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Recombinant Fibromodulin and Decorin Effects on NF-Κb and Tgfβ1 in the 4T1 Breast Cancer Cell Line

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Recombinant Fibromodulin and Decorin Effects on NF-Κb and Tgfβ1 in the 4T1 Breast Cancer Cell Line ONCOLOGY LETTERS 13: 4475-4480, 2017 Recombinant fibromodulin and decorin effects on NF-κB and TGFβ1 in the 4T1 breast cancer cell line LADAN DAWOODY NEJAD1,2, ALIREZA BIGLARI3, TIZIANA ANNESE4 and DOMENICO RIBATTI4,5 1Department of Molecular Medicine and Biochemistry Institute, University of Bern, 3012 Bern, Switzerland; Departments of 2Molecular Medicine and Genetics and 3Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, 45154 Zanjan, Iran; 4Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, I-70124 Bari; 5National Cancer Institute Giovanni Paolo II, I-70126 Bari, Italy Received October 18, 2016; Accepted February 3, 2017 DOI: 10.3892/ol.2017.5960 Abstract. Constitutive activation of nuclear factor-κB NF-κB activity stimulating signals cause dissociation of IκB, (NF-κB) stimulates cell proliferation and metastasis, and allowing NF-κB dimers to locate to the nucleus and alter inhibits apoptosis in breast cancer. Transforming growth gene expression (6). Additionally, NF-κB signaling is essen- factor-β (TGF-β) signaling pathway is deregulated in breast tial for epithelial-mesenchymal transition (EMT), and the cancer progression and metastasis. The aim of the present therapeutic inhibition of NF-κB may be an effective strategy study was to investigate the inhibitory effects of the two small to control tumor invasion and metastasis (7). leucine rich proteoglycans fibromodulin (Fmod) and decorin Transforming growth factor β (TGF-β) is a pleiotropic (Dcn), overexpressed using adenovirus gene transfer, on cytokine that is found in three isoforms (TGF-β1, TGF-β2 NF-κB-activity and TGF-β1-expression in the highly meta- and TGF-β3), which are structurally and functionally static 4T1 breast cancer cell line. The results demonstrate that associated (8). TGF-β isoforms are secreted as biologically Fmod and Dcn overexpression is associated with NF-κB and latent precursor molecules and are stimulated by proteolytic TGF-β1 downregulation, and that Fmod promotes this effect cleavage interactions with integrins or by pH alterations in more effectively compared with Dcn. the local microenvironment; intracellular TGF-β signaling is complex and is activated by numerous signaling Introduction pathways (9). TGF-β1 is involved in the occurrence and development of breast cancer (10) and the TGF-β signaling Breast cancer is the second most prevalent cause of pathway is deregulated in breast cancer progression and cancer-associated mortality in females; metastasis in breast metastasis (11,12). cancer is the primary cause of mortality and is a crucial In invasive breast cancer, certain alterations have been factor in treatment (1-3). Constitutive activation of nuclear observed in the stromal structure, including a reduction factor-κB (NF-κB), a family of transcription factors (4), in the expression of two small leucine rich proteoglycans, stimulates proliferation and metastasis, and inhibits apop- fibromodulin (Fmod) and decorin (Dcn), and the acquisition tosis in breast cancer (5). These proteins form homo- or of TGF-β antagonist activity in vitro and in vivo has been heterodimers and have similar structural characteristics, identified (13-16). Dcn is a dermatansulfate proteoglycan including the Rel homology domain, which is necessary for that reduces the growth of tumors, including gliomas, dimerization, binding to cognate DNA elements and nuclear breast, lung, colon and squamous cell carcinoma, and localization signals (4,6). In non-stimulated cells, NF-κB has an anti-angiogenic role through the binding TGF-β, complexes in an inactive form interact with a monomer of epidermal growth factor receptor (EGFR) and inducing an inhibitory protein called inhibitor of NF-κB (IκB) (6). the expression of p21 (13,17-21). Fmod, a keratan sulfate proteoglycan, functions as a modulator of TGF-β activity in scarless wound healing and is involved in collagen assembly in skin development (22,23). In addition, Fmod exerts a Correspondence to: Mrs. Tiziana Annese, Department of Basic potent TGF-β-antagonist activity, compared with Dcn, in Medical Sciences, Neurosciences and Sensory Organs, Section of the inhibition of neointimal hyperplasia in saphenous vein Human Anatomy and Histology, University of Bari Medical School, graft (15). Fmod is also implicated in the inhibitory effect of 11 Piazza G. Cesare, I-70124 Bari, Italy NF-κB signaling thorough the suppression of IκBα protein in E-mail: [email protected] 3T3‑L1 fibroblasts (24). Key words: adenoviral vector, breast cancer, decorin, fibromodulin, In the current study, the inhibitory effects of Fmod and nuclear factor-κB, transforming growth factor-β1 Dcn overexpression on NF-κB and TGF-β1 were investigated using adenovirus-mediated gene transfer in the 4T1 breast cancer cell line. 4476 NEJAD et al: RECOMBINANT FMOD AND DCN EFFECTS ON NF-κB AND TGF-β1 Materials