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Anticancer Activity of Bispidinone Derivative by Induction of Apoptosis
Biomed Sci Letters 2020;26:336-343
Published online December 31, 2020;  https://doi.org/10.15616/BSL.2020.26.4.336
© 2020 The Korean Society For Biomedical Laboratory Sciences.

Man Gi Lee2,* and Ryong Kwon1,†,**

1Department of Biomedicinal Chemistry, Inje University, Gimhae 50834, Korea
2Department of Sports Healthcare, Inje University, Gimhae 50834, Korea
Correspondence to: Ryong Kwon. Department of Biomedicinal Chemistry, Inje University, Gimhae 50834, Korea.
Tel: +82-10-3044-7412, Fax: +82-55-320-3219, e-mail: kwon7450@naver.com
*Professor, **Researcher.
Received October 29, 2020; Revised December 24, 2020; Accepted December 28, 2020.
 Abstract
The present study was carried out to investigate the possibility that bispidinone derivative makes anticancer drug availability to human cervical carcinoma cell. The B8 has the lowest IC50 value among B8, B9 and B10 which are bispidinone analogue with bromide. According to cytotoxic test through WST-8 assay, B8 shows the most magnificent cytotoxicity effectiveness with 76 μM of IC50 value. In human cervical carcinoma cell treated with B8, it noticeably controlled cellular multiplication by increase of concentration and time. Furthermore, morphological changes like cellular shrink, disruption and nuclear condensation, feature of apoptosis, are observed. Annexin V-FITC/PI double staining assay test proved that B8 can cause apoptosis. Moreover, after treatment with 76 μM of B8, flow cytometry analysis shows that increase of active oxygen species are induced and membrane potential in mitochondria is decreased. Manifestation of Bcl-2 family and caspase cascades protein provides evidence that B8 induces apoptosis through mitochondria and caspase-related pathway. Taken together, we suggested that B8 reduced membrane potential in mitochondria and induce apoptosis through the pathway depended on mitochondria and caspase.
Keywords : Bispidinone, Apoptosis, Mitochondria membrane potential, Reactive oxygen species, Anticancer activity
꽌 濡

꽭룷 궗硫몄 꽭룷媛 二쎌쓬뿉 씠瑜대뒗 諛⑸쾿 以묒쓽 븯굹濡 븘슂븯吏 븡 꽭룷굹 씠긽씠 엳뒗 꽭룷瑜 젣嫄고븯뿬 깮紐낆껜쓽 깮由ъ쟻 洹좏삎쓣 쑀吏븯寃 븳떎. 꽭룷궗硫몄 겕寃 궡遺 寃쎈줈(intrinsic pathway) 쇅遺 寃쎈줈(extrinsic pathway)뿉 쓽빐 븘二 꽭諛븯寃 議곗젅릺뒗 몢 媛吏 硫붿빱땲利섏쓣 媛吏怨 엳뒗뜲, 궡遺 寃쎈줈쓽 寃쎌슦 궛냼利앹씠굹 DNA쓽 넀긽 벑뿉 쓽빐 諛쒖깮븳떎(Ziegler and Kung, 2008). 삉븳 궡遺 寃쎈줈 愿젴맂 誘명넗肄섎뱶由ъ븘쓽 以묒슂븳 뿭븷 以 븯굹뒗 DNA媛 넀긽쓣 諛쏆븯쓣 寃쎌슦 誘명넗肄섎뱶由ъ븘 留(Mitochondria membrane potential, MMP)씠 遺뺢눼릺뼱 꽭룷吏덈줈 cytochrome c媛 諛⑹텧릺怨 씠寃껋 Apaf-1 (apoptotic protease activating factor 1) protein怨 寃고빀븯뿬 apoptosome쓣 삎꽦븳떎(Wang and Youle, 2009). Apoptosome쓽 삎꽦 procaspase瑜 caspase (caspase-3, -7, -9)濡 솢꽦솕떆궎뒗 뿭븷쓣 븯뿬 apoptosis媛 씪뼱굹寃 릺硫 洹 쇅뿉룄 뿬윭 媛吏 떒諛깆쭏쓽 깮꽦 Bcl-2 family, Bax뿉 쓽빐 議곗젅맂떎(Pathak et al., 2009). 쇅遺 寃쎈줈(extrinsic pathway)쓽 寃쎌슦 death signal씠 fas death receptor 넻빐 procaspase-8쓣 caspase-8濡 솢꽦솕떆궎怨 怨꾩냽쟻쑝濡 caspase-3쓣 솢꽦솕븯뿬 꽭룷 궗硫몄씠 씪뼱굹寃 맂떎(Lobrich and Jeggo, 2007).

