• The Korean Journal of Physiology and Pharmacology
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• 제20권3호
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• pp.237-243
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• 2016

Oleanolic acid (OA) has a wide variety of bioactivities such as hepatoprotective, anti-inflammatory and anti-cancer activity and is used for medicinal purposes in many Asian countries. In the present study, the effect of OA on induction of autophagy in human hepatocellular carcinoma HepG2 and SMC7721 cells and the related mechanisms were investigated. MTT assay showed that OA significantly inhibited HepG2 and SMC7721 cells growth. OA treatment enhanced formation of autophagic vacuoles as revealed by monodansylcadaverine (MDC) staining. At the same time, increasing punctuate distribution of microtubule-associated protein 1 light chain 3 (LC3) and an increasing ratio of LC3-II to LC3-I were also triggered by OA incubation. In addition, OA-induced cell death was significantly inhibited by autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) pretreatment. And we found out that OA can suppress the PI3K/Akt1/mTOR signaling pathway. Furthermore, our data suggested that OA-triggered autophagy was ROS-dependent as demonstrated by elevated cellular ROS levels by OA treatment. When ROS was cleared by N-acetylcysteine (NAC), OA-induced LC3-II convertsion and cell death were all reversed. Taken together, our results suggest that OA exerts anticancer effect via autophagic cell death in hepatocellular carcinoma.

• Asian Pacific Journal of Cancer Prevention
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• 제15권16호
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• pp.6939-6944
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• 2014

Sulfasalazine (SSZ) is an anti-inflammatory drug that has been used to treat inflammatory bowel disease and rheumatoid arthritis for decades. Recently, some reports have suggested that SSZ also has anti-cancer properties against human tumors. However, little is known about the effects of SSZ on oral cancer. The aim of this study was to investigate the anti-cancer effects of SSZ in oral squamous cell carcinoma (OSCC) cells and to elucidate the mechanisms involved. The authors investigated the anti-proliferative effect of SSZ using the MTT method in HSC-4 cells (an OSCC cell line). Cell cycle analysis, acidic vesicular organelle (AVO) staining, monodansylcadaverine (MDC) staining and Western blotting were also conducted to investigate the cytotoxic mechanism of SSZ. SSZ significantly inhibited the proliferation of HSC-4 cells in a dose-dependent manner. In addition, SSZ induced autophagic cell death, increased microtubule-associated protein 1 light chain (MAP1-LC; also known as LC) 3-II levels, as well as induced punctate AVO and MDC staining, resulted in autophagic cell death. Furthermore, these observations were accompanied by the inhibition of the Akt pathway and the activation of ERK pathway. These results suggest that SSZ promotes autophagic cell death via Akt and ERK pathways and has chemotherapeutic potential for the treatment of oral cancer.

• 동의생리병리학회지
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• 제23권3호
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• pp.581-589
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• 2009

The water extract of Dohongsamul-tang(DHSMT) has been traditionally used in treatment of ischemic heart and brain diseases in Oriental Medicine. However, little is known about the mechanism by which DHSMT protects C6 glial cells from glucose deprevation induced damages. Therefore, this study was designed to evaluate the protective effects of DHSMT on 2-deoxy-D-glucose induced autophagy of C6 glial cells. Autophagic phenotype is evaluated by fluorescence microscopy and flow cytometry with specific biological staining dyes, including monodansylcadaverine and acridine orange, as well as Western blot analysis with microtubule-associated protein 1 light chain 3(LC3) and Beclin-1. Treatment with 2-deoxy-D-glucose significantly resulted in a decrease of the viability of C6 glial cells and increase of the extracellular LDH release in a dose and time-dependent manner. However, pretreatment with DHSMT protected C6 glial cells from glucose deprivation with 2-deoxy-D-glucose. The author also observed the fact that autophagy phenotype occurred by 2-deoxy-D-glucose in C6 glial cells. Pretreatment with 3-MA, a pharmacological inhibitior of autophagy, abolished the formation of acidic vesicle organelle in C6 glial cells treated with 2-deoxy-D-glucose. However, pretreatment with DHSMT inhibited the formation of autophagic phenotypes, including formation of acidic vesicle organelle, and increase of the expression of LC-3 II Beclin-1 proteins in C6 glial cells treated with 2-deoxy-D-glucose. Taken together, these data suggest that DHSMT is able to protect C6 glial cells from glucose deprivation with marked inhibition of autophagy formation.

