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In Vitro Evaluation of Anti-cancer Properties of Hongyoung on SNU-80 Anaplastic Thyroid Carcinoma Cell Line
Biomed Sci Letters 2023;29:321-329
Published online December 31, 2023;  https://doi.org/10.15616/BSL.2023.29.4.321
© 2023 The Korean Society For Biomedical Laboratory Sciences.

Gaeun Kim* and Eun-Jung Kim†,**

Department of Biomedical Laboratory Science, Sangji University, Wonju 26339, Korea
Correspondence to: Eun-Jung Kim. Department of Biomedical Laboratory Science, Sangji University, Wonju 26339, Korea.
Tel: +82-33-738-7683, Fax: +82-33-738-7683, e-mail: jung0724@sangji.ac.kr
*Undergraduate student, **Professor.
Received November 9, 2023; Revised December 5, 2023; Accepted December 5, 2023.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Anaplastic thyroid cancer has the highest mortality rate of all thyroid cancers and shows low responsiveness to most treatments. Hongyoung, a reddish-colored potato, is an excellent source of dietary polyphenol containing a large amount of anthocyanins, which has anti-cancer and anti-inflammatory effects. This study investigated the effects of Hongyoung extract on apoptosis and invasiveness in SNU-80 anaplastic thyroid cancer cells. The quantification of the total polyphenol content was done by spectrophotometric measurement. Cell growth was measured by using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) 2H tetrazolium, monosodium salt (MTS) assay. Cell cycle was analyzed through FACS analysis. Induction of apoptosis in cells was investigated by annexin V staining using flow cytometer and the expression of caspase-3 and Poly (ADP-ribose) polymerase (PARP) through western blot. mRNA expression and protein activation of matrix metalloproteinases (MMP)-2/-9 were examined by RT-PCR and zymography. As a result, the TPC of Hongyoung was 292.43±8.42 mg gallic acid equivalent (GAE)/100 g dry extract. Hongyoung showed a dose-dependent cell growth inhibition, and the IC50 values was 1,000 μg/mL. sub-G1 phase was more than doubled compared to the control group, and S and G2/M phase arrest were also induced. Hongyoung induced apoptosis by increasing FITC-Annexin V-positive cells and increased the activation of caspase-3 (cleaved caspase-3) and PARP (fragmented PARP). Hongyoung significantly inhibited mRNA expression and protein activation of MMP-2/-9 in phorbol 12-myristate 13-acetate (PMA)-treated SNU-80 cells. Therefore, this study suggests the possibility of development of Hongyoung extract as an anti-cancer agent.
Keywords : Anti-cancer properties, Apoptosis, Hongyoung, SNU-80 anaplastic thyroid cancer cells
꽌 濡

媛묒긽꽑븫 媛옣 쓷븳 궡遺꾨퉬怨 븙꽦 醫낆뼇쑝濡, 諛쒕퀝瑜좎 쟾꽭怨꾩쟻쑝濡 袁몄엳 利앷븯怨 엳떎. 援궡 媛묒긽꽑븫쓽 諛쒕퀝瑜좎 蹂닿굔蹂듭遺 븫벑濡앺넻怨꾩뿉 뵲瑜대㈃ 2000뀈遺꽣 湲됯꺽엳 利앷빐 삤怨 엳쑝硫, 理쒓렐뿉뒗 궓 쟾泥댁뿉꽌 媛옣 留롮씠 諛쒖깮븳 븫쑝濡 蹂닿퀬릺怨 엳떎. 媛묒긽꽑븫 遺꾪솕 젙룄뿉 뵲씪 怨좊텇솕븫, 뿭삎꽦(誘몃텇솕)븫, 湲고(遺꾪솕븫, 쟾씠븫)濡 遺꾨쪟릺怨 엳떎. 씠뱾 以 뿭삎꽦 媛묒긽꽑븫 쟾泥 媛묒긽꽑 븫쓽 2% 誘몃쭔쓣 李⑥븯뒗 뱶臾 醫낆뼇쑝濡, 넂 븙꽦룄濡 씤빐 룊洹 깮議댁쑉 吏꾨떒 썑 3~5媛쒖썡씠硫, 1뀈 깮議댁쑉 20% 誘몃쭔쑝濡 궙 깮議댁쑉쓣 굹궦떎(Amaral et al., 2020; Smallridge and Copland, 2010).