븳렪 怨쇱궛솕닔냼(hydrogen peroxide: H2O2)굹 珥덇낵궛솕 씠삩(superoxide anion: O2-), 닔궛솕 씪뵒移(hydroxyl radical: 쨌OH)怨 媛숈 솢꽦궛냼(reactive oxygen species, ROS)뱾 꽭룷 깮議닿낵 二쎌쓬쓣 議곗젅븯뒗 꽭룷 궡 떊샇 쟾떖 寃쎈줈뿉꽌 以묒슂븳 뿭븷쓣 븳떎(Mine et al., 2001; LeBras et al., 2005). 궛솕쟻 뒪듃젅뒪 꽭룷 궗硫 媛꾩뿉 諛젒븳 愿怨꾧 엳쓬씠 蹂닿퀬릺뿀떎(Fleury et al., 2002). 솢꽦궛냼媛 留롮씠 議댁옱븯硫 꽭룷 궗硫몄씠 씪뼱굹怨 옉寃 議댁옱븯硫 꽭룷쓽 깮議닿낵 利앹떇쓣 뼢긽떆궓떎(Murray, 1994; Pucci et al., 2000). 꽭룷 궗硫몄쓽 쑀룄뿉 誘명넗肄섎뱶由ъ븘쓽 뿭븷씠 以묒슂븯떎(Limon et al., 2000). 솢꽦궛냼뒗 誘명넗肄섎뱶由ъ븘 留 쟾쐞瑜 媛먯냼떆궎怨 cytochrome c瑜 諛⑹텧븯뿬 caspase뱾쓣 솢꽦솕떆耳쒖꽌 꽭룷 궗硫몄씠 씪뼱굹寃 븳떎(Chan et al., 2003; Jiang et al., 2012).

泥쒖뿰 븣移쇰줈씠뱶씤 sparteine怨 援ъ“쟻쑝濡 쑀궗븳 3,7-diazabicyclo[3.3.1]nonan-9-ones (bispidines) 빆遺젙留κ낵 臾댄넻媛, 빆洹좏솢꽦 벑怨 媛숈 깮臾쇳븰쟻 以묒슂꽦 븣臾몄뿉 엯泥 솕븰怨 씠寃껋쓽 빀꽦뿉 留롮 愿떖쓣 媛吏꾨떎(Jaiprakash et al., 2014). 삉븳, bispidines 캡 opioid 닔슜泥댁뿉 넂 移쒗솕젰怨 꽑깮꽦쓣 굹궡뒗 寃껋씠 諛앺議뚮떎(Aline et al., 2019). Bispidines쓽 듅씠븳 援ъ“뒗 씠濡좎쟻씤 愿젏뿉꽌 肉먮쭔 븘땲씪, 듅엳 2,4,6,8-tetraaryl-3,7-diazabicyclo[3.3.1]nonan-9-one (bispidinone) 4媛 aryl 湲곗쓽 議댁옱 븣臾몄뿉 留롮 愿떖쓣 媛吏꾨떎. 諛⑹궗꽦 援щ━ 룞쐞썝냼뿉 븳 吏꾨떒(64Cu) 삉뒗 移섎즺(67Cu)瑜 쐞븳 chelate 由ш컙뱶濡쒖꽌 bispidines씠 留롮씠 씠슜릺怨 엳떎(Kraus et al., 2009). Bispidinone쓽 빆븫솢꽦뿉 븳 옉슜湲곗쟾怨 愿젴맂 깮臾쇳븰쟻 듅꽦 留롮씠 뿰援щ릺吏 븡븯떎.

蹂 뿰援ъ뿉꽌 떎뼇븳 븣移쇰줈씠뱶 遺꾩옄 援ъ“瑜 媛뽯뒗 bispidinone 솕빀臾(mother compounds)뿉꽌 踰ㅼ젨쓽 ortho, meta, para 옄由ъ뿉 釉뚮\쑝濡 移섑솚븯뿬 빀꽦븳 B8, B9, B10 以 B8 (2,4,6,8-tetrakis(2-bromophenyl)-3,7-diazabicyclo[3.3.1]nonan-9-one)쓽 룆꽦씠 媛옣 媛뺥븯떎뒗 寃껋쓣 솗씤븯떎(Table 1). 뵲씪꽌 蹂 뿰援ъ뿉꽌뒗 B8뿉 쓽빐 쑀룄릺뒗 꽭룷궗硫 쁽긽怨 옄꽭븳 옉슜湲곗쟾쓣 諛앺엳怨좎옄 븳떎.

Evaluation of cytotoxicity of bispidinone derivatives

Bispidinone Bispidinone analogue Number IC50 (μM)
B8 76

B9 >80

B10 >80

옱猷 諛 諛⑸쾿

솕븰젣뭹

Phosphate-buffered saline (PBS, pH 7.4) dimethyl sulfoxide (DMSO)뒗 Sigma-Aldrich Chemical Co. (St. Louis, MO, USA)뿉꽌 援щℓ븯怨 Annexin-FITC kit 꽭룷궗硫몄뿉 븳 꽭룷二쇨린 遺꾩꽍쓽 PI/RNase 뿼깋슜븸 BD Pharmingen (San Diego, CA, USA), Eagl챕s minimum essential medium (EMEM), penicillin-streptomycin, Fetal bovine serum (FBS) HyClone Laboratories, InC. (Logan, UT, USA), 洹몃━怨 cell counting Kit-8 Dojindo Molecular Technologies, InC. (Osaka, Japan)뿉꽌 뼸뿀쑝硫 trypsin-EDTA뒗 Gibco-BRL (Invitrogen Co., USA)뿉꽌 援щℓ븯떎.

