Patients With Invasive Ductal Carcinoma Have Reduced Levels of Decorin Expression in Their Breast Tissue Compared to Patients With Fibroadenoma While Plasma Decorin Remains Unchanged.

BACKGROUND: Reduction in the level of tissue decorin is a hallmark of many types of cancer including breast carcinoma. However, reduced decorin expression has also been reported in several types of benign tumors to the extent that it has been proposed as a tissue marker to differentiate malignant from benign tumors. The aim of this study was to investigate the potential role of plasma decorin to distinguish breast cancer from fibroadenoma, the second most common type of benign tumor, after fibrocystic disease. METHODS: From 35 patients recruited in this study, 24 were affected with invasive ductal carcinoma, either grade II (n = 14) or grade III (n = 10). The other 11 patients had fibroadenoma lesions in their breasts. Tissue decorin mRNA and protein levels were assessed with real-time qPCR and Immunohistochemical analysis. ELISA was employed to measure plasma levels of decorin. RESULTS: The mean plasma decorin in cancer patients was measured to be 5.42 ± 1.83 ng/mL while fibroadenoma patients had an average of 4.22 ± 1.17 ng/mL decorin in their plasma. The difference was not significant. However, the mean expression level of decorin mRNA calculated by the 2-ΔΔCt method was 5.6-fold lower in the biopsied tissue specimens of IDC patients versus fibroadenoma, as expected. Consistent reduction in protein abundance was observed in the studied tissue sections. CONCLUSION: We have shown that tissue decorin is a reliable marker, unaffected by patient disease stage, to differentiate IDC from fibroadenoma. However, plasma decorin does not seem to have diagnostic value in this regard.

Increase of segmental mobility through insertion of a flexible linker in split point of firefly luciferase.

The crystal structure of Photinus pyralis luciferase shows a unique molecular architecture consisting of a large N-terminal domain and a small C-terminal domain which is separated by a wide cleft. Protein engineering methods attempts to design the peptide linkers that make a connection between different protein domains or subunits to allow for separating domains and improve kinetics and structural features of proteins. In regard to this; introduction of a flexible linker at split point of luciferase which has a strong self-association activity, may leads to conformational change and improve general flexibility of protein. In this study, two flexible linkers in the split point of luciferase are introduced in order to test the effect of linker on flexibility of luciferase activity. Glycine-rich linkers are introduced into P. pyralis firefly luciferase to make two separate mutant enzymes. Enzymatic properties of mutant and native forms were measured using luminescence assay. Results show that lengthening of luciferase domains through insertion of a flexible linker did not affect bioluminescence emission spectra. Also adding linkers do not have remarkable effect on thermostability. The Km values of mutants were increased compared to native form, indicating lower affinity of mutants toward substrates.

HESA-A Exerts Its Cytoprotective Effects through Scavenging of Free Radicals: An in Vitro Study.

BACKGROUND: Natural medicines have been recently considered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect. METHODS: Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells were treated with different concentrations of HESA-A and H(2)O(2) followed by cell proliferation assays. The antioxidant effect of the HESA-A preparations was evaluated by an antioxidant assay kit. RESULTS: The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H(2)O(2) were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H(2)O(2), respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Antioxidant assay revealed that HESA-A scavenges free radicals. CONCLUSION: The findings indicate that HESA-A had cytoprotective effects in vitro, and that such an effect might be due to antioxidant properties.

Adenovirus-mediated expression of the HO-1 protein within MSCs decreased cytotoxicity and inhibited apoptosis induced by oxidative stresses.

