AGS 인체 위암 세포에서 Akt/mTOR/GSK-3β 신호경로 조절을 통한 개똥쑥 추출물의 Apoptosis 유도 효과

김은지(Eun Ji Kim),김근태(Guen Tae Kim),김보민(Bo Min Kim),임은경(Eun Gyeong Lim),김상용(Sang-Yong Kim),김영민(Young Min Kim) 한국식품영양과학회 2016 한국식품영양과학회지 Vol.45 No.9

개똥쑥은 예로부터 항암, 항바이러스 및 항균의 효능을 지니는 것으로 알려져 왔지만 작용 기작에 대한 내용이 많이 알려지지 않았다. 본 연구에서는 AGS 인체 위암 세포를 대상으로 개똥쑥 추출물(AAE)에 의한 apoptosis 효과와 신호경로 연구를 시행하였다. AAE의 암세포 성장에 미치는 영향을 확인하기 위하여 AGS cell에 AAE를 처리하고 MTT assay와 LDH assay를 수행한 결과 AAE 농도 의존적으로 나타난 세포 성장 억제가 세포 손상에 의한 것임을 확인하였다. 또한, AAE에 의한 암세포 증식 억제 효과가 apoptosis에 의한 것인지 확인하기 위하여 Hoechst 33342 staining과 Annexin Ⅴ-PI staining을 수행한 결과, Hoechst 33342 staining에서 apoptotic body와 세포질 응축이 농도 의존적으로 증가하는 것을 확인하였고, Annexin Ⅴ-PI staining에서 apoptotic cells의 변화가 농도 의존적으로 증가함을 확인하였다. Western blotting의 결과 AAE가 농도 의존적으로 세포 생장에 관여하는 신호 단백질인 p-Akt, p-TSC2, p-mTOR, p-GSK-3β의 발현이 감소함을 확인하였고, anti-apoptotic 단백질인 Bcl-2의 발현이 억제됨으로써 proapoptotic 단백질인 Bax, Bak의 발현이 증가하는 일련의 신호경로를 조절할 수 있다는 것을 확인하였다. 미토콘드리아 막 전위의 탈분극 유도를 확인하기 위한 JC-1 assay 수행 결과, AAE 농도 의존적으로 미토콘드리아 막 전위의 탈분극이 유도됨을 확인하였다. 탈분극에 의한 caspase 활성을 확인하기 위해 caspase-3/7 activity assay를 수행한 결과, AAE 농도 의존적으로 caspase activity 증가를 확인하였다. 또한, apoptosis가 일어나는 일련의 신호경로를 확인하기 위해 apoptosis 상위 단백질인 Akt, mTOR, GSK-3β의 활성을 억제하는 LY294002, Rapamycin, BIO를 각각 AGS cell에 처리하고 세포증식에 미치는 영향과 신호 단백질의 발현 양상을 알아보기 위해 MTT assay, LDH assay, western blotting을 수행하였다. 그 결과 AAE와 LY294002, Rapamycin 처리군에서 세포증식 억제와 LDH 방출량 증가 뿐만 아니라 세포 생장 신호 단백질인 p-mTOR, p-TSC2, p-Akt, p-GSK-3β의 발현이 감소하는 것을 확인하였고, Bcl-2의 발현이 억제됨으로써 Bax와 Bak의 발현을 증가시키는 신호경로를 조절할 수 있다는 것을 확인하였다. 따라서 AGS cell에 개똥쑥 추출물을 처리하였을 때 유도되는 apoptosis 효과는 Akt/mTOR/GSK-3β 경로 활성 억제를 통해 Bcl-2 발현이 감소함에 따라 Bax, Bak를 활성화해 세포질로의 cytochrome C 유리에 따른 caspase 활성으로 이루어진다는 것을 알 수 있었다. Extracts from Artemisia annua Linné (AAE) have various functions (anti-malaria, anti-virus, and anti-oxidant). However, the mechanism of the effects of AAE is not well known. Thus, we determined the apoptotic effects of AAE in AGS human gastric carcinoma cells. In this study, we suggested that AAE may exert cancer cell apoptosis through the Akt/mammalian target of rapamycin (mTOR)/glycogen synthase kinase (GSK)-3β signal pathway and mitochondria-mediated apoptotic proteins. Activation by Akt phosphorylation resulted in cell proliferation through phosphorylation of tuberous sclerosis complex 2 (TSC2), mTOR, and GSK-3β. Thus, de-phosphorylation of Akt inhibited cell proliferation and induced apoptosis through inhibition of Akt, mTOR, phosphorylation of GSK-3β at serine9, and control of Bcl-2 family members. Inhibition of GSK-3β attenuated loss of mitochondrial membrane potential and release of cytochrome C. Bax and pro-apoptotic proteins were activated by their translocation into mitochondria from the cytosol. Translocation of Bax induced outer membrane transmission and generated apoptosis through cytochrome C release and caspase activity. We also measured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase assay, Hoechst 33342 staining, AnnexinⅤ-PI staining, 5,5",6,6"-tetrachloro-1,1",3,3"-tetraethylimidacarbocyanine iodide staining, and Western blotting. Accordingly, our study showed that AAE treatment to AGS cells resulted in inhibition of Akt, TSC2, GSK-3β-phosphorylated, Bim, Bcl-2, and pro-caspase 3 as well as activation of Bax and Bak expression. These results indicate that AAE induced apoptosis via a mitochondrial event through regulation of the Akt/mTOR/GSK-3β signaling pathways.