꽭룷諛곗뼇

American Type Culture Collection (ATCC, Manassas, VA, USA)뿉꽌 뼸 HeLa cells쓣 Fetal bovine serum怨 1% penicillin-streptomycin 꽔뼱 留뚮뱺 EMEM쑝濡 37꼦 (5% CO2)쓽 뒿븳 솚寃쎌뿉꽌 諛곗뼇븯떎.

Bispidinone 슜븸뱾쓽 젣議

2,4,6,8-tetrakis(2-bromophenyl)-3,7-diazabicyclo[3.3.1]nonan-9-one (B8)怨 2,4,6,8-tetrakis(3-bromophenyl)-3,7-diazabicyclo [3.3.1]nonan-9-one (B9), 2,4,6,8-tetrakis(1-bromophenyl)-3,7-diazabicyclo[3.3.1]nonan-9-one (B10) Dr. D. H. Park쓽 떎뿕떎뿉꽌 뼸뿀쑝硫, bispidinone 슜븸 dimethyl sulfoxide (DMSO)뿉 끃뿬 留뚮뱾뿀怨 異붽 씗꽍 떎뿕 吏곸쟾뿉 떎뻾븯떎.

꽭룷쓽 깮議 諛 利앹떇 遺꾩꽍

HeLa cells쓣 96-well plate쓽 媛 well뿉 諛곗 mL떦 5횞104쓽 媛믪쑝濡 怨꾩궛븯뿬 젒醫 썑 諛곗뼇븯뒗 룞븞 media瑜 媛덉븘 二쇱뿀쑝硫 B8쓣 떎뼇븳 냽룄濡 泥섎━븳 썑 48떆媛 룞븞 諛곗뼇븯떎. 삉븳 CCK-8 떆빟 10 關L瑜 媛 well뿉 꽔뼱 異붽濡 2떆媛 룞븞 諛곗뼇븯怨 꽭룷 깮議댁쑉 WST-8瑜 궗슜븯뿬 痢≪젙븯쑝硫, 궡븘엳뒗 꽭룷쓽 optical density뒗 multi-microplate reader (synergy HT, Bio-Tek)濡 450 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. HeLa cells쓽 proliferation assay쓽 寃쎌슦 96-well plate뿉 mL쓽 諛곗떦 5횞104쑝濡 seeding븯怨 B8쓣 76 關M 냽룄濡 泥섎━ 썑 떎뼇븳 떆媛 蹂솕뿉 븳 씉愿묐룄瑜 痢≪젙븯쑝硫, 媛곴컖쓽 떎뿕 3쉶 씠긽 諛섎났븯떎.

꽭룷궗硫몄쓽 삎깭븰쟻 遺꾩꽍

HeLa cells쓣 6-well plate뿉 2횞105 cell/well 냽룄濡 꽔 썑 諛ㅼ깉 遺李⑹떆궎怨, cells뿉 24떆媛, 48떆媛 룞븞 B8(빟 75 關M)怨 븿猿 泥섎━븯떎. 泥섎━릺吏 븡 wells 議곌뎔쑝濡쒖꽌 DMSO (<0.1%) 룞벑븳 뼇쓣 꽔怨 痢≪젙븯떎. 삉븳 꽭룷뿉 븳 寃궗 삎깭븰쟻 蹂솕뒗 Nikon Phase Contrast-2 ELWD 0.3 invert microscope濡 珥ъ쁺븯떎.

Annexin V-FITC/PI瑜 씠슜븳 꽭룷궗硫 遺꾩꽍

HeLa 꽭룷瑜 60 mm-dish뿉 1횞105 꽭룷/mL쓽 냽룄濡 媛븯뿬 24떆媛 諛곗뼇븳 썑뿉 76 關M쓽 B8쓣 룷븿븳 깉濡쒖슫 諛곗濡 媛곴컖 援먯껜븳 썑 媛곴린 떎瑜 떆媛꾨뿉 뵲씪 듃由쎌떊 泥섎━(trypsinization)瑜 씠슜븯뿬 꽭룷瑜 紐⑥떎. 洹몃━怨 썝떖遺꾨━湲곕 씠슜븯뿬 꽭룷 遺쑀븸쓣 씤궛셿異⑹슜븸(PBS)쑝濡 꽭泥숉븳 썑 꽭룷 렆젢(pellet)쓣 100 關L쓽 寃고빀셿異⑹슜븸(binding buffer) 뿉 遺쑀떆궓 떎쓬 5 關L쓽 annexin V-FITC 10 關L쓽 PI (50 關g/mL)瑜 媛븳 썑 떎삩(15~25꼦)쓽 뼱몢슫 怨녹뿉꽌 15遺꾧컙 뿼깋븯떎. 洹몃━怨 씠 꽭룷뱾 488 nm쓽 뙆옣뿉꽌 FACScalibur 쑀꽭룷遺꾩꽍湲(FACScalibur-湲곌린紐⑤뜽, BD Bioscience)瑜 씠슜븯뿬 痢≪젙븯怨 BD Instruments뿉꽌 젣怨듬맂 봽濡쒓렇옩쑝濡 遺꾩꽍븯떎.

