Hong Jae Kim

Baicalein Induces Caspase-dependent Apoptosis Associated with the Generation of ROS and the Activation of AMPK in Human Lung Carcinoma A549 Cells.

Preclinical Research

Folic Acid is Necessary for Proliferation and Differentiation of C2C12 Myoblasts

Folic acid, a water soluble B vitamin, plays an important role in cellular metabolic activities, such as functioning as a cofactor in one‐carbon metabolism for DNA and RNA synthesis as well as nucleotide and amino acid biosynthesis in the body. A lack of dietary folic acid can lead to folic acid deficiency and result in several health problems, including macrocytic anemia, elevated plasma homocysteine, cardiovascular disease, birth defects, carcinogenesis, muscle weakness, and walking difficulty. However, the effect of folic acid deficiency on skeletal muscle development and its molecular mechanisms are unknown. We, therefore, investigated the effect of folic acid deficiency on myogenesis in skeletal muscle cells and found that folic acid deficiency induced proliferation inhibition and cell cycle breaking as well as cellular senescence in C2C12 myoblasts, implying that folic acid deficiency influences skeletal muscle development. Folic acid deficiency also inhibited differentiation of C2C12 myoblasts and induced deregulation of the cell cycle exit and many cell cycle regulatory genes. It inhibited expression of muscle‐specific marker MyHC as well as myogenic regulatory factor (myogenin). Moreover, immunocytochemistry and Western blot analyses revealed that DNA damage was more increased in folic acid‐deficient medium‐treated differentiating C2C12 cells. Furthermore, we found that folic acid resupplementation reverses the effect on the cell cycle and senescence in folic acid‐deficient C2C12 myoblasts but does not reverse the differentiation of C2C12 cells. Altogether, the study results suggest that folic acid is necessary for normal development of skeletal muscle cells.

Mori Folium water extract alleviates articular cartilage damages and inflammatory responses in monosodium iodoacetate‑induced osteoarthritis rats

Mori folium, the leaf of Morus alba L. (Moraceae), has been widely used in traditional medicine for the treatment of various diseases. It has been recently reported that Mori folium possesses potential chondroprotective effects in interleukin (IL)-1β-stimulated human chondrocytes; however, its protective and therapeutic potential against osteoarthritis (OA) in an animal model remains unclear. In this study, as part of an ongoing screening program to evaluate the anti-osteoarthritic potential of Mori folium, the protective effects of a water extract of Mori folium (MF) on cartilage degradation and inflammatory responses in a monosodium iodoacetate (MIA)-induced OA rat model were evaluated. The results demonstrated that administration of MF had a tendency to attenuate the damage to articular cartilage induced by MIA, as determined by knee joint swelling and the histological grade of OA. The elevated levels of matrix metalloproteinases-13 and two bio-markers for the diagnosis and progression of OA, such as the cartilage oligomeric matrix protein and C-telopeptide of type II collagen, were markedly ameliorated by MF administration in MIA-induced OA rats. In addition, MF significantly suppressed the production of pro-inflammatory cytokines, including IL-1β, IL-6 and tumor necrosis factor-α. MF also effectively inhibited the expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2, thus inhibiting the release of NO and prostaglandin E2. Although further work is required to fully understand the critical role and clinical usefulness, these findings indicate that MF may be a potential therapeutic option for the treatment of OA.

Induction of apoptosis by ethanol extract of Citrus unshiu Markovich peel in human bladder cancer T24 cells through ROS-mediated inactivation of the PI3K/Akt pathway

Citrus unshiu peel has been used to prevent and treat various diseases in traditional East-Asian medicine including in Korea. Extracts of C. unshiu peel are known to have various pharmacological effects including antioxidant, anti-inflammatory, and antibacterial properties. Although the possibility of their anti-cancer activity has recently been reported, the exact mechanisms in human cancer cells have not been sufficiently studied. In this study, the inhibitory effect of ethanol extract of C. unshiu peel (EECU) on the growth of human bladder cancer T24 cells was evaluated and the underlying mechanism was investigated. The present study demonstrated that the suppression of T24 cell viability by EECU is associated with apoptosis induction. EECU-induced apoptosis was found to correlate with an activation of caspase-8, -9, and -3 in concomitance with a decrease in the expression of the inhibitor of apoptosis family of proteins and an increase in the Bax:Bcl-2 ratio accompanied by the proteolytic degradation of poly(ADP-ribose) polymerase. EECU also increased the generation of reactive oxygen species (ROS), collapse of mitochondrial membrane potential, and cytochrome c release to the cytosol, along with a truncation of Bid. In addition, EECU inactivated phosphatidylinositol 3-kinase (PI3K) as well as Akt, a downstream molecular target of PI3K, and LY294002, a specific PI3K inhibitor significantly enhanced EECU-induced apoptosis and cell viability reduction. However, N-acetyl cysteine, a general ROS scavenger, completely reversed the EECU-induced dephosphorylation of PI3K and Akt, as well as cell apoptosis. Taken together, these findings suggest that EECU inhibits T24 cell proliferation by activating intrinsic and extrinsic apoptosis pathways through a ROS-mediated inactivation of the PI3K/Akt pathway.