HCT116 대장암세포에서 AKT/mTOR/GSK-3β 신호경로 조절을 통한 벌 사상자 추출물(CME)의 apoptosis 및 cell cycle arrest 효과

임은경(Eun Gyeong Lim),김근태(Guen Tae Kim),김보민(Bo Min Kim),김은지(Eun Ji Kim),하성호(Sung Ho Ha),김상용(Sang-Yong Kim),김영민(Young Min Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.6

벌 사상자[Cnidium monnieri (L.) Cusson]는 중국과 한국에 분포하는 일년생 초본으로, 화농성피부염 및 여성의 생식기 질환의 치료에 널리 사용되고 있다. 이 외에도 면역기능개선과 천식 등에 대한 효과는 보고된 바 있으나 아직까지 암과 관련된 연구는 많이 이루어지지 않았다. 이에 따라 본 연구에서는 인간 대장암 세포인 HCT116 세포주에서 벌 사상자 에탄올 추출물(CME)의 apoptosis 및 세포주기정지 유도 효과에 대하여 알아보고자 하였으며, 이러한 효과가 AKT/mTOR/GSK-3β 신호경로의 조절을 통하여 이루어지는지 확인하고자 하였다. MTT assay와 LDH assay 결과, 벌 사상자 에탄올 추출물에 의하여 HCT116 세포의 세포생존율이 감소하였으며, 세포독성효과가 나타났다. 또한 벌 사상자 에탄올 추출물의 농도의존적으로 apoptotic body의 수와 apoptosis 비율이 증가하였으며, G1기에서 세포주기정지 유도 효과가 관찰되었다. 세포의 성장과 증식 및 분열에 관련된 단백질인 Akt는 mTOR, p53, GSK-3β와 같은 신호단백질들의 발현을 조절하는 것으로 보고되었다. 벌 사상자 에탄올 추출물을 처리하였을 때, Akt와 mTOR 단백질의 인산화가 저해되었으며, 이에 따라 하위 신호조절 단백질인 GSK-3β, Bcl-2 family, Caspase-3, PARP의 발현이 조절되었다. 또한 Akt와 GSK-3β, mTOR 저해제 처리를 통하여 CME에 의한 apoptosis 효과가 AKT/mTOR/GSK-3β 신호경로를 통하여 이루어지는 것을 확인하였다. 결론적으로, 본 연구를 통하여HCT116 대장암 세포주에서 벌 사상자 에탄올 추출물이 암세포의 apoptosis 및 세포주기정지 유도에 효과적임을 확인하였다. The Cnidium monnieri (L.) Cusson is an annual plant distributed in China and Korea. The fruit of C. monnieri is used as a medicinal herb that is effective for the treatment of carbuncle and pain in female genitalia. However, the anti-cancer effects of CME have not yet been reported. In this study, we assessed the apoptotic effects and cell cycle arrest effects of ethanol extracts from C. monnieri on HCT116 colon cancer cells. The results of an MTT assay and LDH assay demonstrated a decrease in cell viability and the cytotoxic effects of CME. In addition, the number of apoptotic body and the apoptotic rate were increased in a dose-dependent manner through Hoechst 33342 staining and Annexin V-PI double staining. In addition, cell cycle arrest occurred at the G1 phase by CME. Protein kinase B (Akt) plays an important role in cancer cell survival, growth, and division. Akt down-regulates apoptosis-mediated proteins, such as mammalian target of rapamycin (mTOR), p53, and Glycogen Synthase kinase-3β (GSK-3β). CME could regulate the expression levels of p-Akt, p-mTOR, p-GSK-3β, Bcl-2 family members, caspase-3, and PARP. Furthermore, treatment with CME, LY294002 (PI3K/Akt inhibitor), BIO (GSK-3β inhibitor), and Rapamycin (mTOR inhibitor) showed that apoptotic effects occurred through the regulation of the AKT/mTOR/GSK-3β signaling pathway. Our results demonstrated CME could induce apoptosis and cell cycle arrest in HCT116 colon cancer cells.