꽭룷 궡 솢꽦궛냼 痢≪젙

솢꽦궛냼 醫낆쓽 깮꽦 삎愿 吏떆빟씤 2,7-dichlorodihydrofluorescein (H2DCFDA)瑜 궗슜븯뿬 痢≪젙븯떎. H2DCFDA뒗 ROS쓽 깮꽦쓣 넻빐 넂 끃깋 삎愿묐Ъ吏덉씤 2,7-dichlorofluorescein (DCF)濡 궛솕븳떎. B8 (76 關M)怨 븿猿 泥섎━븯嫄곕굹 泥섎━븯吏 븡 HeLa cells (1횞105 cells in a 60 mm dish)쓣 듃由쎌떊 泥섎━ 썑 썝떖遺꾨━븯뿬 닔吏묓븯떎. 떆猷뚮 李④슫 PBS濡 꽭泥숉븯怨 37꼦, 븫떎뿉꽌 30遺 룞븞 2 關L쓽 H2DCFDA瑜 泥섎━븯뿬 뿼깋븯떎. 긽쟻 삎愿묎컯룄뒗 FACSCalibur flow cytometer쑝濡 痢≪젙븯怨, 520 nm쓽 씉愿묐룄 FL-1 channel (green)濡 留욎텛뼱 Cell Quest pro software濡 遺꾩꽍븯떎.

誘명넗肄섎뱶由ъ븘 留 쟾쐞(뼰顆m)쓽 痢≪젙

誘명넗肄섎뱶由ъ븘 留 쟾쐞쓽 蹂솕뒗 삎愿 probe씤 rhodamine 123 (RH-123; Molecular Probes)쓣 씠슜븯뿬 痢≪젙븯떎. B8 (76 關M)쓣 泥섎━븯嫄곕굹 泥섎━븯吏 븡 HeLa cells (1횞105 cells in 60 mm dish)뿉 듃由쎌떊 泥섎━ 썑 썝떖遺꾨━븯뿬 닔吏묓븯떎. 떆猷뚮 李④슫 PBS濡 꽭泥숉븯怨, rhodamine intensities瑜 5 關L 꽔怨 뿼깋븳 썑 FACSCalibur flow cytometer뿉꽌 痢≪젙媛믪쓣 FL-1 channel濡 留욎떠 愿李고븯怨 cell Quest pro software濡 遺꾩꽍븯떎.

異붿텧臾 젣議 諛 western blot 遺꾩꽍

B8 (76 關M)쓣 HeLa cells뿉 븿猿 泥섎━븯嫄곕굹 泥섎━븯吏 븡 HeLa cells (1횞105 cells in 150 mm dish)쓽 珥 꽭룷 異붿텧臾쇨낵 꽭룷吏 遺꾪쉷 PBS 泥섎━ 썑 썝떖遺꾨━븯뿬 닔吏묓븯떎. 異붿텧臾쇱쓣 4꼦뿉꽌 5遺꾧컙 25,000 g뿉꽌 썝떖遺꾨━븯뿬 留뚮뱾뼱吏 긽벑븸쓽 떒諛깆쭏 븿웾 Bradford 떒諛깆쭏 遺꾩꽍쑝濡 痢≪젙븯떎. 紐⑤뱺 꽭룷 異붿텧臾(25 關g)쓣 쟾湲곗쁺룞븯뿬 nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA) 긽쑝濡 삷寃쇰떎. Bcl-2, procaspase-8, procaspase-9, 棺-actin (1: 1,000; Cell Signaling Technology, Danvers, MA, USA), procaspase-3 (1: 300, Santa Cruz Biotechnology, Santa Cruz, CA, USA) 洹몃━怨 Bax (1: 1,000, BD Biosciences Pharmingen)媛 primary polyclonal antibodies濡 궗슜릺뿀떎.

넻怨 遺꾩꽍

媛 떎뿕 3踰 諛섎났 떎뿕븳 뜲씠꽣쓽 룊洹좎 짹 SD 媛믪쑝濡 몴쁽븯떎. One-way analysis濡 넻怨꾩쟻 遺꾩꽍쓣 븯怨, P<0.05뿉꽌 쑀쓽븳 李⑥씠瑜 蹂댁떎. Microsoft Excel 2016 (Roselle, IL, USA)濡 넻怨 遺꾩꽍븯怨 룄몴瑜 洹몃졇떎.

