ABSTRACT
Human embryonic stem cell (hESC) and human-induced pluripotent stem cell (hiPSC) technologies have a critical role in regenerative strategies for personalized medicine. Both share the ability to differentiate into almost any cell type of the human body. The study of their properties and clinical applications requires the development of robust and reproducible cell culture paradigms that direct cell differentiation toward a specific phenotype in vitro and in vivo. Our group evaluated the potential of mouse ESCs (mESCs), hESCs, and hiPSCs (collectively named pluripotent stem cells, PSCs) to analyze brain microenvironments through the use of embryoid body (EB)-derived cells from these cell sources. EB are cell aggregates in 3D culture conditions that recapitulate embryonic development. Our approach focuses on studying the midbrain dopaminergic phenotype and transplanting EB into the substantia nigra pars compacta (SNpc) in a Parkinson's disease rodent model. Here, we describe cell culture protocols for EB generation from PSCs that show significant in vivo differentiation toward dopaminergic neurons.
Subject(s)
Induced Pluripotent Stem Cells , Pluripotent Stem Cells , Animals , Cell Differentiation/genetics , Embryoid Bodies , Embryonic Stem Cells , Humans , Mesencephalon , MiceABSTRACT
The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24 h exposure to ENN B1 at levels of 2.5-10 µmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5-1.7 µmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4 h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.
Subject(s)
Depsipeptides/toxicity , Intracellular Membranes/drug effects , Lysosomes/pathology , Mycotoxins/toxicity , Permeability/drug effects , Animals , Chaperone-Mediated Autophagy/drug effects , Fibroblasts , Gene Deletion , HSC70 Heat-Shock Proteins/drug effects , HSC70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/drug effects , HSP90 Heat-Shock Proteins/metabolism , Hydrogen-Ion Concentration/drug effects , Lysosomal-Associated Membrane Protein 2/drug effects , Lysosomal-Associated Membrane Protein 2/genetics , Lysosomal-Associated Membrane Protein 2/metabolism , Mice , Molecular Chaperones/drug effects , Molecular Chaperones/metabolismABSTRACT
BACKGROUND: Breast cancer is the second most common cancer worldwide. N, N, N', N'-Tetrakis (2-pyridylmethyl)-ethylenediamine (TPEN) is a lipid-soluble zinc metal chelator that induces apoptosis in cancer cells through oxidative stress (OS). However, the effectiveness and the mechanisms involved in TPENinduced cell death in mammary adenocarcinoma cells in vitro and in vivo are still unclear. OBJECTIVE: This study aimed to evaluate the cytotoxic effect of TPEN in mouse embryonic fibroblasts (MEFs, as normal control cells) and mammary adenocarcinoma cancer cells (TS/A cells) in vitro and in a mammary tumor model in vivo. METHODS: Cells were treated with TPEN (0-3 µM), and changes in nuclear chromatin and DNA, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species (ROS) levels were determined by both fluorescence microscopy and flow cytometry. Cell proliferation and the cell cycle were also analyzed. Cellular markers of apoptosis were evaluated by Western blot. Finally, the effect of TPEN in a mammary adenocarcinoma tumor model in vivo was determined by immunohistological analyses. RESULTS: TPEN induced apoptosis in TS/A cells in a dose-dependent manner, increasing nuclear chromatin condensation, DNA fragmentation, cell cycle arrest and ΔΨm loss. Additionally, TPEN increased dichlorofluorescein fluorescence (DCF+) intensity, indicative of ROS production; increased DJ-1-Cys106-sulfonate expression, a marker of intracellular H2O2 stress; induced p53 and PUMA upregulation; and activated caspase-3. Moreover, TPEN induced mammary cancer cell elimination and tumor size reduction in vivo 48 h after treatment through an OS-induced apoptotic mechanism. CONCLUSION: TPEN selectively induces apoptosis in TS/A cells through an H2O2-mediated signaling pathway. Our findings support the use of TPEN as a potential treatment for breast cancer.