Eung-Ryoung Lee

Differentiation and Transplantation of Functional Pancreatic Beta Cells Generated from Induced Pluripotent Stem Cells Derived from a Type 1 Diabetes Mouse Model

The nonobese diabetic (NOD) mouse is a classical animal model for autoimmune type 1 diabetes (T1D), closely mimicking features of human T1D. Thus, the NOD mouse presents an opportunity to test the effectiveness of induced pluripotent stem cells (iPSCs) as a therapeutic modality for T1D. Here, we demonstrate a proof of concept for cellular therapy using NOD mouse-derived iPSCs (NOD-iPSCs). We generated iPSCs from NOD mouse embryonic fibroblasts or NOD mouse pancreas-derived epithelial cells (NPEs), and applied directed differentiation protocols to differentiate the NOD-iPSCs toward functional pancreatic beta cells. Finally, we investigated whether the NPE-iPSC-derived insulin-producing cells could normalize hyperglycemia in transplanted diabetic mice. The NOD-iPSCs showed typical embryonic stem cell-like characteristics such as expression of markers for pluripotency, in vitro differentiation, teratoma formation, and generation of chimeric mice. We developed a method for stepwise differentiation of NOD-iPSCs into insulin-producing cells, and found that NPE-iPSCs differentiate more readily into insulin-producing cells. The differentiated NPE-iPSCs expressed diverse pancreatic beta cell markers and released insulin in response to glucose and KCl stimulation. Transplantation of the differentiated NPE-iPSCs into diabetic mice resulted in kidney engraftment. The engrafted cells responded to glucose by secreting insulin, thereby normalizing blood glucose levels. We propose that NOD-iPSCs will provide a useful tool for investigating genetic susceptibility to autoimmune diseases and generating a cellular interaction model of T1D, paving the way for the potential application of patient-derived iPSCs in autologous beta cell transplantation for treating diabetes.

Sustained ERK activation is involved in the kaempferol-induced apoptosis of breast cancer cells and is more evident under 3-D culture condition

In order to determine the effects of a variety of flavonoids, we applied differing amounts of several flavonoids to human breast cancer cells. Kaempferol treatment resulted in significant reduction of cell viability in the MCF-7 cells, although it exerted only minor effect on the cell viability of MDA-MB-231 or mammary epithelial HC-11 cells. Kaempferol was demonstrated to induce sustained ERK activation concomitantly with MEK1 and ELK1 activation, and this kaempferol-induced apoptosis was suppressed by treatment with PD98059, the overexpression of a kinase-inactive ERK mutant, or ERK siRNA. Kaempferol treatment was shown to profoundly induce the generation of fluorescent DCF in the MCF-7 cells, and treatment with N-acetyl cysteine suppressed kaempferol-induced PARP cleavage. Moreover, because breast cancer is associated with increased collagen synthesis and accumulation, we utilized a collagen-based 3D culture method. Under the 3-dimensional culture condition employed herein, kaempferol treatment was shown to result in a significant reduction in cell viability, an effect which occurred in a dose-dependent manner. Compared with what was observed under conventional 2D culture condition, we observed more evident apoptotic cell death and ERK activation as the result of kaempferol treatment in a collagen-based 3D culture environment. Similar to the case of conventional 2D cultured cells, the addition of PD98059 significantly suppressed intracellular ROS production. Collectively, these results show that the sustained activation of the ERK signaling pathway is markedly involved in kaempferol-induced apoptosis of breast cancer MCF-7 cells, and that this effect is more evident under 3D culture condition.

Effect of flavonoids on human health: old subjects but new challenges.

Flavonoids are highly diversified plant pigments that are present in a wide range of fruits, vegetables, nuts, and beverages. They are regularly consumed in the human diet and have various biological activities including anti-inflammatory, anti-cancer, and anti-viral properties. The flavonoids maybe one of the safest non-immunogenic drugs because they are small organic compounds which have been normally absorbed by the human body for long time. During the past decades, the patents on their health effects have inflated very much and the yearly number of the patents is on an increasing trend. This review summarizes the current patents on the health effects of various flavonoids, and suggests the possible expectation that a wide variety of diseases are successful treated with newly-developed specific flavonoids or their derivatives in the near future. In recent patents, specific flavonoids were described to function as anti-oxidants, enzyme inhibitors, hormones, or immune modulators. Moreover, the recent patents also tried to provide the molecular mechanism of the flavonoid compounds on treating or preventing various human diseases. Recent mechanistic studies in molecular level make it possible that specific flavonoids are identified to have a wide range of biological properties that can contribute to the beneficial effects on human health.