뿭삎꽦 媛묒긽꽑븫(Anaplastic thyroid cancer, ATC)쓽 諛쒖깮 湲곗쟾 뿬쟾엳 紐낇솗엳 諛앺졇 엳吏 븡吏留, 遺遺꾩 湲곗〈쓽 遺꾪솕 媛묒긽꽑븫뿉꽌 깉遺꾪솕瑜 넻빐 諛쒖깮븳떎怨 븣젮졇 엳떎(Paes and Ringel, 2008; Park, 2010; Volante et al., 2021). 삉븳 ATC뒗 鍮좊Ⅸ 꽦옣怨 媛뺥븳 移⑥쑄 諛 쟾씠 媛숈 뒫젰쓣 媛뽮퀬 엳뼱 뼱뼚븳 移섎즺踰뺤뿉룄 슌졆븳 諛섏쓳쓣 蹂댁씠吏 븡쑝硫, 떎닔쓽 솚옄뿉寃 洹뱀떖븳 넻利, 뵾濡, 샇씉怨ㅻ 벑쓽 떖媛곹븳 遺옉슜怨 빀蹂묒쬆쓣 쑀諛쒗븯뿬 궣쓽 吏덉쓣 븯씫떆궓떎怨 蹂닿퀬릺怨 엳떎(Amaral et al., 2020; Nagaiah et al., 2011; O'Neill and Shaha, 2013; Smallridge and Copland, 2010). 뿭삎꽦 媛묒긽꽑븫쓽 깉濡쒖슫 移섎즺踰뺤뿉 븳 뿰援ш 뿬쟾엳 吏냽릺怨 엳쑝硫, 씠뱾 以 듅엳 泥쒖뿰臾쇱쓣 씠슜븳 移섎즺踰뺤씠 遺옉슜怨 빀蹂묒쬆쓣 理쒖냼솕븯怨 移섎즺 슚怨쇰 洹밸솕븷 닔 엳湲 븣臾몄뿉 二쇰ぉ諛쏄퀬 엳떎(Ferrari et al., 2020; Jaskula-Sztul et al., 2011; Lan et al., 2017; Li et al., 2022; Saini et al., 2018; Sharifi-Rad et al., 2020; Shin et al., 2019; Yeo et al., 2014; Yeo et al., 2014; Yu et al., 2013).

媛먯옄(Solanum tuberosum Linnaeus)뒗 踰, 諛, 삦닔닔 븿猿 꽭怨 4 옉臾쇰줈 븣젮졇 엳쑝硫, 吏諛, 鍮꾪誘, 떒諛깆쭏, 꽟쑀吏 諛 湲고 留롮 쁺뼇냼 븿猿 깂닔솕臾쇱씠 뭾遺빐 썙瑜븳 떇웾옄썝쑝濡 씠슜릺뼱 삤怨 엳떎. 쁽옱 쟾꽭怨꾩쟻쑝濡 솴깋怨 諛깆깋쓽 愿닿꼍쓣 媛吏 媛먯옄媛 씪諛섏쟻씠吏留, 넂 쑀쟾쟻 떎뼇꽦쑝濡 씤빐 쟻깋, 옄깋 벑 떎뼇븳 깋긽쓽 쑀깋媛먯옄룄 議댁옱븳떎(Burlingame et al., 2009; Yeo et al., 2014). 쑀깋媛먯옄뒗 솴깋 諛 諛깆깋쓣 씈뒗 씪諛 媛먯옄蹂대떎 뤃由ы럹 븿웾씠 3諛 뜑 넂쑝硫, carotenoid, anthocyanin怨 媛숈 깋냼濡 씤빐 떎뼇븳 깋쓣 씈怨 엳떎(Li et al., 2022; Petropoulos et al., 2019; Rasheed et al., 2022; Rytel et al., 2014). 듅엳 anthocyanin 떎瑜 쑀깋媛먯옄뱾蹂대떎룄 쟻깋怨 옄깋쓣 씈뒗 媛먯옄뿉꽌 븿웾씠 뜑슧 넂 寃껋쑝濡 븣젮졇 엳떎(Jansen and Flamme, 2006; Park et al., 2009). 理쒓렐 쑀깋媛먯옄쓽 빆궛솕, 빆븫, 빆룎뿰蹂씠 벑쓽 떎뼇븳 깮由ы솢꽦뿉 븳 뿰援ш 솢諛쒗엳 吏꾪뻾릺怨 엳떎(Jang and Yoon, 2012; Kang and Choung, 2008; Lee et al., 2017; Park et al., 2008; Park et al., 2007; Petropoulos et al., 2019; Rasheed et al., 2022; Tsang et al., 2018). 븯吏留 븘吏 뿭삎꽦 媛묒긽꽑븫꽭룷뿉꽌쓽 쑀깋媛먯옄뿉 쓽븳 빆븫 슚怨 뿰援щ뒗 蹂닿퀬맂 諛붽 뾾떎.