Effects of Platycodon grandiflorum on the Induction of Autophagy and Apoptosis in HCT-116 Human Colon Cancer Cells

Platycodon grandiflorum (PG) has been known to possess many biological effects, including anti-inflammatory and anti-allergy activity and anti-obesity and hyperlipidemia effects. However, little research has been conducted regarding its anticancer effects, with the exception of its ability to stimulate apoptosis in skin cells. There has also been no study regarding PG-induced autophagy. The modulation of autophagy is recognized as one of the hallmarks of cancer cells. Depending on the type of cancer and the context, autophagy can suppress or help cancer cells to overcome metabolic stress and the cytotoxicity of chemotherapy. Therefore, the present study was designed to investigate whether or not extracts from PG-induced cell death were connected with autophagy and apoptosis in HCT-116 human colon cancer cells. PG stimulation decreased cell proliferation in a doseand time-dependent manner and induced apoptosis, which was partially dependent on the activation of caspases. PG treatment also resulted in the formation of autophagic vacuoles simultaneously with regulation of autophagy-related genes. Interestingly, a PG-mediated apoptotic effect was further triggered by pretreatment with the autophagy inhibitors 3-methyladenin and bafilomycin A1. However, cell viability recovered quite well with bafilomycin A1 treatment. These findings show that PG treatment promotes both autophagy and apoptosis and that PG-induced autophagic response might play a role in the autophagic cell death of HCT-116 cells.Platycodon grandiflorum (PG) has been known to possess many biological effects, including anti-inflammatory and anti-allergy activity and anti-obesity and hyperlipidemia effects. However, little research has been conducted regarding its anticancer effects, with the exception of its ability to stimulate apoptosis in skin cells. There has also been no study regarding PG-induced autophagy. The modulation of autophagy is recognized as one of the hallmarks of cancer cells. Depending on the type of cancer and the context, autophagy can suppress or help cancer cells to overcome metabolic stress and the cytotoxicity of chemotherapy. Therefore, the present study was designed to investigate whether or not extracts from PG-induced cell death were connected with autophagy and apoptosis in HCT-116 human colon cancer cells. PG stimulation decreased cell proliferation in a dose- and time-dependent manner and induced apoptosis, which was partially dependent on the activation of caspases. PG treatment also resulted in the formation of autophagic vacuoles simultaneously with regulation of autophagy-related genes. Interestingly, a PG-mediated apoptotic effect was further triggered by pretreatment with the autophagy inhibitors 3-methyladenin and bafilomycin A1. However, cell viability recovered quite well with bafilomycin A1 treatment. These findings show that PG treatment promotes both autophagy and apoptosis and that PG-induced autophagic response might play a role in the autophagic cell death of HCT-116 cells.

Reactive oxygen species-dependent apoptosis induction by water extract of Citrus unshiu peel in MDA-MB-231 human breast carcinoma cells

BACKGROUND/OBJECTIVES Although several recent studies have reported the anti-cancer effects of extracts or components of Citrus unshiu peel, which has been used for various purposes in traditional medicine, the molecular mechanisms for their effects remain unclear. In the present study, the anti-cancer activity of a water-soluble extract of C. unshiu peel (WECU) in MDA-MB-231 human breast carcinoma cells at the level of apoptosis induction was investigated. MATERIALS/METHODS Cytotoxicity was evaluated using the MTT assay. Apoptosis was detected using DAPI staining and flow cytometry analyses. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, caspase activity and Western blotting were used to confirm the basis of apoptosis. RESULTS The results indicated that WECU-induced apoptosis was related to the activation of caspase-8, and -9, representative initiator caspases of extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3 accompanied by proteolytic degradation of poly(ADP-ribose) polymerase and down-regulation of the inhibitors of apoptosis protein family members. WECU also increased the pro-apoptotic BAX to anti-apoptotic BCL-2 ratio, loss of mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm. Furthermore, WECU provoked the generation of ROS, but the reduction of cell viability and induction of apoptosis by WECU were prevented when ROS production was blocked by antioxidant N-acetyl cysteine. CONCLUSIONS These results suggest that WECU suppressed proliferation of MDA-MB-231 cells by activating extrinsic and intrinsic apoptosis pathways in a ROS-dependent manner.

