Seung-Kyou Lee

Intranasal delivery of the cytoplasmic domain of CTLA-4 using a novel protein transduction domain prevents allergic inflammation

CTLA-4 is a negative regulator of T-cell activation, and its inhibitory effects can be accomplished either by competition with CD28 or by transmitting negative signals through its intracellular domain. To utilize the cytoplasmic domain of CTLA-4 to suppress allergic inflammation, we fused it to a novel protein-transduction domain in the human transcriptional factor Hph-1. Transduction efficiency was verified in vitro and in vivo after ocular, intranasal and intradermal administration. After transduction into T cells, the Hph-1–ctCTLA-4 fusion protein inhibited the production of interleukin (IL)-2, and downregulated CD69 and CD25. Intranasal administration of Hph-1–ctCTLA-4 resulted in markedly reduced infiltration of inflammatory cells, secretion of T helper type 2 (TH2) cytokines, serum IgE levels and airway hyper-responsiveness in a mouse model of allergic airway inflammation. These results indicated that Hph-1–ctCTLA-4 constitutes an effective immunosuppressive protein drug for potential use in the treatment of allergic asthma, via nasal administration.

Mesenchymal Stem Cells Pretreated with Delivered Hph‐1‐Hsp70 Protein Are Protected from Hypoxia‐Mediated Cell Death and Rescue Heart Functions from Myocardial Injury

Mesenchymal stem cell (MSC) therapy for myocardial injury has inherent limitations due to the poor viability of MSCs after cell transplantation. In this study, we directly delivered Hsp70, a protein with protective functions against stress, into MSCs, using the Hph‐1 protein transduction domain ex vivo for high transfection efficiency and low cytotoxicity. Compared to control MSCs in in vitro hypoxic conditions, MSCs delivered with Hph‐1‐Hsp70 (Hph‐1‐Hsp70‐MSCs) displayed higher viability and anti‐apoptotic properties, including Bcl2 increase, reduction of Bax, JNK phosphorylation and caspase‐3 activity. Hsp70 delivery also attenuated cellular ATP‐depleting stress. Eight animals per group were used for in vivo experiments after occlusion of the left coronary artery. Transplantation of Hph‐1‐Hsp70‐MSCs led to a decrease in the fibrotic heart area, and significantly reduced the apoptotic positive index by 19.5 ± 2%, compared to no‐treatment controls. Hph‐1‐Hsp70‐MSCs were well‐integrated into the infarcted host myocardium. The mean microvessel count per field in the infarcted myocardium of the Hph‐1‐Hsp70‐MSC‐treated group (122.1 ± 13.5) increased relative to the MSC‐treated group (75.9 ± 10.4). By echocardiography, transplantation of Hph‐1‐Hsp70‐MSCs resulted in additional increases in heart function, compared to the MSCs‐transplanted group. Our results may help formulate better clinical strategies for in vivo MSC cell therapy for myocardial damage. STEM CELLS 2009;27:2283–2292

Tautomycetin inhibits growth of colorectal cancer cells through p21cip/WAF1 induction via the extracellular signal–regulated kinase pathway

Tautomycetin is an antifungal antibiotic retaining potent immunosuppressive function. We have identified the roles of tautomycetin on cellular proliferation and transformation of colorectal cancer cells. The proliferation and anchorage-independent growth of HCT-15, HT-29, and DLD-1 colorectal cancer cells were efficiently inhibited without induction of apoptosis by 150 nmol tautomycetin. These growth inhibitory effects were dependent on p21Cip/WAF induction via the extracellular signal–regulated kinase pathway, and the tautomycetin effects were abolished in HCT-116 colon cells and eight other types of cells that did not induce p21Cip/WAF by 150 nmol tautomycetin. The crucial role of p21Cip/WAF1 in the extracellular signal–regulated kinase pathway–dependent antiproliferative responses by tautomycetin was confirmed by using p21Cip/WAF1 gene–deleted HCT-116 cells. The growth inhibitory effect of tautomycetin was acquired by regulation of Raf-1 activity through inhibition of protein phosphatase type 1 and protein phosphatase type 2A with high preference toward protein phosphatase type 1. Tautomycetin could be a potential drug for colorectal cancer. [Mol Cancer Ther 2006;5(12):3222–31]

Alleviation of rheumatoid arthritis by cell-transducible methotrexate upon transcutaneous delivery

Rheumatoid arthritis (RA) is a systemic autoimmune disease that is initiated and maintained by various inflammatory/immune cells and their cytokines, leading to cartilage degradation and bone erosion. Despite its potent therapeutic efficacy on RA, the oral administration of methotrexate (MTX) provokes serious adverse systemic complications, thus necessitating the local application of MTX. Here, we show that transcutaneous MTX (TC-MTX) can efficiently penetrate joint skin ex vivo and in vivo, and that TC-MTX can significantly improve the various inflammatory symptoms associated with RA. Further, TC-MTX preserved the joint-structures in mice with collagen-induced arthritis (CIA), which was also confirmed by three-dimensional micro-computed tomography scan. TC-MTX markedly decreased the secretion of inflammatory cytokines both in the serum and in inflamed joints of CIA mice. Further, its therapeutic potential is comparable to that of etanercept, a biological agent that block tumor necrosis factor (TNF)-α. Importantly, the systemic cytotoxicity of TC-MTX was not detected. Thus, TC-MTX can be a new therapeutic modality for RA patients without systemic complications.