寃 怨

Bispidinone 쑀룄泥대뱾쓽 꽭룷룆꽦 슚怨

떎뼇븳 븣移쇰줈씠뱶 遺꾩옄 援ъ“瑜 媛뽯뒗 bispidinone 솕빀臾(mother compounds)뿉꽌 踰ㅼ젨쓽 ortho, meta, para 옄由ъ뿉 釉뚮\쑝濡 移섑솚븯뿬 빀꽦븳 B8, B9, B10 以 B8 (2,4,6,8-tetrakis(2-bromophenyl)-3,7-diazabicyclo[3.3.1]nonan-9-one)쓽 룆꽦씠 媛옣 媛뺥븯떎뒗 寃껋쓣 WST-8 遺꾩꽍쓣 넻븯뿬 솗씤븯떎(Table 1). 냽룄(40, 60, 80, 洹몃━怨 120 關M)媛 利앷븯룄濡 꽕젙븯뿬 48 h 룞븞 諛곗뼇븯硫 議곌뎔뿉 鍮꾪빐 냽룄媛 利앷븿뿉 뵲씪 꽭룷쓽 깮議대뒫젰씠 媛먯냼븯떎(Fig. 1A). B8씠 겙 꽭룷룆꽦 슚怨쇰 蹂댁뿬二쇱뿀쑝硫 IC50 媛믪씠 76 關M엫쓣 솗씤븯떎. HeLa cells뿉 B8쓣 泥섎━븯쓣 븣 媛숈 냽룄뿉꽌 떆媛꾩뿉 뵲瑜 꽭룷 利앹떇쓣 솗씤쓣 쐞빐 0~60 h源뚯 12떆媛 媛꾧꺽쑝濡 꽕젙븯怨 IC50 媛믪씤 76 關M濡 냽룄瑜 媛숆쾶 뻽쓣 븣쓽 寃곌낵(Fig. 1B)瑜 솗씤븯떎. 議곌뎔 떆媛꾩씠 利앷븿뿉 뵲씪 꽭룷쓽 利앹떇씠 솢諛쒗엳 씪뼱굹뒗 諛섎㈃ B8쓣 泥섎━븳 寃쎌슦 利앹떇븯吏 븡怨 젏李 媛먯냼븯뒗 寃쏀뼢쓣 蹂댁떎.

Fig. 1. Effect of B8 on cytotoxicity and proliferation of HeLa cells. (A) HeLa cells were treated with 0, 40, 80 μM of B8 for 48 h. (B) HeLa cells were treated with 76 μM of B8 for various times (0, 12, 24, 36, 48, 60 h). Cell viability was measured by WST-8 assay. Results are expressed as mean ± SD, n=3. *P<0.05, significantly different from the control at the same level.

B8뿉 쓽븳 HeLa cells쓽 삎깭븰쟻 蹂솕 愿李

꽭룷쓽 삎깭븰쟻 蹂솕뒗 꽭룷 궗硫몄쓽 以묒슂븳 듅吏뺤쓣 굹궡湲 븣臾몄뿉 B8쓣 泥섎━븳 HeLa cells怨 泥섎━븯吏 븡 HeLa cells쓽 蹂솕瑜 쁽誘멸꼍쑝濡 愿李고븯떎. B8쓣 泥섎━븯吏 븡 HeLa cells쓣 culture plates뿉꽌 24, 48 h 룞븞쓽 蹂솕瑜 愿李고븯쓣 븣 24 h뿉꽌쓽 議곌뎔(Fig. 2A)怨 48 h쓽 議곌뎔(Fig. 2C) 꽭룷 궗硫멸낵 愿젴맂 삎깭媛 留롮씠 蹂댁씠吏 븡븯怨 媛吏瑜 六쀬쑝硫 옒 옄씪뒗 諛섎㈃뿉, B8쓣 76 關M쓽 냽룄濡 24 h 룞븞 泥섎━븯쓣 븣(Fig. 2B) 씪遺쓽 HeLa cells씠 plates뿉꽌 뼥뼱졇 遺쑀븯怨 엳뿀쑝굹 꽭룷 궗硫몄쓽 듅吏뺤씠 슌졆븯寃 蹂댁씠吏 븡븯떎. 48 h 룞븞 B8쓣 76 關M쓽 냽룄濡 泥섎━뻽쓣 븣(Fig. 2D)쓽 寃쎌슦 꽭룷 궗硫몄쓽 삎깭븰쟻 듅吏뺤씤 꽭룷뱾씠 닔異뺣릺怨 뙆愿대릺뒗 紐⑥뒿怨 留롮 꽭룷媛 룞洹몃 紐⑥뼇쑝濡 遺쑀븯怨 엳쓬쓣 蹂댁떎.