Fabrication and characterization of PLGA loaded nanoparticles of Umbelliferone β-d-galactopyranoside in the treatment of lipopolysaccharide-induced acute lung inflammation via PI3K/Akt/Mtor pathway

( D Chauhan ),( V Kumar ) 대한결핵 및 호흡기학회 2019 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.127 No.-

Purpose: Acute Lung injury (ALI) which is included by a strong pulmonary inflammation and its causes the mortality and morbidity in critically ill patients. Phosphoinositide 3- kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathways is consider as the singling pathway, who activates the diverse cellular function viz., survival, cell expansion, vesicular transport and proliferation. Umbelliferone β-d-galactopyranoside (UFG) plays an important protective role in the expansion of inflammation, oxidative stress and cancer. The present research was initiated to develop a suitable delivery system for UFG with an intention to enhance its therapeutic efficacy against acute lung inflammation induced lipopolysaccharide in mice via alteration of PI3K/Akt/Mtor inhibitor. Methods: UFG-loaded polymeric nanoparticles prepared by sonication were scrutinized for average size, zeta potential, and drug release potency in animals. The rodent was divided into different groups and counted in the bronchoalveolar lavage fluid (BALF). Antioxidant parameters and proinflammatory cytokines were measured. The concentration of PI3K, P-PI3K, mTOR, P-mTOR Akt and P-Akt were determined, respectively. Result: UFG-loaded poly(d,l-lactide-co-glycolide) nanoparticles (UFG-PLGA-NP) with particle size of 187.1 nm and polydispersity index 0.16 were uniform in nature with 82.5% release of the total amount of drug after 48 h. UFG marked suppressed the inflammation number in the BALF (78.5%) as well as reduced the oxidative stress via improved the status of endogenous antioxidant parameters such as SOD (45.5%) and MPO (53.4%); pro-inflammatory cytokines viz., TNF-α (442.3%), IL-6 (48.3%) and IL-1β (54.4%) . ALI control group mice confirmed the change protein levels of PI3K/Akt/mTOR pathway in lung as compared to normal control, which was significantly down-regulated by the UFG-PLGA-NPs. In the histological study, we found that the UFG-PLGA-NPs substantially reduced the LPS-induced neutrophils in lung tissue. Conclusion: UFG-PLGA-NPs attenuating the acute lung inflammation developed during the LPS induced lung cancer via play an effective role to inhibit the PI3K/Akt/mTOR pathway.