Induction of Apoptosis by Citrus unshiu Peel in Human Breast Cancer MCF-7 Cells: Involvement of ROS-Dependent Activation of AMPK

The fruit of Citrus unshiu MARKOVICH used for various purposes in traditional medicine has various pharmacological properties including antioxidant, anti-inflammatory, and antibacterial effects. Recently, the possibility of anti-cancer activity of the extracts or components of this fruit has been reported; however, the exact mechanism has not yet been fully understood. In this study, we evaluated the anti-proliferative effect of water extract of C. unshiu peel (WECU) on human breast cancer MCF-7 cells and investigated the underlying mechanism. Our results showed that reduction of MCF-7 cell survival by WECU was associated with the induction of apoptosis. WECU-induced apoptotic cell death was related to the activation of caspase-8 and -9, representative initiate caspases of extrinsic and intrinsic apoptosis pathways, respectively, and increase in the Bax : Bcl-2 ratio accompanied by cleavage of poly(ADP-ribose) polymerase (PARP). WECU also increased the mitochondrial dysfunction and cytosolic release of cytochrome c. In addition, AMP-activated protein kinase (AMPK) and its downstream target molecule, acetyl-CoA carboxylase, were activated in a concentration-dependent manner in WECU-treated cells. In contrast, compound C, an AMPK inhibitor, significantly inhibited WECU-induced apoptosis, while inhibiting increased expression of Bax and decreased expression of Bcl-2 by WECU and inhibition of WECU-induced PARP degradation. Furthermore, WECU provoked the production of reactive oxygen species (ROS); however, the activation of AMKP and apoptosis by WECU were prevented, when the ROS production was blocked by antioxidant N-acetyl cysteine. Therefore, our data indicate that WECU suppresses MCF-7 cell proliferation by activating the intrinsic and extrinsic apoptosis pathways through ROS-dependent AMPK pathway activation.

Sargassum serratifolium attenuates RANKL-induced osteoclast differentiation and oxidative stress through inhibition of NF-κB and activation of the Nrf2/HO-1 signaling pathway

Sargassum serratifolium C. Agardh is a marine brown alga that has long been used as an ingredient for food and medicine by many people living along Asian coastlines. Recently, various beneficial effects of extracts or compounds isolated from S. serratifolium have been reported, but their efficacies against bone destruction are unclear. Therefore, in this study, we investigated the inhibitory property of an ethanol extract of S. serratifolium (EESS) on osteoclast differentiation by focusing on the receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis model using RAW 264.7 macrophages. Our results demonstrated that EESS reduced RANKL-induced osteoclast differentiation in RAW 264.7 cells, by inhibiting tartrate-resistant acid phosphatase (TRAP) activity and destroying the F-actin ring formation. EESS also attenuated RANKL-induced expressions of key osteoclast-specific genes, such as nuclear factor of activated T cells cytoplasmic 1 (NFATC1), TRAP, cathepsin K and matrix metalloproteinase-9. These effects were mediated by impaired nuclear translocation of nuclear factor (NF)-κB and suppression of IκB-α degradation. In addition, EESS effectively inhibited the production of reactive oxygen species (ROS) by RANKL, which was associated with enhanced expression of nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Overall, our findings provide evidence that EESS suppresses RANKL-induced osteoclastogenesis and oxidative stress through suppression of NF-κB and activation of Nrf2/HO-1 signaling pathway, indicating that S. serratifolium has a potential application the prevention and treatment of osteoclastogenic bone disease.

Anti-oxidant and Anti-microbial Activities of Herb-combined Remedies used in Traditional Korean Medicine for Treating Breast Cancer

Eun-Ok Choi 1,2 , Da Hee Son 3 , Min Young Kim 1,4 , Hyun Hwang-Bo 1,5 , Hong Jae Kim 1,6 , Jin-Woo Jeong 1 , Su Hyun Hong, Cheol Park and Yung Hyun Choi* Anti-Aging Research Center, Dongeui University, Busan 614-714, Korea 2 Department of Food and Nutrition, College of Human Ecology, Pusan National University, Busan 609-735, Korea Department of Biochemistry, Dongeui University College of Korean Medicine, Busan 614-052, Korea Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Korea 5 Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, Korea Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 609-735, Korea Department of Molecular Biology, College of Natural Sciences and Human Ecology, Dongeui University, Busan 614-714, Korea

Single Dose Oral Toxicity Test of Ethanol Extracts of Schisandrae fructus and Mori folium, and their Mixture in ICR Mice

Eun Ok Choi 1,2 , Da Hye Kwon 2 , Min Young Kim 1 , Hyun Hwang-Bo 2 , Hong Jae Kim 2 , Kyu Im Ahn 1 , Jin-Woo Jeong, Ki Won Lee, Ki Young Kim, Sung Goo Kim, Young Whan Choi, Su Hyun Hong, Cheol Park and Yung Hyun Choi* Department of Biochemistry, Dongeui University College of Korean Medicine, Busan 614-052, Korea 2 Anti-Aging Research Center, Dongeui University, Busan 614-714, Korea Research Institute, Bio-Port Korea INC, MarineBio-industry Development Center, Busan 619-912, Korea Department of Horticultural Bioscience, College of Natural Resource and Life Sciences, Pusan National University, Miryang 627-706, Korea 5 Department of Molecular Biology, College of Natural Sciences and Human Ecology, Dongeui University, Busan 614-714, Korea