Fig. 2. Induction of morphological change in HeLa cells treated with B8. HeLa cells were untreated with B8 for 24 h (A) and 48 h (C). HeLa were treated with B8 (76 μM) for 24 h (B) and 48 h (D). Magnification, x160. All the experiments were performed in triplicate and gave similar results.

B8뿉 쓽븳 ROS MMP levels쓽 蹂솕

B8뿉 쓽븯뿬 쑀룄릺뒗 ROS level쓽 蹂솕瑜 솗씤븯湲 쐞븯뿬 B8쓣 76 關M쓽 냽룄濡 泥섎━븯뿬 0, 1, 2, 4, 8 h 룞븞쓽 蹂솕瑜 꽭룷 닾怨쇱꽦 뿼猷뚯씤 H2DCF-DA 삎愿 뿼猷뚮 궗슜븯뿬 痢≪젙븯떎(Fig. 3A). B8쓣 泥섎━븯쓣 븣 H2DCF-DA쓽 룊洹 삎愿묎컪 29.27짹1.69 (0 h)뿉꽌 74.24짹1.64 (2 h)濡 利앷븯怨 4떆媛 썑 媛먯냼븯뒗 寃쏀뼢쓣 蹂댁떎. 씠濡 씤빐 B8씠 ROS쓽 깮꽦뿉 쁺뼢쓣 以떎뒗 寃껋쓣 븣 닔 엳뿀떎. ROS쓽 깮꽦 誘명넗肄섎뱶由ъ븘 留 쟾쐞쓽 遺뺢눼 뿰愿릺뼱 엳怨 誘명넗肄섎뱶由ъ븘 留됱쓽 깉遺꾧레 삉븳 꽭룷궗硫몄쓽 以묒슂븳 듅吏뺤쑝濡 뿬寃⑥뒗뜲, rhodamine 123 dye쓽 삎愿 듅꽦쓣 씠슜빐 깮泥대Ъ吏덉씠굹 洹멸쾬쓣 몮윭떥뒗 二쇰 솚寃쎌쓽 꽦吏덉쓣 議곗궗븯湲 쐞빐 궗슜븯뿬 MMP쓽 넀떎 뿬遺瑜 솗씤븯떎(Fig. 3B). 洹 寃곌낵 76 關M쓽 B8쓣 泥섎━븳 썑 룊洹 rhodamine 123쓽 삎愿묒씠 73.67짹1.98 (0 h)뿉꽌 66.94짹1.64 (12 h), 53.45짹1.87 (24 h) 諛 39.25짹1.60 (48 h)濡 젏李 媛먯냼븯뿬 B8씠 泥섎━맂 HeLa cells뿉꽌 誘명넗肄섎뱶由ъ븘媛 꽭룷 궗硫몄뿉 愿뿬븿쓣 蹂댁떎.

Fig. 3. The effect of B8 on ROS generation and MMP level on HeLa cells. (A) Cells treated with B8 were incubated with DCF-DA. And the intracellular levels of ROS were determined by measuring the level of DCF fluorescence. (B) Cells were incubated with 76 μM of B8 for varying lengths of time (0~48 h) and then analyzed by JC-1 derived fluorescence. Results are the mean ± SD, n=3. *P< 0.05, means significantly differ from the control at the same level.

B8뿉 쓽븳 꽭룷 궗硫 쑀룄 뿬遺 솗씤

B8뿉 쓽븳 꽭룷 궗硫몄쓽 쑀룄 뿬遺瑜 븣븘蹂닿린 쐞븯뿬 Annexin V-FITC/PI 씠以 뿼깋 遺꾩꽍踰뺤쓣 궗슜븯뿬 HeLa cells뿉 B8쓣 泥섎━븯吏 븡 꽭룷 76 關M쓽 냽룄濡 泥섎━븳 cells뿉 븳 24, 48 h 룞븞쓽 蹂솕瑜 愿李고븯떎(Fig. 4). Fig. 4뿉 굹 엳뒗 媛濡쒖텞 Annexin V-FITC, 꽭濡쒖텞 PI瑜 쓽誘명븯硫 媛슫뜲쓽 以묒떖쓣 湲곗쑝濡 삤瑜몄そ 븘옒, 쐞媛 媛곴컖 꽭룷궗硫 援ш컙(early apoptotic cells (Annexin Vpositive/PInegative)怨 late apoptotic cells (Annexin Vpositive/PIpositive)씠硫 쇊履 쐞 븘옒뒗 媛곴컖 necrosis (Annexin Vnegative/PIpositive) live cells (Annexin Vnegative/PInegative)쓣 쓽誘명븳떎. 24 h 룞븞 B8쓣 泥섎━븯吏 븡 議곌뎔(Fig. 4A) B8쓣 泥섎━븳 HeLa cells (Fig. 4C)뿉 鍮꾪빐 꽭룷궗硫 援ш컙씠 빟 20% 利앷븯뒗 寃껋쓣 蹂댁쑝硫, 48 h 룞븞 B8쓣 泥섎━븯吏 븡 議곌뎔(Fig. 4B)怨 B8쓣 泥섎━븳(Fig. 4D) 寃껋쓽 꽭룷궗硫 援ш컙 李⑥씠뒗 빟 30% 利앷븯떎. 씠濡 씤빐 HeLa cells뿉꽌 B8씠 꽭룷 궗硫몄쓣 쑀룄븿쓣 븣 닔 엳뿀떎.