• 동의생리병리학회지
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• 제24권6호
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• pp.942-949
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• 2010

Morus alba L., a kind of Oriental medicinal herbs, has been traditionally used to treat pulmonary asthma and congestion. According to recent studies, extracts of M. alba L. have showed anti-inflammatory, anti-oxidant, anti-tumor and hypoglycemic effects. However, the molecular mechanisms on how it acts as a death-inducer in cancer cells have not been fully understood. In this study, we investigated the cell death effects of methylene chloride extracts of M. alba L. (MEMA) in A549 human lung carcinoma cells. It was shown that MEMA induced the apoptotic cell death proved by increased sub-G1 phase cell population, apoptotic body formation and chromatin condensation. MEMA treatment induced the expression of death receptor-related proteins such as death receptor (DR) 4, DR5, Fas and FasL, which further triggered the activation of caspase-8 and the cleavage of Bid in a concentration-dependent manner. However, MEMA reduced anti-apoptotic Bcl-2 and Bcl-xL expression which contributed to the loss of mitochondrial membrane potential (MMP), and the activations of caspase-9 and caspase-3. Meanwhile, the morphological study indicated a characteristic finding of autophagy, such as the formation of autophagosomes in MEMA-treated cells. Furthermore, markers of autophagy, namely, the increased MDC-positive cells, conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II and increased beclin-1 accumulation, were observed. Taken together, these findings demonstrated that MEMA triggered both autophagy and apoptosis in A549 cancer cells. They might suggest that M. alba L. could be a prospective clinical application to treat human lung cancers.

• Molecules and Cells
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• 제46권4호
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• pp.245-255
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• 2023

This study aimed to exploring the pathophysiological mechanism of 7α,25-dihydroxycholesterol (7α,25-DHC) in osteoarthritis (OA) pathogenesis. 7α,25-DHC accelerated the proteoglycan loss in ex vivo organ-cultured articular cartilage explant. It was mediated by the decreasing extracellular matrix major components, including aggrecan and type II collagen, and the increasing expression and activation of degenerative enzymes, including matrix metalloproteinase (MMP)-3 and -13, in chondrocytes cultured with 7α,25-DHC. Furthermore, 7α,25-DHC promoted caspase-dependent chondrocyte death via extrinsic and intrinsic pathways of apoptosis. Moreover, 7α,25-DHC upregulated the expression of inflammatory factors, including inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E₂, via the production of reactive oxygen species via increase of oxidative stress in chondrocytes. In addition, 7α,25-DHC upregulated the expression of autophagy biomarkers, including beclin-1 and microtubule-associated protein 1A/1B-light chain 3 via the modulation of p53-Akt-mTOR axis in chondrocytes. The expression of CYP7B1, caspase-3, and beclin-1 was elevated in the degenerative articular cartilage of mouse knee joint with OA. Taken together, our findings suggest that 7α,25-DHC is a pathophysiological risk factor of OA pathogenesis that is mediated a chondrocyte death via oxiapoptophagy, which is a mixed mode of apoptosis, oxidative stress, and autophagy.

• 운동영양학회지
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• 제16권2호
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• pp.93-100
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• 2012

Alzheimer's disease (AD) is the most common cause of dementia in adults. Microtubule associated protein tau is abnormally phosphorylated in AD and aggregates as paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). NFTs are the most common intraneuronal inclusion in the brains of patients with AD and have been implicated in mediating neuronal cell death and cognitive deficit. Aberrant phosphorylation of tau is an early pathological event in AD, but the underlying mechanisms are unclear. MAP kinases are a family of Serine/Threonine (Ser/Thr) kinases that involved hyper - phosphorylation of tau in AD. The purpose of this study was to investigate the effect of treadmill exercise on phosphorylation of tau level and activation of MAPKs including JNK, ERK, p38-MAPK. To address this, Tg mouse model of AD, Tg-NSE/hTau 23, which expresses human tau 23 in the brain, was chosen. Animals were subjected to treadmill exercise for 12 weeks from 24 months of age. Treadmill exercise in Tg group improved cognitive function compared with Tg-SED group in watermaze test. In addition, treadmill exercised Tg mice significantly reduced the activation of JNK54/46, p38-MAPK and tau (Ser404, Ser202, Thr231), and increased activation of ERK44/42 in cerebral cortex. These results suggest that treadmill exercise may provide a therapeutic potential to alleviate the tau pathology like AD.

• 대한화장품학회지
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• 제48권4호
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• pp.365-372
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• 2022

셀로비오스(cellobiose)는 식물 등에 널리 분포하는 셀롤로오스의 분해 과정에서 생성되는 이당류이다. 환원당인 셀로비오스는 2 개의 β-1,4 결합으로 결합한 구조를 가지며, 식물 등에 존재하는 천연물로서, 건강식품이나 식품의 감미료, 화장품 원료, 의약품 원료로서 활용되어지고 있다. 본 연구에서는 다양하게 유용될 셀로비오스를 기능성 화장품인 미백 화장품 소재로서의 가능성을 확인하고자, 자가포식 활성화 및 멜라닌 생성저해 효능을 검토하였다. 인간 유래 멜라닌 생성 세포로 알려진 MNT-1 세포에서 셀로비오스의 농도에 따른 독성 실험을 진행한 결과 20 mg/mL이하에서 세포독성이 없음을 확인하였고, 공초점 현미경을 통해 셀룰비오스 처리에 따른 자가포식 표지자인 microtubule-associated protein 1 light chain 3 (LC3) puncta의 증가가 확인되었다. 또한 LC3의 형태가 LC3-I에서 LC3-∏로 변환됨을 확인하였으며, 농도와 시간에 따라 LC3-II양이 증가하는 것을 확인하였다. 셀로비오스를 동일 세포에서 20 mg/mL 농도로 7 일간 처리하여 멜라닌 생성량을 분석한 결과, 50% 이상 멜라닌 생성 감소를 확인하였다. 추가적으로, 멜라닌 생성 관련 단백질TYR 및 TYRP1의 발현량을 western blot법을 이용하여 분석한 결과, 농도의존적으로 발현이 저해되어 멜라닌 생성이 감소함을 확인하였다. 이러한 연구 내용을 기반을 토대로 셀로비오스를 함유한 화장품 크림제형을 확보하여 4 주 동안 제형의 변화를 확인 해본 결과 성상이 고온에서 액채형태로 변화하였지만 pH는 변화하지 않았음을 확인하였다. 결론적으로, 자가포식 활성화 및 멜라닌 생성 억제 과정에 셀로비오스가 관여함을 확인하였으며, 이를 통해 미백 화장품 소재로서의 활용 가능성을 제안하고자 한다.