洹몃윭誘濡 蹂 뿰援ъ뿉꽌뒗 쑀깋媛먯옄 솉쁺 뿉깂삱 異붿텧臾쇱쓽 빆븫 슚怨쇰 SNU-80 뿭삎꽦 媛묒긽꽑븫꽭룷뿉꽌 룊媛븳 寃곌낵瑜 蹂닿퀬옄 븳떎.

옱猷 諛 諛⑸쾿

솉쁺 뿉깂삱 異붿텧臾 議곗젣

蹂 떎뿕뿉꽌 궗슜븳 솉쁺 냽珥뚯쭊씎泥뿉꽌 넻긽떎떆沅뚯쓣 떎떆븯뒗 솗궛醫낅쵖(媛뺣쫱, 븳援)뿉꽌 援ъ엯븯떎. 援ъ엯븳 솉쁺 猿띿쭏쓣 젣嫄고븯怨, 愿닿꼍쓣 3 mm 닔以쑝濡 옄瑜 썑, 떆猷 100 g뿉 80% 뿉깂삱 1,000 mL쓣 泥④븯뿬 24떆媛 룞븞 긽삩 異붿텧븯떎. 옍瑜섎Ъ 룞씪븳 諛⑸쾿쑝濡 2쉶 諛섎났빐 異붿텧븯쑝硫, 異붿텧맂 슜븸 紐⑤몢 빀븯뿬 뿬怨쇳븯떎. 뿬怨쇰맂 슜븸 60꼦 rotary vacuum evaporator (EYELA N-1300, Tokyo, Japan)濡 媛먯븬 냽異뺥븳 썑, 룞寃곌굔議곌린(EYELA FDU-1200, Tokyo, Japan)濡 룞寃곌굔議 븯떎. 룞寃곌굔議 븳 솉쁺 뿉깂삱 異붿텧臾쇱 dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA)뿉 씗꽍븯뿬 50 mg/mL 냽룄濡 以鍮꾪븯뿬 궗슜븯떎.

꽭룷 諛 떆빟

SNU-80 뿭삎꽦 媛묒긽꽑븫꽭룷二쇰뒗 븳援꽭룷二쇱뻾(Korea Cell Line Bank, Seoul, Korea)뿉꽌 遺꾩뼇 諛쏆븘 궗슜븯떎. 꽭룷뒗 RPMI-1640 (Gibco-BRL Life technologies Inc., Grand Island, NY, USA) 諛곗뿉 10% fetal bovine serum (Gibco-BRL), 1% penicillin/streptomycin (Gibco-BRL)쓣 泥④븯뿬 37꼦, 5% CO2 議곌굔쓽 諛곗뼇湲곗뿉꽌 諛곗뼇븯떎. 洹몃━怨 듅蹂꾪븳 뼵湲됱씠 뾾뒗 떆빟 紐⑤몢 Sigma-Aldrich궗쓽 理쒓퀬 닚룄 벑湲 젣뭹쓣 궗슜븯떎.

珥 뤃由ы럹 븿웾 痢≪젙

珥 뤃由ы럹 븿웾 Folin-ciocalteu 諛⑸쾿쓣 씠슜빐 痢≪젙븯떎. Gallic acid瑜 몴以臾쇱쭏濡 궗슜븯怨, 0, 50, 100, 250, 500, 750, 1,000 關g/mL濡 씗꽍븯뿬 以鍮꾪븯떎. 96-well plate뿉 씗꽍맂 gallic acid 50 mg/mL쓽 異붿텧臾쇱쓣 well 떦 4 關L뵫 꽔怨, 룞웾쓽 Folin-Ciocalte첬s reagent (Sigma-Aldrich) 180 關L쓽 利앸쪟닔瑜 泥④븯뿬 떎삩뿉꽌 5遺 諛섏쓳븯떎. 2% Na2CO3 (DUKSAN Science, Seoul, Korea) 12 關L瑜 泥④빐 40꼦 諛곗뼇湲곗뿉꽌 30遺 룞븞 븫諛섏쓳븯怨, microplate reader (Molecular Devices, Sunnydale, CA, USA)瑜 씠슜븯뿬 700 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. 珥 뤃由ы럹 븿웾 몴以臾쇱쭏씤 gallic acid濡쒕꽣 뼸 寃웾怨≪꽑쓣 씠슜븯뿬 援ы븯떎.