Fig. 4. B8 induced apoptosis in HeLa cells. Untreated with B8 for 24 h (A) and 48 h (C), or treated with B8 (76 μM) for 24 h (B) and 48 h (D). Data were obtained by flow cytometric analysis of Annexin V-FITC/PI double-staining assay. All the experiments were performed in triplicate and gave similar results.

Western blot 遺꾩꽍

꽭룷 궗硫멸낵 愿젴릺뒗 떒諛깆쭏뱾쓽 諛쒗쁽쓣 議곗궗븯뿬 B8씠 뼱뼡 硫붿빱땲利섏쓣 넻빐 꽭룷 궗硫몄쓣 쑀룄븯뒗吏瑜 western blot analysis쓣 궗슜븯뿬 솗씤븯떎(Fig. 5). Fig. 5A뿉꽌 Bcl-2 愿젴맂 떒諛깆쭏 mitochondrial-mediated pathway媛 썝솢븯寃 씪뼱굹湲 쐞빐 以묒슂븳 뿭븷쓣 븳떎(Gross et al., 1999). Anti-apoptotic protein씤 Bcl-2쓽 諛쒗쁽 떆媛꾩씠 吏궓뿉 뵲씪 媛먯냼뻽쑝硫, pro-apoptotic protein씤 Bax쓽 諛쒗쁽 떆媛꾩씠 吏궓뿉 뵲씪 利앷븯뒗 寃쏀뼢쓣 蹂댁怨, procaspases-3, 9쓽 諛쒗쁽 삉븳 利앷븯떎. Bax/Bcl-2 鍮꾩쑉쓣 솗씤븳 寃곌낵 48 h뿉꽌 겕寃 利앷븯뒗 寃껋쓣 솗씤븷 닔 엳뿀떎(Fig. 5C). Fig. 5A쓽 寃곌낵뿉 쓽븯硫 꽭룷 궗硫몄 궡遺 寃쎈줈濡 씪뼱궓쓣 솗씤븯怨, 삉븳 procaspase-8쓽 諛쒗쁽(Fig. 5B)씠 媛먯냼븯뒗 寃쏀뼢쓣 蹂댁엫쑝濡쒖뜥 B8 Fas signaling apoptosis 寃쎈줈瑜 넻븯뿬 꽭룷 궗硫몄쓣 쑀룄븷 媛뒫꽦룄 엳쓬쓣 솗씤븯떎.

Fig. 5. Induction of protein activation in B8-induced apoptosis on HeLa cells. HeLa cells were treated with 76 μM B8 for 0. 24 and 48 h. the expression of proteins was analyzed by western blotting. (A) Protein expression levels of Bcl-2, Bax, procaspase-9, procaspase-3. (B) Protein expression of levels of procaspase-8. (C) Ratio of Bax/Bcl-2. Results are expressed as mean ± SD, n=3. *P<0.05, significantly different from the control at the same level.
怨 李

理쒓렐뿉 bispidinone뿉 븳 븫 移섎즺踰뺢낵 愿젴맂 옄猷뚭 諛쒗몴릺뿀怨 씠寃껋쓽 꽭룷룆꽦 슚怨쇰뒗 留롮씠 븣젮졇 엳쑝誘濡 븫쓣 移섎즺븯湲 쐞븳 諛⑸쾿쑝濡 벐씪 닔 엳쓣 寃껋쑝濡 깮媛곹븳떎(Predebon et al., 2019). Bispidinone뿉 엳뒗 븣移쇰줈씠뱶 以 鍮꾪誘 A굹 씛媛쒕몄쓽 럹濡쒕が 벑쓽 깮臾쇳솢꽦쓣 媛吏뒗 뵒뀒瑜댄렂(diterpene) 떊泥댁뿉꽌 삁븸 닚솚쓣 媛쒖꽑떆耳쒖<硫, 떖由 븞젙, 媛뺣젰븳 빆궛솕 옉슜怨 빆븫 옉슜쓣 븯뒗 벑 떎뼇븳 빟由 옉슜怨 젙꽌瑜 븞젙떆궎뒗뜲 룄쓣 二쇰뒗 寃껋쑝濡 蹂닿퀬릺뼱 엳怨 猷⑦ 벑怨 媛숈 媛뺣젰븳 깮由 옉슜쓣 二쇰뒗 듅꽦쓣 媛吏뒗 븣移쇰줈씠뱶媛 議댁옱븯湲 븣臾몄뿉 깮臾쇳븰쟻쑝濡 愿떖쓣 留롮씠 媛吏寃 릺뿀떎. B8 떎瑜 bispidinone 쑀룄泥대뱾(B9, B10)蹂대떎 궙 냽룄뿉꽌 HeLa cells쓽 利앹떇쓣 뼲젣븯뿬 B8씠 뼱뼡 寃쎈줈瑜 嫄곗퀜 꽭룷媛 二쎌쓬뿉 씠瑜대뒗吏뿉 븳 궗떎쓣 떎뼇븳 諛⑸쾿쓣 궗슜븯뿬 꽭룷궗硫 쑀룄 寃쎈줈瑜 솗씤븯떎(Kiechle and Zhang, 2002; Otsuki et al., 2003).

