extracellular matrix (ECM) to control the infection caused by Mycobacterium tuberculosis (M. tuberculosis). A predominant role of RD-1-encoded secretory proteins, early secreted antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in the formation of granulomas has recently been emphasized. However, the precise molecular events that induce the formation of these granulomatous structures are yet to be elucidated. Macrophages use integrins to adhere to fibronectin (FN) as a major component of the ECM. The major goal of this study was to investigate whether recombinant M. tuberculosis antigens can modulate integrin-mediated macrophage adhesion.MethodsDifferentiated THP-1 cell line was stimulated with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins and evaluated for alterations in the expression levels of α5β1 and α4β1 by semiquantitative real-time polymerase chain reaction. The role of these recombinant antigens in the cytoskeleton rearrangement was determined by adhesion assay and immunofluorescent microscopy.ResultsOur data showed that ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to FN through α4β1 integrin. An increased expression level of α4β1 integrin in comparison with α5β1 integrin in differentiated THP-1 cells was also observed. Results of immunofluorescence studies showed that recombinant proteins-treated THP-1 cells form well-organized stress fibers and focal contacts containing vinculin compared with untreated THP-1 cells.ConclusionIncreased expression level of α4β1 in differentiated THP-1 cells could suggest the important role of α4β1 integrin in adhesion and focal contact formation of macrophages exposed to M. tuberculosis antigens

Distinct High-Profile Methylated Genes in Colorectal Cancer

Background: Mutations and promoters ’ methylation of a set of candidate cancer genes (CAN genes) are associated with progression of colorectal cancer (CRC). We hypothesized that these genes ’ promoters are inactivated through epigenetic silencing and may show a different profile in high-risk populations. We investigated the status of CAN gene methylation and CHD5 protein expression in African American CRC tissue microarrays (TMA) using immunohistochemical staining. Methodology/Principal Findings: The promoter methylation status of the CAN genes was studied by methylation-specific PCR (MSP) in 51 Iranians (a white population) and 51 African Americans (AA). Microsatellite instability (MSI) was analyzed as well. The differential frequency of methylation for each gene was tested by chi-square analysis between the two groups based on matched age and sex. CHD5 protein expression was evaluated in moderate to well differentiated and poorly differentiated carcinomas compared to matched normal tissue using TMA. In addition, the correlation between these epigenetic biomarkers and various clinicopathological factors, including, age, location, and stage of the disease were analyzed. Seventy-seven and 34 % of tumors were distal in Iranian and African American patients, respectively. In both populations, the percentage of methylation was.65 % for SYNE1, MMP2, APC2, GPNMB, EVL, PTPRD, and STARD8, whereas methylation was,50 % for LGR6, RET, CD109, and RNF. The difference in methylation between the two populations was statistically significant for CHD5, ICAM5 and GPNMB. Thirty-one percent AA tumors showed MSI-H, compared to 28 % i

Impact of BRAF, MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study

We have identified an alternative pathway of tumorigenesis in sporadic colon cancer, involving microsatellite instability due to mismatched repair methylation, which may be driven by mutations in the BRAF gene (V600E). Colorectal cancer (CRC) is the most common cancer in the world, and African Americans show a higher incidence than other populations in the United States. We analyzed sporadic CRCs in Omani (of African origin, N = 61), Iranian (of Caucasian origin, N = 53) and African American (N = 95) patients for microsatellite instability, expression status of mismatched repair genes (hMLH1, hMSH2) and presence of the BRAF (V600E) mutation. In the Omani group, all tumors with BRAF mutations were located in the left side of the colon, and for African Americans, 88% [7] of tumors with BRAF mutations were found in the right side of the colon. In African Americans, 31% of tumors displayed microsatellite instability at two or more markers (MSI-H), while this rate was 26% and 13% for tumors in the Iranian and Omani groups, respectively. A majority of these MSI-H tumors were located in the proximal colon (right side) in African American and Iranian subjects, whereas most were located in the distal colon (left side) in Omani subjects. Defects in hMLH1 gene expression were found in 77% of MSI-H tumors in both African Americans and Iranians and in 38% of tumors in Omanis. BRAF mutations were observed in all subjects: 10% of tumors in African Americans (8/82), 2% of tumors in Iranians (1/53), and 19% of tumors in Omanis (11/59). Our findings suggest that CRC occurs at a younger age in Omani and Iranian patients, and these groups showed a lower occurrence of MSI-H than did African American patients. Our multivariate model suggests an important and significant role of hMLH1 expression and BRAF mutation in MSI-H CRC in these populations. The high occurrence of MSI-H tumors in African Americans may have significant implications for treatment, since patients with MSI-H lesions display a different response to chemotherapeutic agents such as 5-fluorouracil