Improvement of ventricular remodeling and regulation of PI3K/Akt/mTOR signaling pathway in rats with heart failure by polysaccharides from Stropharia rugosoannulata

Zhang Gang,Sun Xiaofeng,Yu Kun,Zhang Xiwen,Yong Hui 대한독성 유전단백체 학회 2024 Molecular & cellular toxicology Vol.20 No.3

Background Heart failure is a syndrome of impaired cardiac circulation caused by the inability of the heart to provide adequate blood supply. Objective This study aimed to evaluate the improvement of ventricular remodeling and the regulation of PI3K/Akt/mTOR signaling pathway in rats with heart failure by polysaccharides from Stropharia rugosoannulata. Results In high dose group of polysaccharides from Stropharia rugosoannulata, the levels of LVESD, LVEDD, LVMI, CI, PVCA, CVF, and MMP-2 and MMP-9 mRNA expression in rats with heart failure were significantly decreased, while the levels of LVEF, LVFS were significantly elevated (P<0.05). In high dose group of polysaccharides from Stropharia rugosoannulata, the levels of PI3K, p-Akt/Akt, p-mTOR/mTOR, and cleaved-caspase 3 expression and TIMP-1 mRNA expression were significantly elevated, while Bax, p65, Bcl2 protein expression and serum levels of PRA, AngII, ALD, IL-6, TNF-α, ST2 and NT-proBNP were significantly decreased (P<0.05). Conclusions Polysaccharides from Stropharia rugosoannulata can improve ventricular remodeling and cardiac function, reduce inflammatory response and protect cardiac function in rats with heart failure in a dose-dependent manner, and the mechanism of action may be related to the regulation of MMPs/TIMPs balance, inhibition of RAAS system and activation of PI3K/Akt/mTOR signaling pathway. Background Heart failure is a syndrome of impaired cardiac circulation caused by the inability of the heart to provide adequate blood supply. Objective This study aimed to evaluate the improvement of ventricular remodeling and the regulation of PI3K/Akt/mTOR signaling pathway in rats with heart failure by polysaccharides from Stropharia rugosoannulata. Results In high dose group of polysaccharides from Stropharia rugosoannulata, the levels of LVESD, LVEDD, LVMI, CI, PVCA, CVF, and MMP-2 and MMP-9 mRNA expression in rats with heart failure were significantly decreased, while the levels of LVEF, LVFS were significantly elevated (P<0.05). In high dose group of polysaccharides from Stropharia rugosoannulata, the levels of PI3K, p-Akt/Akt, p-mTOR/mTOR, and cleaved-caspase 3 expression and TIMP-1 mRNA expression were significantly elevated, while Bax, p65, Bcl2 protein expression and serum levels of PRA, AngII, ALD, IL-6, TNF-α, ST2 and NT-proBNP were significantly decreased (P<0.05). Conclusions Polysaccharides from Stropharia rugosoannulata can improve ventricular remodeling and cardiac function, reduce inflammatory response and protect cardiac function in rats with heart failure in a dose-dependent manner, and the mechanism of action may be related to the regulation of MMPs/TIMPs balance, inhibition of RAAS system and activation of PI3K/Akt/mTOR signaling pathway.

Hep3B 간암세포에서 개똥쑥추출물로부터 Akt-mTOR-GSK3β 신호경로에 의한 apoptosis 효과

김은지(Eun Ji Kim),김근태(Guen Tae Kim),김보민(Bo Min Kim),임은경(Eun Gyeong Lim),하성호(Sung Ho Ha),김상용(Sang-Yong Kim),김영민(Young Min Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.7