The capacity of mesenchymal stem cells (MSCs) to survive and engraft in the target tissue may lead to promising therapeutic effects. However, the fact that the majority of MSCs die during the first few days following transplantation complicates cell therapy. Hence, it is necessary to strengthen the stem cells to withstand the rigors of the microenvironment to improve the efficacy of cell therapy. In this study, we manipulated MSCs to express a cytoprotective factor, heme oxygenase-1 (HO-1), to address this issue. Full-length cDNA of human HO-1 was isolated and cloned into TOPO vector by TOPO cloning reaction. Then, the construct was ligated to gateway adapted adenovirus expression vector by LR recombination reaction. Afterwards, the recombinant virus expressing HO-1 was produced in appropriate mammalian cell line and used to infect MSCs. The HO-1 engineered MSCs were exposed to hypoxic and oxidative stress conditions followed by evaluation of the cells' viability and apoptosis. Transient expression of HO-1 was detected within MSCs. It was observed that HO-1 expression could protect MSCs against cell death and the apoptosis triggered by hypoxic and oxidative stress conditions. The MSCs-HO-1 retained their ability to differentiate into adipogenic, chondrogenic, or osteogenic lineages. These findings could be applied as a strategy for prevention of graft cell death in MSCs-based cell therapy and is a good demonstration of how an understanding of cellular stress responses can be used for practical applications.

Isolation, Cloning and High- Level Expression of Neutrophil Gelatinase-Associated Lipocalin Lipocalin2 by Baculovirus Expression System through Gateway Technology.

OBJECTIVES: Lipocalin 2 (Lcn2) is a 25-kDa glycoprotein that has initially been extracted from neutrophil granules. Expression of Lcn2 is induced under various pathophysiological conditions. It is also known as an early marker of kidney and heart injury. High-level expression of recombinant Lcn2 neutrophil gelatinase-associated (NGAL) in insect cells was the aim of this study. MATERIALS AND METHODS: Lcn2 gene was isolated from HepG2 cell line. The PCR product was cloned into TOPO vector to construct TOPO-Lcn2. Then Lcn2 was transferred to Gateway adapted Baculovirus DNA by LR recombination reaction. The recombinant Baculovirus DNA was transfected into insect cell line. Expression of recombinant Lcn2 was detected by RT-PCR, ELISA and western blot analysis. RESULTS: Insertion of Lcn2 into pENTR/D-TOPO vector was confirmed by using PCR. The accuracy of the nucleotides sequence was verified by DNA sequencing. Transfer of the Lcn2 cDNA into the Baculovirus destination vector by LR recombination reaction was confirmed by amplification of a band of about 860 bp length by using forward Lcn2 primer and V5 reverse primer. Next, Lcn2 protein was detected as a prominent band with approximate molecular weight of 30 kDa in SDS-PAGE and western blot analysis. ELISA results revealed high-level expression of Lcn2 by Spodoptera frugiperda (Sf9) cells. CONCLUSION: High-level expression of Lcn2 protein in insect cells is promising for future potential applications. Recombinant Lcn2 might be used for producing monoclonal or polyclonal antibodies and as potential therapeutic agent. Large scale expression and purification are next steps that are on the way.

Neutrophil gelatinase-associated lipocalin: a new antioxidant that exerts its cytoprotective effect independent on Heme Oxygenase-1.

Neutrophil Gelatinase-Associated Lipocalin (NGAL/Lcn2), a member of the lipocalin family, has a variety of functions. There are extensive studies examining the expression of NGAL under harmful conditions. However, its precise function remains poorly understood. Heme Oxygenase 1 (HO-1) is an enzyme with well-established cytoprotective effects. Previous work showed that NGAL induces expression of HO-1. Interestingly, the same stimuli induced the expression of both NGAL and HO-1. The current study was designed to (1) determine whether NGAL exerts its cytoprotective effect through HO-1 and (2) compare NGAL and HO-1 with each other in terms of their protective role against oxidative stress. The current data indicate that NGAL exerts its cytoprotective effect independent of HO-1 and protects cells against oxidative stress more efficiently than HO-1. The data also strongly suggest that induction of NGAL under harmful conditions is a compensatory response to ameliorate oxidative stress-mediated toxicity. These findings may suggest new applications of NGAL, particularly when oxidative stress is a major factor.

The expression of heme oxygenase-1 in human-derived cancer cell lines.