Evolution of glycophosphatidyl inositol anchors in carcinoembryonic antigen family members : functional implications

Carcinoembryonic antigen (CEA) family members in humans can be divided into two subgroups according to the type of anchorage to the cell membrane: Glycophosphatidyl inositol (GPI)-linked and transmembrane (TM) linked. The mode of membrane anchorage can profoundly affect the biological functions of CEA family molecules, including the ability to inhibit differentiation. All information so far, suggests that the GPI-anchored CEA family members evolved recently presumably from a more primordial TM-anchored family member such as CEACAM1. The results of this study indicate that very few mutations in the TM domain are required to effect this change. The introduction of a stop codon in the CEACAM1 TM domain at the position corresponding to that of all GPI-linked CEA family members plus two more amino acid changes in this domain resulted in highly efficiently GPI-processed protein. The GPI-anchored mutant CEACAM1 protein is still capable of mediating cell-cell adhesion but, unlike TM-linked CEACAM1, could block myogenic differentiation. Screening of genomic DNA from various mammalian groups by PCR showed convergent evolution of GPI anchorage in the CEA family, in that it evolved twice independently by different packages of mutations during the primate radiation. The same group of GPI-generating mutations as in human CEA family members evolved in the common ancestor of primitive primate groups of tarsiers and New World monkeys, and a novel group of GPI producing mutations evolved later in Cebidae family of New World monkeys. These independent groups of mutations that gave rise to the same structural feature imply adaptive evolution of GPI-anchor acquisition in CEA family, This supports the notion that GPI-anchoring of CEA family members is functionally important in vivo and therefore, has been positively selected during evolution. A chimeric cDNA of the novel GPI anchor-generating TM domain linked to the extracellular domains of human CEACAMI-4L was efficiently processed

Effect of soluble Carcinoembryonic antigen on myogenic and enterocytic differentiation

Introduction: Carcinoembryonic antigen (CEA), a glycophosphatidyl inositol (GPI) anchored glycoprotein, is over-expressed in various cancers, including colorectal carcinomas. Inhibition of cell differentiation can cause cancer. Previous studies show transfection and over-expression of CEA gene in myoblasts or colonocytes leads to inhibition of cell differentiation. We investigated whether soluble CEA has a role in inhibition of cell differentiation.Materials and Methods: Monolayer cultures of L6, C2C12, Caco-2 and CHO cell lines were grown in dulbecco's modification of eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). For differentiation induction C2C12 and L6 cell lines media, was changed to DMEM containing 2% horse serum. CEA concentration was measured in harvested media by ELISA assay. H&E staining, CK assay and RT-PCR for myogenin gene were used for C2C12 differentiation experiments. Results: The results showed although commercial pure CEA and LS-180 conditioned media (contain high level of CEA) have inhibitory effects on Caco-2 differentiation, but it can also inhibit differentiation of control groups. We observed the effect of high concentration of CEA (5μg/ml) on L6 differentiation is identical with the effect of BSA as control. C2C12 differentiation was inhibited in response to LS-180 conditioned media at morphological level and myogenin expression. CK activity was significantly lower in LS-180 conditioned media (P= 0.0012) and CHO (P= 0.0002) conditioned media groups in comparison to control group.Conclusion: Our finding shows soluble CEA antigen did not have a significant effect on cell differentiation