개똥쑥 추출물은 항박테리아, 항바이러스 그리고 항산화효과를 포함한 다양한 기능을 가지고 있는 것으로 잘 알려져 있다. 그러나, 개똥쑥 항증식 작용기전은 알려지지 않았다. 따라서, 우리는 Hep3B 간암 세포에서 AAE추출물의 apoptotic 효과를 알아보고자 한다. 본 연구의 목적은 AAE가 인체 간암 세포주(Hep3B)의 증식에 미치는 효과를 분석하고 이에 대한 apoptosis의 효과를 조사하는데 있다. 인산화에 의해 활성화된 Akt는 TSC2, mTOR 그리고 GSK-3β의 인산화를 유도하여 세포증식을 유도한다. 본 연구에서, 우리는 AAE가Akt-mTOR-GSK3β 신호경로와 mitochondria를 매개하는 apoptotic 단백질을 통한 암세포의 apoptosis 유도할 것이라고 추측하였다. 이를 위해, 먼저 AAE가 처리농도에 따라 세포증식에 미치는 효과를 분석하였다. AAE처리는 세포증식을 억제시켰을 뿐만 아니라 젖산 탈수소 효소의 방출을 유도하였다. 이러한 결과는 MTT assay, LDH assay로 확인하였다. 또한 Hoechst 33342 staining, Annexin Ⅴ- PI staining, JC-1 staining 그리고 Western blotting을 통해 apoptosis 효과를 확인하였다. 본 연구에서는 간암세포에 AAE의 처리가 Akt, TSC2, GSK-3β-phosphorylated, Bim, Bcl-2, pro-caspase 3의 억제와 Bak, Bax 활성을 유도한다는 것을 확인하였다. 이러한 결과는 AAE가 Akt-mTOR-GSK-3β 신호경로를 통해 intrinsic apoptosis를 유도한다는 것을 나타낸다. Extracts from Artemisia annua Linné (AAE) have been known to possess various functions, including anti-bacterial, anti-virus, and anti-oxidant effects. However, the mechanism of those effects of AAE is not well-known. The aim of this study was to analyze the inhibitory effects of AAE on cell proliferation of the human hepatoma cell line (Hep3B) and to examine its effects on apoptosis. Activation by phosphorylation of Akt is cell proliferation through the phosphorylation of TSC2, mTOR, and GSK-3β. We suggested that AAE may exert cancer cell apoptosis through Akt/mTOR/GSK-3β signal pathways and mitochondria-mediated apoptotic proteins. For this, we examined the effects of extracts of AAE on cell proliferation according to treatment concentration. Treatment with AAE not only reduced cell viability, but also resulted in the induced release of lactate dehydrogenase (LDH). These results were determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) assay. Furthermore, we determined the effects of apoptosis through Hoechst 33342 staining, annexinⅤ-propidium iodide (PI) staining, 5,5‘, 6,6’-tetrachloro-1,1‘,3,3’-tetraethyl-imidacarbocyanine iodide (JC-1) staining, and Western blotting. Our study showed that the treatment of liver cancer cells with AAE resulted in the inhibition of Akt, TSC2, GSK-3β-phosphorylated, Bcl-2, and pro-caspase 3 and the activation of Bim, Bax, Bak, and cleaved PARP expressions. These results indicate that AAE induced apoptosis by means of a mitochondrial event through the regulate of Akt/mTOR/GSK-3β signaling pathways.