Self-renewal of embryonic stem cells through culture on nanopattern polydimethylsiloxane substrate.

Embryonic stem (ES) cells can undergo continual proliferation and differentiation into cells of all somatic cell lineages in vitro; they are an unlimited cell source for regenerative medicine. However, techniques for maintaining undifferentiated ES cells are often inefficient and result in heterogeneous cell populations. Here, we determined effects of nanopattern polydimethylsiloxane (PDMS) as a culture substrate in promoting the self-renewal of mouse ES (mES) cells, compared to commercial plastic culture dishes. After many passages, mES cells efficiently maintained their undifferentiated state on nanopattern PDMS, but randomly differentiated on commercial plastic culture dishes, as indicated by partially altered morphologies and decreases in alkaline phosphatase activity and stage-specific expression of embryonic antigen-1. Under nanopattern PDMS conditions, we found increased activities of STAT3 and Akt, important proteins involved in maintaining the self-renewal of mES cells. The substrate-cell interactions also enhanced leukemia inhibitory factor (LIF)-downstream signaling and inhibited spontaneous differentiation, concomitant with reduced focal adhesion kinase (FAK) signaling. This reduction in FAK signaling was shown to be important for promoting mES cell self-renewal. Thus, our data demonstrates that nanopattern PDMS contributes to maintaining the self-renewal of mES cells and may be applicable in the large-scale production of homogeneously undifferentiated mES cells.

Involvement of caspase-9 in autophagy-mediated cell survival pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been considered for use in the prevention and treatment of cancer malignancy. FR122047 (FR) is known to have an anti-inflammatory effect, but the anticancer activity of the chemical has not yet been identified. In the present study, we could find that treatment of breast cancer MCF-7 cells with FR led to apoptosis accompanying with apparent activation of caspases. Treatment of caspase-specific inhibitors revealed that FR-induced apoptosis was caspase-8-dependent and inhibition of caspase-9 activity resulted in unexpected, marked enhancement of cell death. Knockdown of caspase-9 expression by specific siRNA caused increased susceptibility to FR-induced cell death, consistent with the results obtained with treatment of caspase-9 inhibitor. Inhibition of caspase-9 blocked the autophagic process by modulating lysosomal pH and acid-dependent cathepsin activities and augmented cell death due to blockage of cytoprotective autophagy. MCF-7 cells treated with sulforaphane, an autophagy-inducing drug, also showed marked accumulation of LC3-II, and co-treatment with caspase-9 inhibitor brought about increased susceptibility to sulforaphane-induced cell death. Different from the cases with FR or sulforaphane, etoposide- or doxorubicin-induced cell death was suppressed with co-treatment of caspase-9 inhibitor, and the drugs failed to induce significant autophagy in MCF-7 cells. Taken together, our data originally suggest that inhibition of caspase-9 may block the autophagic flux and enhance cell death due to blockage of cytoprotective autophagy.

Regulation of apoptosis by modified naringenin derivatives in human colorectal carcinoma RKO cells

Flavonoids are micronutrients that are widely detected in foods of plant origin and have been ascribed pharmacological properties. Several biological functions of flavonoids have been thus far identified, whereas there currently exists a lack of evidence to support the relationship between the structure‐activity relationship and apoptosis‐inducing activity. In an attempt to determine the importance of the OH group or substitution of the 5‐ or 7‐carbon in the diphenylpropane skeleton of flavonoids, we selected 14 different flavonoids with different structures, particularly with regard to the 5‐ or 7‐carbon, and found that naringenin treatment caused a slight decrease in the cell viability of the human colorectal carcinoma RKO cells. Next, in order to characterize the effects of specific substitutions of the 7‐carbon of naringenin on apoptosis‐regulatory activities, and in an attempt to develop anti‐proliferative flavonoid derivatives that would be more effective against colon cancer, we originally synthesized several modified naringenin derivatives (MNDs) including 7‐O‐benzyl naringenin (KUF‐1) and 7‐O‐(m‐metoxybenzyl) naringenin (KUF‐2). Treatment with KUF‐1 or KUF‐2 resulted in significant apoptosis‐inducing effects concomitant with losses in mitochondrial membrane potential, caspase activation, intracellular ROS production, and sustained ERK activation. Our data show that KUF‐1 or KUF‐2 regulate the apoptosis of RKO cells via intracellular ROS production coupled with the concomitant activation of the ERK signaling pathway, thereby implying that hydroxylation or substitution at C7 is critical for the apoptosis‐inducing activity of flavonoids. J. Cell. Biochem. 104: 259–273, 2008.