HeLa cells쓽 삎깭븰쟻 遺꾩꽍뿉 쓽븯뿬 B8뿉 쓽빐 꽭룷겕湲곌 以꾩뼱뱾뿀쑝硫 닔異뺢낵 遺꾩뿴 媛숈 꽭룷궗硫 쑀삎씠 愿李곕릺뿀떎. ROS쓽 깮꽦怨 MMP瑜 痢≪젙븯湲 쐞빐 rhodamine 123 뿼깋踰뺤쓣 궗슜뻽떎. Rh-123 삎愿묐Ъ吏덉 mitochondrial potential뿉 뵲씪 깋씠 蹂븯뒗 꽦吏덉쓣 궗슜븯뿬 痢≪젙븯떎. 痢≪젙븳 寃곌낵 B8씠 꽭룷 궗硫몄쓣 쑀룄뻽쑝硫, 씠뒗 꽭룷 媛 ROS쓽 깮꽦怨 MMP쓽 쑀쓽븳 媛먯냼瑜 留ㅺ컻븿쓣 蹂댁떎. 洹몃━怨 Annexin V-FITC/PI 씠以묒뿼깋 遺꾩꽍踰뺤쓣 궗슜븳 寃곌낵 B8뿉 쓽빐 꽭룷 궗硫몄씠 씪뼱궓쓣 솗떊븷 닔 엳뿀떎.

HeLa cells뿉꽌 B8씠 뼱뼡 寃쎈줈瑜 넻빐 꽭룷 궗硫몄쓣 씪쑝궎뒗吏 븣븘蹂닿린 쐞빐 western blot analysis쓣 븯떎. 꽭룷궗硫 寃쎈줈뿉꽌 以묒슂븳 뿭븷쓣 떞떦븯뒗 떒諛깆쭏뿉뒗 Bax Bcl-2媛 엳쑝硫, Bax (pro-apoptotic protein)뒗 꽭룷吏덈줈 cytochrome c쓽 諛⑹텧쓣 洹밸솕떆궎怨 Bcl-2 (anti-apoptotic protein)뒗 諛⑹텧쓣 뼲젣떆궎뒗 뿭븷쓣 떞떦븳떎. 蹂 뿰援ъ뿉꽌 Bcl-2쓽 諛쒗쁽씠 젏李⑥쟻쑝濡 媛먯냼븯怨 Bax쓽 諛쒗쁽씠 利앷븯뒗 寃껋쓣 솗씤븯怨 Bcl-2 family protein씠 꽭룷 궗硫몄쓣 議곗젅븯뒗 뿴뇿 뿭븷쓣 븯怨 엳쓬쓣 利앸챸븯떎. 삉븳 誘명넗肄섎뱶由ъ븘 궡遺뿉 엳뒗 cytochrome c뒗 Apaf-1怨 caspase-9濡 씤빐 apoptosome쓽 삎꽦쓣 洹밸솕떆궎怨, caspase-3쓽 솢꽦쓣 利앷떆耳 꽭룷 궗硫몄쓣 씪쑝궓떎(Fulda and Debatin, 2006; Saitoh et al, 2004; Ghobrial et al., 2005). B8 ROS쓽 깮꽦쓣 利앷떆궎怨 誘명넗肄섎뱶由ъ븘 留 쟾쐞瑜 媛먯냼떆耳 꽭룷 궗硫몄쓣 珥됱쭊떆耳곕떎. B8뿉 쓽빐 caspase cascades媛 솢꽦솕릺뼱 誘명넗肄섎뱶由ъ븘媛 愿뿬릺뒗 寃쎈줈瑜 넻븯뿬 꽭룷 궗硫몄씠 쑀룄맖쓣 븣 닔 엳뿀떎. 삉븳 B8 procaspase-8쓽 諛쒗쁽쓣 媛먯냼떆耳 쇅遺 寃쎈줈瑜 넻빐 꽭룷 궗硫몄쓣 씪쑝궗 닔 엳뒗 媛뒫꽦룄 솗씤븯떎.

ACKNOWLEDGEMENT

None.

CONFLICT OF INTEREST

No a conflict of interest exist in other authors or my institution.

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