Sung-Ho Kim

Proteomic Analysis of Differently Expressed Proteins in a Mouse Model for Allergic Asthma

Allergic asthma is associated with persistent functional and structural changes in the airways and involves many different cell types. Many proteins involved in allergic asthma have been identified individually, but complete protein profiles (proteome) have not yet been reported. Here we have used a differential proteome mapping strategy to identify tissue proteins that are differentially expressed in mice with allergic asthma and in normal mice. Mouse lung tissue proteins were separated using two-dimensional gel electrophoresis over a pH range between 4 and 7, digested, and then analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS). The proteins were identified using automated MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. This approach identified 15 proteins that were differentially expressed in the lungs of mice with allergic asthma and normal mice. All 15 proteins were identified by MS, and 9 could be linked to asthma-related symptoms, oxidation, or tissue remodeling. Our data suggest that these proteins may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy.

Knockdown of Bcl-xL Enhances Growth-Inhibiting and Apoptosis-Inducing Effects of Resveratrol and Clofarabine in Malignant Mesothelioma H-2452 Cells

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma. Graphical Abstract

Activated Charcoal Suppresses Breeding of the House Dust Mite, Dermatophagoides Pteronyssinus, in Culture

House dust mite sensitized asthmatics are advised to practice allergen avoidance. Charcoal pillows are used in Korea with unsubstantiated claims regarding their efficacy in alleviating asthma symptoms. We tested the effects of activated charcoal on breeding of house dust mites in culture. Twenty live adult house dust mites (Dermatophagoides pteronyssinus) were inoculated, 10 replicates, on culture media containing 0%, 1%, 3%, 5%, 10%, and 20% activated charcoal and incubated at 25℃ and a relative humidity of 75%. After four weeks, the mean numbers of live house dust mites were 286, 176, 46, 16, 7, and 0 for the 0%, 1%, 3%, 5%, 10%, and 20% charcoal-containing culture media, respectively. Thus, activated charcoal suppresses breeding of house dust mites and offers a new promising method for house dust mite control.

Silibinin attenuates radiation-induced intestinal fibrosis and reverses epithelial-to-mesenchymal transition

Radiotherapy is a common treatment for cancer patients, but its use is often restricted by the tolerance of normal tissue. As cancer patients live longer, delayed radiation effects on normal tissue have become a concern. Radiation-induced enteropathy, including inflammatory bowel disease and fibrosis, are major issues for long-term cancer survivors. To investigate whether silibinin attenuates delayed radiation-induced intestinal injury in mice, we focused on intestinal fibrotic changes. Silibinin improved delayed radiation injuries in mice in association with decreased collagen deposition within the intestines and deceased transforming growth factor (TGF)-β1 levels in the intestine and plasma. Treating mice bearing CT26 mouse colon cancer tumors with both silibinin and radiation stimulated tumor regression more than radiation alone. We also investigated the effect of silibinin on the radiation-induced epithelial-to-mesenchymal transition (EMT), the primary mechanism of fibrosis. We assessed changes in E-cadherin, N-cadherin, and α-smooth muscle actin expression, and demonstrated that silibinin attenuates radiation-induced EMT. Irradiating intestinal epithelial cells increased TGF-β1 levels, but silibinin suppressed TGF-β1 expression by inhibiting Smad2/3 phosphorylation. These results suggest silibinin has the potential to serve as a useful therapeutic agent in patients with radiation-induced intestinal fibrosis.

Cariporide Enhances the DNA Damage and Apoptosis in Acid-tolerable Malignant Mesothelioma H-2452 Cells

The Na+/H+ exchanger is responsible for maintaining the acidic tumor microenvironment through its promotion of the reabsorption of extracellular Na+ and the extrusion of intracellular H+. The resultant increase in the extracellular acidity contributes to the chemoresistance of malignant tumors. In this study, the chemosensitizing effects of cariporide, a potent Na+/H+-exchange inhibitor, were evaluated in human malignant mesothelioma H-2452 cells preadapted with lactic acid. A higher basal level of phosphorylated (p)-AKT protein was found in the acid-tolerable H-2452AcT cells compared with their parental acid-sensitive H-2452 cells. When introduced in H-2452AcT cells with a concentration that shows only a slight toxicity in H-2452 cells, cariporide exhibited growth-suppressive and apoptosis-promoting activities, as demonstrated by an increase in the cells with pyknotic and fragmented nuclei, annexin V-PE(+) staining, a sub-G0/G1 peak, and a G2/M phase-transition delay in the cell cycle. Preceding these changes, a cariporide-induced p-AKT down-regulation, a p53 up-regulation, an ROS accumulation, and the depolarization of the mitochondrial-membrane potential were observed. A pretreatment with the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 markedly augmented the DNA damage caused by the cariporide, as indicated by a much greater extent of comet tails and a tail moment with increased levels of the p-histone H2A.X, p-ATMSer1981, p-ATRSer428, p-CHK1Ser345, and p-CHK2Thr68, as well as a series of pro-apoptotic events. The data suggest that an inhibition of the PI3K/AKT signaling is necessary to enhance the cytotoxicity toward the acid-tolerable H-2452AcT cells, and it underlines the significance of proton-pump targeting as a potential therapeutic strategy to overcome the acidic-microenvironment-associated chemotherapeutic resistance.

Geranylgeranylacetone Ameliorates Intestinal Radiation Toxicity by Preventing Endothelial Cell Dysfunction

Radiation-induced intestinal toxicity is common among cancer patients after radiotherapy. Endothelial cell dysfunction is believed to be a critical contributor to radiation tissue injury in the intestine. Geranylgeranylacetone (GGA) has been used to treat peptic ulcers and gastritis. However, the protective capacity of GGA against radiation-induced intestinal injury has not been addressed. Therefore, we investigated whether GGA affects intestinal damage in mice and vascular endothelial cell damage in vitro. GGA treatment significantly ameliorated intestinal injury, as evident by intestinal crypt survival, villi length and the subsequently prolonged survival time of irradiated mice. In addition, intestinal microvessels were also significantly preserved in GGA-treated mice. To clarify the effect of GGA on endothelial cell survival, we examined endothelial function by evaluating cell proliferation, tube formation, wound healing, invasion and migration in the presence or absence of GGA after irradiation. Our findings showed that GGA plays a role in maintaining vascular cell function; however, it does not protect against radiation-induced vascular cell death. GGA promoted endothelial function during radiation injury by preventing the loss of VEGF/VEGFR1/eNOS signaling and by down-regulating TNFα expression in endothelial cells. This finding indicates the potential impact of GGA as a therapeutic agent in mitigating radiation-induced intestinal damage.