A549 폐암 세포주에서 AMPK/Akt/mTOR 신호 경로를 통한 소나무 담쟁이덩굴 에탄올 추출물의 Apoptosis 유도효과

조경조,남건희,박예슬,김상용,구봉성,김영민 한국생물공학회 2019 KSBB Journal Vol.34 No.3

Lung cancer can be caused by several environmentalfactors, such as smoking and urban and industrialpollution. Lung cancer also has a higher recurrence rate and alower cure rate than other types of cancer. Parthenocissustricuspidata (Siebold & Zucc.) Planch Extract (PTE) is knownto have antioxidant, antidiabetic, and anti-inflammatory effects;however, its efficacy as an anticancer agent has not beenreported. The purpose of this study was to investigate the effectof PTE on apoptosis-related proteins in A549 lung cancer cellsand to determine how apoptosis is induced through the AMPactivatedprotein kinase (AMPK)/Akt/mTOR signaling pathway. AMPK is activated by metabolic stress, such as glucosedeficiency, and induces apoptosis through the intrinsic pathwaythat activates tumor suppressor p53, thereby regulating the bcl-2 family protein, releasing cytochrome c from the mitochondria,and activating caspase. In addition, Akt plays an important rolein the metabolism, growth, and survival of cancer cells. Inhibitionof Akt induces apoptosis by regulating key signaling molecules,such as the mammalian target of rapamycin and tumor suppressorp53. In this study, we examined the effect of PTE on theapoptosis of A549 cells and determined how the regulation ofapoptosis proteins through the intrinsic pathway is achieved. Our results demonstrated that PTE regulates bcl-2 proteins andinduces apoptosis through the intrinsic pathway. In addition,AMPK and Akt inhibitors were treated to determine whetherthese apoptotic effects were dependent on AMPK and Akt.

Flavonoid myricetin inhibits TNF-α-stimulated production of inflammatory mediators by suppressing the Akt, mTOR and NF-κB pathways in human keratinocytes

Lee, D.H.,Lee, C.S. North-Holland ; Elsevier Science Ltd 2016 european journal of pharmacology Vol.784 No.-

<P>Flavonoid myricetin has been shown to exhibit anti-inflammatory and anti-oxidant effects. Nevertheless, the effect of myricetin on the TNF-alpha-stimulated production of inflammatory mediators in keratinocytes has not been studied. Using human keratinocytes, we examined the effect of myricetin on the TNF-alpha-stimulated production of inflammatory mediators in relation to the Akt, mTOR and NF-kappa B pathways, which regulate the transcription genes involved in immune and inflammatory responses. TNF-alpha-stimulated production of the inflammatory mediators and reactive oxygen species in keratinocytes, and activation of the Akt, mTOR and NF-kappa B pathways in HaCaT cells and primary keratinocytes. Myricetin, Akt inhibitor, Bay 11-7085 (an inhibitor of NF-kappa B activation), rapamycin (mTOR inhibitor) and N-acetylcysteine attenuated TNF-alpha-induced activation of Akt, mTOR and NF-kappa B. Myricetin and N-acetylcysteine attenuated the TNF-alpha-stimulated production of cytokines and chemokines, and production of reactive oxygen species in keratinocytes. The results show that myricetin may reduce TNF-alpha-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of the Akt, mTOR and NF-kappa B pathways. The effect of myricetin appears to be associated with inhibition of the production of reactive oxygen species. Further, myricetin appears to attenuate the proinflammatory mediator-induced inflammatory skin diseases. (C) 2016 Elsevier B.V. All rights reserved.</P>

Plumbagin inhibits proliferation and promotes apoptosis of ovarian granulosa cells in polycystic ovary syndrome by inactivating PI3K/Akt/mTOR pathway

Cai Zhaowei,He Shaojuan,Li Tao,Zhao Li,Zhang Kerong 한국통합생물학회 2020 Animal cells and systems Vol.24 No.4

Polycystic ovary syndrome (PCOS) is recognized as a general endocrine disease and reproductive disorder. Although evidence indicates that PCOS has a complex etiology and genetic basis, the pathogenic mechanisms and signal pathway in PCOS remain unclear. In this study, the normal structure of follicle and corpus luteum were observed, and no cyst nor hyperemia was observed under the light microscopic study with hematoxylin and eosin (H&E) staining. Eestosterone and progesterone were evaluated by radioimmunoassay in rat serum. The alterations of proliferative ability and cell cycle distribution of each group were assessed by Cell Counting Kit-8 (CCK8) assay and flow cytometry. The protein expression of p-mTOR/mTOR, p-PI3K/PI3K, p-AKT/AKT, and GAPDH were analyzed by western blotting. Both doses of PLB could benefit the ovarian morphology and polycystic property. PLBinduced a suppress effect on the proliferation of rat ovarian granulosa cells. In addition, PLB also induced concentrationdependent apoptosis in rat ovarian granulosa cells. The rat ovarian granulosa cells treated with PLB that the expression levels of p-AKT, p-mTOR, and p-PI3K were significantly decreased in a concentration-dependent manner. PLB not only plays a critical role in attenuating the pathology and polycystic property changes in the ovary but can also induce rat ovarian granulosa cell apoptosis through the PI3K/Akt/mTOR signal pathway. This study showed the innovative role of PLB in the pathogenesis of PCOS and provides a new therapeutic modality for the treatment of PCOS.

