ABSTRACT
Although chronic wounds are common and continue to be a major cause of morbidity and mortality, treatments for these conditions are lacking and often ineffective. A large body of evidence exists demonstrating the therapeutic potential of mesenchymal stem cells (MSCs) for repair and regeneration of damaged tissue, including acceleration of cutaneous wound healing. However, the exact mechanisms of wound healing mediated by MSCs are unclear. In this study, we examined the role of MSC exosomes in wound healing. We found that MSC exosomes ranged from 30 to 100-nm in diameter and internalization of MSC exosomes resulted in a dose-dependent enhancement of proliferation and migration of fibroblasts derived from normal donors and chronic wound patients. Uptake of MSC exosomes by human umbilical vein endothelial cells also resulted in dose-dependent increases of tube formation by endothelial cells. MSC exosomes were found to activate several signaling pathways important in wound healing (Akt, ERK, and STAT3) and induce the expression of a number of growth factors [hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), nerve growth factor (NGF), and stromal-derived growth factor-1 (SDF1)]. These findings represent a promising opportunity to gain insight into how MSCs may mediate wound healing.
Subject(s)
Exosomes/metabolism , MAP Kinase Signaling System/physiology , Mesenchymal Stem Cells/cytology , Neovascularization, Physiologic/physiology , Wound Healing/physiology , Aged , Cell Movement/physiology , Cell Proliferation/physiology , Cells, Cultured , Chemokine CXCL12/metabolism , Coculture Techniques , Diabetes Mellitus , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/metabolism , Hepatocyte Growth Factor/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Insulin-Like Growth Factor I/metabolism , Male , Nerve Growth Factor/metabolism , Proto-Oncogene Proteins c-akt/metabolism , STAT3 Transcription Factor/metabolismABSTRACT
Topical application of therapeutic agents has been a mainstay in Dermatology for the treatment of skin disorders but is not commonly used for systemic delivery. For a topically applied agent to reach distant body sites it must first overcome the barrier function of the skin and then penetrate into deeper structures before reaching the systemic circulation. This has limited the use of topically applied agents to those having specific charge, solubility and size restrictions. Pretreatment of the skin with ablative fractional laser appears to enhance the uptake of some topically applied drugs but the ability to effectively deliver agents to distant sites is largely unproven. In this report we used a fractional ablative Erb:YAG (Erbium/Yttrium Aluminum Garnet) laser to facilitate the transfer of bone marrow stem cells through the skin in a murine bone marrow transplant model. Chimerism could be detected in the peripheral blood of recipient C57BL/6 mice that were pretreated with ablative fractional laser and had topically applied enhanced green fluorescent protein (GFP) labeled bone marrow cells from syngeneic donor transgenic mice. This study indicates that fractional laser can be used to deliver stem cells through the skin and remain functionally intact.
Subject(s)
Ablation Techniques/methods , Bone Marrow Transplantation/methods , Lasers, Solid-State , Skin , Animals , Female , Green Fluorescent Proteins/genetics , Male , Mice , Mice, Inbred C57BLABSTRACT
FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R. Previously, using virtual screening and functional testing, we identified a lead compound (INT2-31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo. INT2-31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2-31 significantly inhibited cell proliferation and viability (range 0.05-10 µM). More importantly, 15 mg/kg of INT2-31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo. Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2-31) has potential anti-neoplastic therapeutic effects in human melanoma.
Subject(s)
Focal Adhesion Kinase 1/metabolism , Melanoma/physiopathology , Protein Kinase Inhibitors/pharmacology , Purine Nucleosides/pharmacology , Receptor, IGF Type 1/metabolism , Signal Transduction/drug effects , Apoptosis/drug effects , Blotting, Western , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Flow Cytometry , Humans , Immunohistochemistry , Immunoprecipitation , In Situ Nick-End Labeling , Melanoma/drug therapy , Melanoma/metabolism , Protein Kinase Inhibitors/therapeutic use , Purine Nucleosides/therapeutic use , RNA, Small Interfering/genetics , Signal Transduction/physiologyABSTRACT
INTRODUCTION: Esophageal cancer remains an aggressive disease with poor survival rates. FAK and IGF-1R are two important tyrosine kinases important for cell survival signaling and found to be upregulated in esophageal cancer. Our hypothesis is that a novel small molecule compound that disrupts FAK and IGF-1R protein-protein interactions (PPIs) would decrease the growth of human esophageal cancer. METHODS: The compound INT2-31 (NSC344553) was identified from a virtual high throughput screen to bind to FAK and disrupt PPIs. The in vitro effects of this compound, +/- 5-FU chemotherapy, on cell signaling, viability and apoptosis in human esophageal cancer cells (KYSE 70, 140) and a direct esophageal cancer xenograft was evaluated. RESULTS: INT2-31 caused a disruption of PPIs between FAK and IGF-1R starting at a concentration of 1µM. It also caused a dose dependent inhibition of cell viability and induction of apoptosis at low micromolar doses. These effects were associated with decreased AKT and ERK1/ERK2 phosphorylation. INT2-31 treatment, when administered via IP injection, at 50mg/kg, resulted in an in vivo decrease in tumor growth in a direct xenograft. Furthermore, treatment with 5-FU chemotherapy combined with INT2-31 resulted in a synergistic increase in apoptosis and decrease in tumor growth compared to 5-FU or INT2-31 alone. CONCLUSIONS: A novel compound that disrupts the PPIs of FAK and IGF-1R results in decreased tumor proliferation and increased apoptosis. These effects appear to be mediated through downregulation of p-AKT and p-ERK. This compound deserves further study as a novel treatment strategy in patients with esophageal cancer.