꽭룷 깮議댁쑉 痢≪젙

꽭룷 깮議댁쑉쓣 議곗궗븯湲 쐞빐, EZ-3000 assay kit (DoGENBIO, Seoul, Korea)쓣 씠슜븯뿬 痢≪젙븯떎. SNU-80 꽭룷瑜 96-well plate뿉 well떦 2 횞 104媛쒕줈 seeding븯怨 12떆媛 룞븞 諛곗뼇븳 썑, 솉쁺 뿉깂삱 異붿텧臾쇱쓣 500, 750, 1,000, 1,500, 2,000 關g/mL쓽 냽룄濡 泥섎━븯떎. 洹몃━怨 37꼦, 5% CO2 議곌굔쓽 諛곗뼇湲곗뿉꽌 24, 48, 72떆媛 룞븞 諛곗뼇븯떎. 洹 씠썑 MTS 슜븸쓣 꽭룷諛곗뼇븸뿉 10 關L/well쓣 泥④븯怨 1떆媛 룞븞 37꼦 諛곗뼇湲곗뿉꽌 諛섏쓳븯뿬 microplate reader (Molecular Devices, Sunnydale, CA, USA)瑜 씠슜븯뿬 450 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎.

꽭룷二쇨린 遺꾩꽍

SNU-80 꽭룷瑜 60 mm culture dish뿉 1 횞 106 cells/well濡 遺꾩<븯怨 18떆媛 룞븞 諛곗뼇븳 썑, 500, 750, 1,000 關g/mL쓽 냽룄濡 48떆媛 룞븞 諛곗뼇븯떎. 洹 썑, 꽭룷瑜 trypsin 泥섎━븯怨 phosphate-buffered saline (PBS)濡 꽭泥숉븳 썑, 70% ethanol濡 4꼦뿉꽌 12떆媛 怨좎젙븯떎. 怨좎젙맂 꽭룷瑜 PBS濡 꽭泥숉븯怨 100 關g/mL RNase A瑜 37꼦뿉 30遺 룞븞 泥섎━븯떎. 50 關g/mL Propidium Iodide濡 뿼깋븯怨 CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences, CA, USA)瑜 씠슜븯뿬 꽭룷二쇨린瑜 솗씤븯떎.

Reverse transcription-polymerase chain reaction (RT-PCR)

꽭룷濡쒕꽣 TRIzol 떆빟(Invitrogen Co., Carlsbad, CA, USA)쓣 궗슜븯뿬 珥 RNA瑜 異붿텧븯떎. 1 關g쓽 RNA瑜 ReverTra ACE PCR RT master mix kit (TOYOBO Co., Osaka, Japan)쓣 씠슜븯뿬 PCR쓣 닔뻾븯떎. 蹂 떎뿕뿉꽌뒗 MMP-2/-9 諛 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (Table 1)쓣 궗슜븯怨, housekeeping 쑀쟾옄씤 GAPDH 쑀쟾옄瑜 internal control濡 궗슜븯떎. 利앺룺맂 PCR 깮꽦臾쇱 0.5 關g/mL ethidium bromide (Sigma-Aldrich)媛 룷븿맂 1.5% agarose (Sigma-Aldrich) gel뿉 쟾湲곗쁺룞븯뿬 UV light 긽뿉꽌 솗씤븯떎.

Primer sets for RT-PCR

Gene Primer sequence (5' - 3') Annealing temperature (꼦) PCR product size (bp)
MMP-2 F : GCGACAAGAAGTATCGCTTC 58 390
R : TGCCAAGGTCAATGTCAGGA
MMP-9 F : CCATTTCGACGATGACGAGTT 58 530
R : CTTGTCGCTGTCAAAGTTCGAG
GAPDH F : GAAGGTGAAGGTCGGAGT 58 226
R : GAAGATGGTGATGGGATTTC