The Impact of Thymidylate Synthase and Methylenetetrahydrofolate Reductase Genotypes on Sensitivity to 5-Fluorouracil Treatment in Colorectal Cancer Cells

5-fluorouracil (5-FU) is one of the major components of many standard regimens for chemotherapy of colorectal cancer (CRC) and some other malignancies. Given the known relationship between thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) activity and 5-FU metabolism, this study investigated the impact of selected functional polymorphisms of the TS and MTHFR genes on chemotherapy resistance in 5 human CRC cell lines. HCT116, SW1116, HT29/219, LS180, and Caco-2 CRC cells were cultured as monolayer and their chemosensitivity to 5-FU, oxaliplatin, and irinotecan was determined by MTT assay. Genomic DNA was extracted from the cultured cells, and a 6-bp insertion or deletion (6-bp ins/del) polymorphism in 3´-UTR of the TS gene was determined by the PCR-RFLP method. Genotyping of MTHFR 677 C/T and 1298A/C single nucleotide polymorphism (SNP) was also performed by MS-PCR and PCR-RFLP, respectively. Caco-2 with the homozygous TS 6-bp ins/ins and MTHFR 677 T/T and 1298 C/C genotype, was the most 5-FU resistant cell line. HCT116 with the homozygous TS 6-bp del/del and MTHFR 1298 A/A and heterozygous MTHFR 677 C/T genotype was the least 5-FU resistant cell. LS180, the second most 5-FU resistant cell line, was heterozygous for all three polymorphic sits. HT29/219 and SW1116 cells with homozygous TS 6-bp ins/ins and heterozygous MTHFR 677 C/T and 1298 A/C genotypes had intermediate 5-FU sensitivity. The results indicate that TS 3´-UTR 6-bp insertion and MTHFR 677T and 1298C alleles increase 5-FU resistance in CRC cells. No relationship was observed between TS and MTHFR genotypes and oxaliplatin or irinotecan sensitivity in these cells

Methylenetetrahydrofolate reductase C677T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status

AIM: To evaluate joint effects of Methylentetrahydrofolate reductase (MTHFR) C677T genotypes, and serum folate/vitamin B12 concentrations on promoter methylation of tumor-associated genes among Iranian colorectal cancer patients

Effects of novel and conventional thermal treatments on the physicochemical properties of iron-loaded double emulsions

In this work, changes in different physicochemical properties of iron-loaded double emulsions (DEs) were monitored under the influence of novel (microwave and ohmic) and conventional heat treatments. Microwave heating led to destabilization and obvious phase separation. As compared to control samples, heat treatment increased particle size, light absorbance, centrifugal instability, iron expulsion from the internal aqueous phase, color difference, chemical instability of the lipid phase, release of iron in simulated gastrointestinal fluids and iron bioavailability in cell line SW742. Emulsion viscosity and whiteness index decreased after heat treatment, whereas the zeta-potential remained unchanged. There was a negative correlation between physicochemical stability and heat treatment intensity. Conventional heating resulted in greater stability than ohmic heating. Yoghurt samples (as model systems) were fortified with either iron-loaded or iron-free DEs. Our results showed that the presence of iron in the aqueous phase resulted in significant lipid oxidation during storage

The Roles of microRNA miR-185 in Digestive Tract Cancers

Digestive tract cancers represent a serious public health issue. In recent years, evidence has accumulated that microRNA miR-185 is implicated in the pathogenesis of this group of highly malignant tumors. Its expression variations correlate with clinical features, such as tumor size, lymph node metastasis, tumor node metastatic stage, survival, recurrence and response to adjuvant therapy, and have diagnostic and prognostic potential. In this review, we compile, evaluate and discuss the current knowledge about the roles of miR-185 in digestive tract cancers. Interestingly, miR-185 is apparently involved in regulating both tumor suppressive and oncogenic processes. We look at downstream effects as well as upstream regulation. In addition, we discuss the utility of miR-185 for diagnosis and its potential concerning novel therapeutic approaches