AGS 위암세포주에서 Akt/mTOR 신호경로를 통한 영릉향 부탄올 분획물에서의 Apoptosis 유도 효과

이효재,서지원,김상현,이창열,김상용,김영민 한국식품영양과학회 2022 한국식품영양과학회지 Vol.51 No.3

영릉향(Lysimachia foenum-graecum)은 두통, 치통, 염증 완화에 효과가 있는 한약재로 항산화, 항비만, 항염 효과가 있는 것으로 알려져 있다. 그러나 위암에서 영릉향의 항암 효과는 보고되지 않았으며, 본 연구에서는 AGS 인체 위암 세포에서 영릉향 부탄올 분획물(LFB)의 apoptosis 효과를 확인하고자 하였다. MTT assay를 통해 세포 증식 억제 효과를, Annexin V & Dead Cell staining을 통해 LFB의 apoptosis 유도 효과를 확인하였다. 또한, western blotting을 통해 LFB 처리가 농도 의존적으로 종양 형성과 암세포의 생존에 있어서 중요한 역할을 하는 p-Akt와 p-mTOR의 활성 수준을 감소시키는 것을 확인하였다. 더해서 LFB 처리가 농도 의존적으로 intrinsic pathway의 신호 단백질을 조절하여 apoptosis를 유도하는 것을 확인하였다. 그리고 caspase-3/7 activity assay를 통해 LFB가 농도 의존적으로 effecter caspase인 caspase-3의 발현을 증가시키는 것을 확인하였다. 마지막으로 LY294002(Akt inhibitor)와 rapamycin(mTOR inhibitor)을 처리하여 apoptosis 관련 신호 단백질의 발현 조절을 확인하였다. 그 결과 LFB가 Akt와 mTOR의 발현을 억제하여 이를 경유하는 signaling pathway를 통해 apoptosis를 유도하는 것을 확인하였다.

HCT116 대장암세포에서 Akt-mTOR 신호경로를 통한 개똥쑥 추출물(AAE)의 세포주기 억제 효과

김보민(Bo Min Kim),김근태(Guen Tae Kim),임은경(Eun Gyeong Lim),김은지(Eun Ji Kim),김상용(Sang Yong Kim),하성호(Sung Ho Ha),김영민(Young Min Kim) 한국생물공학회 2015 KSBB Journal Vol.30 No.5

In this study, extract from Artemisia annua in L.(AAE) is known as a medicinal herb that is effective against cancer. The cell cycle is regulated by the activation of cyclindependent kinase (CDK)/cyclin complex. We will focus on regulation of CDK2 by cyclin E. cyclin E is associated with CDK2 to regulate progression from G1 into S phase. Akt is known to play an important role in cell proliferation and cell survival. Activation of Akt increases mTOR activity that promotes cell proliferation and cancer growth. In this study, we investigated that AAE-induced cell cycle arrest at G1/S phase in HCT116 colon cancer. Treatment of AAE shows that reduced activation of Akt decreases mTOR/Mdm2 activity and then leads to increase the activation of p53. The active p53 promotes activation of p21. p21 induces inactivation of CDK2/ cyclin E complex and occurs cell cycle arrest at G1/S phase. We treated LY294002 (Akt inhibitor) and Rapamycin (mTOR inhibitor) to know the relationship between the signal transduction of proteins associated with cell cycle arrest. These results suggest that AAE induces cell cycle arrest at G1/S phase by Akt/mTOR pathway in HCT116 colon cancer cell.