BACKGROUND: Heme oxygenase-1 (HO-1) is a cytoprotective and antiapoptotic enzyme, which has been involved in maintaining cellular homeostasis, and plays an important protective role by modulating oxidative injury. Up-regulation of (HO-1) has contributed to tumorogenicity of some cancers. In this study we investigated the expression pattern of the HO-1, in five different human-derived cancer cell lines with high incidence in Iran. METHODS: Total cell RNA were extracted from HepG2 (hepato carcinoma), A549 (lung adenocarcinoma), MCF-7 (breast cancer), K562 (myeloid leukemia) and LS174T (colon cancer) cell lines. Human embryonic kidney (HEK293) cell line was used as a control. cDNAs were synthesized and expression of HO-1 was examined using RT-PCR. RESULTS: The expression of HO-1 was not detected in the control cell line (HEK293), but it was observed to express following ultraviolet (UV) exposure indicating that HO-1 is not constantly expressed. The examined cancer cell lines constitutively expressed different variety of HO-1 on mRNA level. Strong expression of HO-1 was observed in HepG2, MCF-7 and A549 cells. A moderate expression of HO-1 was observed in K562 cells, and LS174T cells showed no expression of HO-1. CONCLUSION: Heme oxygenase-1 could be considered as a new marker in the diagnosis of some cancers, especially hepatomacarcinoma. Our results also suggest that up-regulation of HO-1 may contribute to tumorogenicity of some cancers. Therefore, the combination of gene-silencing effect of HO-1 and chemotherapy might be considered as a new modality for the treatment of cancers in which the expression HO-1 is up-regulated.

Neutrophil gelatinase-associated lipocalin induces the expression of heme oxygenase-1 and superoxide dismutase 1, 2.

Lipocalin-2 (Lcn2, NGAL) is a member of the lipocalin super family with diverse function such as the induction of apoptosis, the suppression of bacterial growth, and modulation of inflammatory response. Much interest has recently been focused on the physiological/pathological role of the lipocalin-2 that is considered to be a novel protective factor against oxidative stress. However, its precise biological roles in this protection are not fully understood. In this report we intended to test the effect of lipocalin-2 on the expression of heme oxygenase ((1, 2)) and superoxide dismutase ((1, 2)) which are two strong antioxidants. NGAL was cloned to pcDNA3.1 plasmid by using genetic engineering method. The recombinant vector was transfected to CHO and HEK293T to establish stable cell expressing NGAL and the expression of HO-1, 2 and SOD(1, 2) were compared with appropriate controls by RT-PCR and western blot. On the other hand, expression of NGAL was suppressed by siRNA transfection in order to study the effect of lipocalin-2 on mentioned genes/proteins. The results showed that the expression of HO-1 and SOD(1, 2) enzymes were higher in cells expressing recombinant lipocalin-2 compared with the control cells. Although the expression of HO-1 was lower in NGAL silencing cells, the expression of SOD(1) and SOD(2) were higher. Our data suggest that NGAL is a potent inducer of HO-1 and somewhat SOD(1) and SOD(2) and it appears that part of antioxidant property of NGAL could be attributed to the induction of HO-1 and SOD(1, 2).

Lipocalin 2 regulation by thermal stresses: protective role of Lcn2/NGAL against cold and heat stresses.

Environmental temperature variations are the most common stresses experienced by a wide range of organisms. Lipocalin 2 (Lcn2/NGAL) is expressed in various normal and pathologic conditions. However, its precise functions have not been fully determined. Here we report the induction of Lcn2 by thermal stresses in vivo, and its role following exposure to cold and heat stresses in vitro. Induction of Lcn2 in liver, heart and kidney was detected by RT-PCR, Western blot and immunohistochemistry following exposure of mice to heat and cold stresses. When CHO and HEK293T cells overexpressing NGAL were exposed to cold stress, cell proliferation was higher compared to controls. Down-regulatrion of NGAL by siRNA in A549 cells resulted in less proliferation when exposed to cold stress compared to control cells. The number of apoptotic cells and expression of pro-apoptotic proteins were lower in the NGAL overexpressing CHO and HEK293T cells, but were higher in the siRNA-transfected A549 cells compared to controls, indicating that NGAL protects cells against cold stress. Following exposure of the cells to heat stress, ectopic expression of NGAL protected cells while addition of exogenous recombinant NGAL to the cell culture medium exacerbated the toxicity of heat stress specially when there was low or no endogenous expression of NGAL. It had a dual effect on apoptosis following heat stress. NGAL also increased the expression of HO-1. Lcn2/NGAL may have the potential to improve cell proliferation and preservation particularly to prevent cold ischemia injury of transplanted organs or for treatment of some cancers by hyperthermia.