Cytoprotective Effect of Eriodictyol in UV-irradiated Keratinocytes via Phosphatase-dependent Modulation of both the p38 MAPK and Akt Signaling Pathways

Although flavonoids exhibit a variety of beneficial biological activities, the exact molecular mechanism of the cellular effects is still not fully explained. In this study, we investigated the molecular mechanism of cytoprotective effect of eriodictyol in UV-irradiated keratinocytes. We found that treatment with eriodictyol effectively suppressed the UV-induced cell death of the keratinocytes, concomitant with the inhibition of pro-caspase-3 or pro-caspase-9 cleavage and the suppression of cytochrome C release. The phosphorylation of p38 MAPK was suppressed during UV-induced apoptosis of the keratinocytes and eriodictyol could reverse the down-regulation of p38 MAPK upon UV irradiation. Inhibition of p38 MAPK activity by SB202190, or over-expression of dominant-negative mutant form of p38 MAPK resulted in suppression of cytoprotective effect of the flavonoid. PP2A appeared to participate in the regulation of both p38 MAPK and Akt activities by directly associating with the kinases. UV treatment stimulated not only the phosphatase activity, but also its association with p38 MAPK or Akt. Interestingly, eriodictyol reversed the increase in PP2A activity and the association between the proteins. Taken together, these findings suggest that eriodictyol may lead to protection of keratinocytes from UV-induced cytotoxicity by modulating both the p38 MAPK and Akt signaling pathways in a phosphatase-dependent manner.

Combined treatment of 3-hydroxyflavone and imatinib mesylate increases apoptotic cell death of imatinib mesylate-resistant leukemia cells

Imatinib mesylate, a Bcr/Abl tyrosine kinase inhibitor, is widely used in treating chronic myeloid leukemia. However, drug-resistance of leukemia cells becomes an emergent problem. Herein, various flavonoids were screened for applicability in leukemia treatment, and 3-hydroxyflavone (3-HF) was found to be most effective in reducing cancer cell viability. The combination of 3-HF and imatinib mesylate resulted in significant apoptotic cell death in imatinib mesylate-resistant leukemia cells. Combined treatment resulted in apparent activation of caspases and decrease of the oncoprotein phosphor-Bcr/Abl in leukemia cells. Our results suggest that this combined treatment is beneficial in imatinib mesylate-resistant chronic myelogenous leukemia.

Analysis of flavonoid contents and expression of flavonoid biosynthetic genes in Populus euramericana Guinier in response to abiotic stress

Gene encoding the key flavonoid biosynthetic enzyme, chalcone synthase, in Populus euramericana Guinier (PeCHS) was cloned and characterized. PeCHS preferentially uses feruloyl- CoA as a substrate. Expression of the flavonoid biosynthetic genes, phenylalanine ammonimum lyase from P. euramericana (PePAL), PeCHS, chalcone isomerase from P. euramericana (PeCHI), and, flavonol synthase from P. euramericana (PeFLS) from P. euramericana in response to abiotic stresses such as wounding and UV-irradiation were analyzed. These genes were induced under the stress conditions. Most genes were expressed at an early stage of the stress response. In addition, total flavonoid content increased after UV-irradiation.

Role of BI-1 (TEGT)-mediated ERK1/2 activation in mitochondria-mediated apoptosis and splenomegaly in BI-1 transgenic mice

Bax Inhibitor-1 (BI-1) is an evolutionally conserved apoptotic suppressor and belongs to the BI-1 family of proteins, which contain BI-1-like transmembrane domains. As their cellular functions and regulatory mechanisms remain incompletely understood, we compared their anti-apoptotic properties. Forced expression of BI-1 resulted in the most effective suppression of stress-induced apoptosis, compared with other family members, together with significant extracellular signal-regulated kinase (ERK)1/2 activation. BI-1-mediated ERK1/2 activation led to the suppression of mitochondria-mediated reactive oxygen species (ROS) production. Involvement of the ERK signaling pathway in BI-1-induced anti-apoptotic effects was confirmed by knockdown studies with ERK- or BI-1-specific siRNA. Moreover, we produced transgenic (TG) mice overexpressing BI-1, and the relationship between ERK1/2 activation and the suppression of ROS production or apoptosis was confirmed in mouse embryonic fibroblast (MEF) cells derived from these mice. Interestingly, we found that BI-1 TG mice showed splenomegaly and abnormal megakaryopoiesis. Taken together, our results suggest that BI-1-induced ERK1/2 activation plays an important role in the modulation of intracellular ROS generation and apoptotic cell death and may also affect autoimmune response.