ABSTRACT
Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features, despite some controversies in age-related studies. GDF11 has been poorly investigated in cancer, particularly in those with stemness capacity, such as hepatocellular carcinoma (HCC), one of the most aggressive cancers worldwide. Here, we focused on investigating the effects of GDF11 in liver cancer cells. GDF11 treatment significantly reduced proliferation, colony and spheroid formation in HCC cell lines. Consistently, down-regulation of CDK6, cyclin D1, cyclin A, and concomitant upregulation of p27 was observed after 24â¯h of treatment. Interestingly, cell viability was unchanged, but cell functionality was compromised. These effects were potentially induced by the expression of E-cadherin and occludin, as well as Snail and N-cadherin repression, in a time-dependent manner. Furthermore, GDF11 treatment for 72â¯h induced that cells were incapable of sustaining colony and sphere capacity in the absent of GDF11, up to 5â¯days, indicating that the effect of GDF11 on self-renewal capacity is not transient. Finally, in vivo invasion studies revealed a significant decrease in cell migration of hepatocellular carcinoma cells treated with GDF11 associated to a decreased proliferation judged by Ki67 staining. Data show that exogenous GDF11 displays tumor suppressor properties in HCC cells.
Subject(s)
Bone Morphogenetic Proteins/pharmacology , Cell Movement/drug effects , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic , Growth Differentiation Factors/pharmacology , Neovascularization, Pathologic/prevention & control , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , Cadherins/genetics , Cadherins/metabolism , Cell Differentiation/drug effects , Cell Line, Tumor , Chick Embryo , Chorioallantoic Membrane/blood supply , Chorioallantoic Membrane/drug effects , Cyclin A/genetics , Cyclin A/metabolism , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Growth Differentiation Factors/genetics , Growth Differentiation Factors/metabolism , Hep G2 Cells , Humans , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Occludin/genetics , Occludin/metabolism , Signal Transduction , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolism , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Spheroids, Cellular/pathologyABSTRACT
Biosensor technology has great potential for the detection of cancer through tumor-associated molecular biomarkers. In this work, we describe the immobilization of the recombinant humanized anti-HER2 monoclonal antibody (trastuzumab) on a silver nanostructured plate made by pulsed laser deposition (PLD), over a thin film of Au(111). Immobilization was performed via 4-mercapto benzoic acid self-assembled monolayers (4-MBA SAMs) that were activated with coupling reagents. A combination of immunofluorescence images and z-stack analysis by confocal laser scanning microscopy (CLSM) allowed us to detect HER2 presence and distribution in the cell membranes. Four different HER2-expressing breast cancer cell lines (SKBR3 +++, MCF-7 +/-, T47D +/-, MDA-MB-231 -) were incubated during 24 h on functionalized silver nanostructured plates (FSNP) and also on Au(111) thin films. The cells were fixed by means of an ethanol dehydration train, then characterized by atomic force microscopy (AFM) and surface-enhanced Raman scattering (SERS). SERS results showed the same tendency as CLSM findings (SKBR3 > MCF-7 > T47D > MDA-MB-231), especially when the Raman peak associated with phenylalanine amino acid (1002 cm-1) was monitored. Given the high selectivity and high sensitivity of SERS with a functionalized silver nanostructured plate (FSNP), we propose this method for identifying the presence of HER2 and consequently, of breast cancer cells.