• Clinical and Experimental Reproductive Medicine
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• 제28권1호
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• pp.33-40
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• 2001

Objective: This study was to establish the human embryonic stem (ES) cells derived from frozen-thawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. Methods: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. Results: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and ${\beta}$-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. Conclusion: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.

• Asian Pacific Journal of Cancer Prevention
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• 제15권8호
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• pp.3613-3618
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• 2014

Stathmin, also called oncoprotein 18, is a founding member of the family of microtubule-destabilizing proteins that play a critical role in the regulation of mitosis. At the same time stathmin has been recognized as one of responsible factors in cancer cells. The aim of this study was to assess stathmin status, its correlations with clinicopathological parameters and its role as a progosnostic marker in EC patients. The protein and mRNA levels of stathmin were examined byimmunohistochemistry (IHC) and in situ hybridization in 100EC tissues and adjacent noncancerous tissues. mRNA and protein expression of stathmin in three EC cell lines(EC9706, ECa109, EC1 commonly used in research) were also analyzed using immunocytochemistry, western blot and in situ hybridization. The prognostic value of Stathmin expression within the tumor tissues were assessed by Cox regression and Kaplan-Meier analysis. We showed that stathmin expression was significantly higher in EC tissues than in adjacent noncancerous tissues. High stathmin immunostaining score in the EC was positively correlated with tumor differentiation, Tumor invasion, Lymph node metastases, and TNM stage. In addition, we demonstrated that three EC cell lines examined, were constitutively expressing a high level of stathmin. Of those, EC-1 showed the strongest mRNA and protein expression for the stathmin analyzed. Kaplan-Meier analysis showed that significantly longer 5-year survival rate was seen in EC patients with high Stathmin expression, compared to those with low expression of Stathmin expression. Furthermore, multivariate Cox proportional hazard analyses revealed that Stathmin was an independent factors affecting the overall survival probability. In conclusion, our data provide a basis for the concept that stathmin might be associated with EC development and progression. High levels of Stathmin expression in the tumor tissues may be a good prognostic marker for patients with EC.

• Asian Pacific Journal of Cancer Prevention
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• 제15권9호
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• pp.3927-3932
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• 2014

Background: Epidermal growth factor receptor (EGFR) mutations and echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) define specific molecular subsets of lung adenocarcinomas with distinct clinical features. Our purpose was to analyze clinical features and prognostic value of EGFR gene mutations and the EML4-ALK fusion gene in lung adenocarcinoma. Patients and Methods: EGFR gene mutations and the EML4-ALK fusion gene were detected in 92 lung adenocarcinoma patients in China. Tumor marker levels before first treatment were measured by electrochemiluminescence immunoassay. Results: EGFR mutations were found in 40.2% (37/92) of lung adenocarcinoma patients, being identified at high frequencies in never-smokers (48.3% vs. 26.5% in smokers; P=0.040) and in patients with abnormal serum carcinoembryonic antigen (CEA) levels before the initial treatment (58.3% vs. 28.6%, P=0.004). Multivariate analysis revealed that a higher serum CEA level before the initial treatment was independently associated with EGFR gene mutations (95%CI: 1.476~11.343, P=0.007). We also identified 8 patients who harbored the EML4-ALK fusion gene (8.7%, 8/92). In concordance with previous reports, younger age was a clinical feature for these (P=0.008). Seven of the positive cases were never smokers, and no coexistence with EGFR mutation was discovered. In addition, the frequency of the EML4-ALK fusion gene among patients with a serum CEA concentration below 5ng/ml seemed to be higher than patients with a concentration over 5ng/ml (P=0.021). No significant difference was observed for time to progression and overall survival between EML4-ALK-positive group and EML4-ALK-negative group or between patients with and without an EGFR mutation. Conclusions: The serum CEA level before the initial treatment may be helpful in screening population for EGFR mutations or EML4-ALK fusion gene presence in lung adenocarcinoma patients.