and methods TGF-β1 and GAPDH were generated via five serial dilutions with cDNA. Cq values from each gene were measured from Recombinant adenovirus construct. The recombinant the curve and were quantified relative to GAPDH as the control adenovirus (Rad) Fmod and Dcn expression cassettes housekeeping gene (27). All experiments were performed in were constructed by Dr Paul Kingston (Gene Therapy three independent experiments with 60˚C as the annealing Unit, University of Manchester, Manchester, UK), and temperature. The amplification process included 95˚C for contain the major immediate-early murine cytomegalovirus 5 min, followed by 35 cycles at 95˚C for 10 sec and 60˚C for enhancer/promoter, Woodchuck hepatitis virus regulatory 30 sec. The primers were as follows: Mouse TGF-β1 forward, element and a fragment of the rabbit smooth muscle myosin 5'-GGT AAC CGG CTG CTG ACC-3; mouse TGF-β1 reverse, heavy chain promoter, which produces increased transgene 5'-GCC CTG TAT TCC GTC TCC TTG-3'; mouse GAPDH expression compared with other expression vectors. Rad forward, 5'-GGC CTT CCG TGT TCC TAC-3'; mouse GAPDH vectors are E1/E3‑deleted first‑generation adenoviruses that reverse, 5'-TGT CAT CAT ACT TGG CAG GTT-3'. have a recombinant transgene and promoter inserted instead of possessing an E1 region (13). The efficiency of these vectors Nuclear extract and NF‑κB transbinding assay. A total of 1x106 was confirmed in a previous study (13). cells/ml 4T1 cells were collected and nuclear extracts were isolated using the Nuclear Extract kit from Active Motif GmbH Cell culture. The highly metastatic 4T1 breast cancer cell line (Regensburg, Germany) according to the manufacturer's was obtained from Pasture Institute of Iran (Tehran, Iran) protocol. Nuclear extracts were stored at ‑80˚C until they were and cultured in RPMI-1,640 (Sigma-Aldrich; Merck KGaA, used, and their concentration was measured using a Bradford Darmstadt, Germany) supplemented with 10% fetal bovine assay. NF‑κB DNA binding activity was determined using an serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, Trans‑AM P65‑NF‑κB ELISA‑based kit (cat number 40096; MA, USA) in 5% CO2 at 37˚C. Cells were not allowed to be Active Motif GmbH), which is a sensitive assay that measures >80% confluent, and 4x105 cells were treated with Rad-Fmod, the quantity of activated NF‑κB in the nuclear extracts from the Rad-Dcn or Rad-LacZ at a multiplicity of infection (MOI) 4T1 breast cancer cell line, prior to and following Fmod and of 1,000, which was considered an appropriate MOI for Dcn expression. In total, 10 µg nuclear extract was added to a this adenovirus (25). Cells were incubated for 4 h at 37˚C biotinylated oligonucleotide containing the NF‑κB consensus and medium was regularly replaced with fresh RPMI-1,640 site attached to the streptavidin‑coated 96‑well plates. Plates (Sigma-Aldrich; Merck KGaA). After 72 h, cells were used for were washed with wash buffer (cat number 40096; Active further analysis. Uninfected cells cultured in the same condi- Motif GmbH) to remove all the unbound reagents; to visualize tions were used as a negative control. NF‑κB DNA binding, an anti‑p65 primary antibody (dilution, 1:2,000; cat number 40096; Active Motif GmbH) was added for Reverse transcription‑polymerase chain reaction (RT‑PCR). 1 h at room temperature without agitation, followed by a goat The 4T1 cell line total RNA was extracted using an RNeasy anti‑rabbit secondary antibody conjugated with horseradish Mini kit (Qiagen GmbH, Hilden, Germany) according to the peroxidase (dilution, 1:5,000; cat number 40096; Active Motif manufacturer's protocol. RNA was reverse-transcribed with GmbH) at room temperature without agitation. Subsequent to a OneStep RT‑PCR kit (Qiagen GmbH, Germany) at 50˚C an incubation for 1 h at room temperature, 100 µl developing for 30 min with initial PCR activation at 95˚C for 15 min. solution (cat number 40096; Active Motif GmbH) was added to cDNA was amplified by 35 cycles, each consisting of three all wells for 5 min at room temperature protected from direct steps: Denaturation at 94˚C for 45 sec, annealing at 63˚C for light. The blue color development in the sample wells was 45 sec, extension at 72˚C for 1 min and final extension at 72˚C monitored until it turned medium to dark blue. Subsequently, for 10 min. Specific primers (Metabion GmbH, Steinkirchen, 100 µl stop solution (cat number 40096; Active Motif GmbH) Germany) for Dcn (forward primer, 5'-CCC AGA AGT TCC was added and the blue color turned yellow. Finally, the absor‑ TGA TGA C-3'; reverse primer, 5'-CAG AGC GCA CGT AGA bance value was ascertained using a spectrophotometer at a CAC-3'), Fmod (forward primer, 5'-TGA AGG CAG CAC CTG wavelength of 450 nm. For the p65 positive control: 2.5 µg of ACC GC-3'; reverse primer, 5'-ACG CCT TGG CTT CTC CTG Jurkat nuclear extract was provided (1 µl of nuclear extract in CC-3') and β-actin as a control (forward primer, 5'-ATA TCG 19 µl of complete lysis buffer per well according to the Active CTG CGC TGG TCG TC-3'; reverse primer, 5'-AGG ATG GCG Motif kit protocol).
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