Gelatin zymography

떎뼇븳 냽룄쓽 솉쁺 異붿텧臾쇨낵 醫낆뼇 珥됱쭊옄濡 옒 븣젮吏 phorbol 12-myristate 13-acetate (PMA)瑜 0.5 關M 냽룄濡 떒룆 삉뒗 룞떆뿉 泥섎━븳 SNU-80 (1 횞 106) 꽭룷瑜 24떆媛 룞븞 諛곗뼇븳 썑, 긽痢듭븸쓣 紐⑥븘 Ultra-4-Centrifugal Filter Unit (Merck Millipore, Temecula, CA, USA)쓣 궗슜븯뿬 냽異뺥븯떎. 20 關g 냽룄쓽 떒諛깆쭏 non-reducing sample buffer (0.5 M Tris-Cl, pH 6.8, 5% SDS, 20% glycerol, 1% bromphenol blue) 븿猿 샎빀븯뿬 1% gelatin씠 룷븿맂 10% sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gel뿉꽌 쟾湲곗쁺룞 븯떎. 쟾湲곗쁺룞 썑, washing buffer (2.5% triton X-100)濡 SDS瑜 젣嫄고븯怨, incubation buffer (1 M Tris-Cl pH 7.5, 1 M CaCl2, 5 M NaCl, 0.02% NaN3, 0.2 mM ZnCl2, 2.5% triton X-100)濡 37꼦뿉꽌 12떆媛 룞븞 諛섏쓳븯떎. Gel coomassie brilliant blue (7% glacial acetic acid, 40% methanol, 0.25% coomassie blue)濡 30遺 룞븞 뿼깋븳 썑, destaining solution (7% glacial acetic acid, 40% methanol)쑝濡 깉깋븯뿬 white band쓣 솗씤븯떎.

Annexin V 뿼깋

SNU-80 꽭룷瑜 60 mm culture dish뿉 1 횞 106 cells/well濡 遺꾩<븯怨 12떆媛 룞븞 諛곗뼇븳 썑, 솉쁺쓣 떎뼇븳 냽룄濡 泥섎━븯뿬 48떆媛 諛곗뼇븯떎. 꽭룷瑜 紐⑥븘 PBS濡 꽭泥숉븳 썑, Annexin V-FITC Apoptosis kit (Biovision Inc., Milpitas, CA, USA)瑜 궗슜븯뿬 꽭룷瑜 뿼깋븯怨, BD FACSAriaIII Cell Sorter (BD Biosciences, San Jose, CA, USA) 遺꾩꽍湲곕 씠슜븯뿬 痢≪젙븯떎.

Western Blot analysis

솉쁺 異붿텧臾쇱쓣 냽룄 蹂꾨줈 泥섎━븳 SNU-80 꽭룷瑜 紐⑥븘 PBS濡 꽭泥숉븯怨 lysis buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris-Cl pH 8.0)쓣 泥④븯뿬 4꼦뿉꽌 15遺꾧컙 諛섏쓳븳 썑, 12,000 rpm쑝濡 10遺꾧컙 썝떖遺꾨━븯뿬 긽痢듭븸쓣 痍⑦븯떎. Bradford踰뺤쓣 씠슜븯뿬 떒諛깆쭏쓣 젙웾븳 뮘, 50 關g쓽 떒諛깆쭏쓣 SDS-PAGE gel쓣 씠슜븯뿬 遺꾨━븯떎. 洹 썑, poly-vinylidene difluoride membrane뿉 transfer븯뿬 5% skim milk媛 븿쑀맂 TBS-T buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.1% Tween-20)瑜 씠슜븯뿬 떎삩뿉꽌 1떆媛 諛섏쓳븯떎. Caspase-3 (#9662; Cell signaling Technology, Danvers, MA, USA), PARP (#9542; Cell signaling Technology), GAPDH (#AC002; Cell signaling Technology)瑜 1:1,000쑝濡 씗꽍븳 썑 泥④븯뿬 4꼦뿉꽌 12떆媛 諛섏쓳븯쑝硫, goat anti-mouse horseradish peroxidase(HRP)-conjugated IgG secondary antibody (#SA001; GenDEPOT, Barker, TX, USA) goat anti-rabbit HRP-conjugated IgG secondary antibody (#SA002; GenDEPOT)瑜 1:5,000쑝濡 씗꽍븯뿬 긽삩뿉꽌 1떆媛 諛섏쓳븯떎. 洹 썑 enhanced chemilluminescence (ECL) kit (Amersham pharmacia Biotech Ltd., Amersham, UK)瑜 씠슜븯뿬 痢≪젙븯떎.

Statistical Analysis

꽭룷 깮議댁쑉 슚怨, RT-PCR, Gelatinase 솢꽦, Western Blot 洹몃━怨 꽭룷二쇨린 遺꾩꽍 Student's t-test瑜 떎떆븯떎. 떎뿕寃곌낵뒗 3쉶 諛섎났 떎뿕쓣 넻븯뿬 mean 짹 SD濡 굹궡뿀쑝硫, 媛 떎뿕援 媛꾩쓽 넻怨꾪븰쟻 遺꾩꽍 쑀쓽븳 寃곌낵瑜 뼸뿀떎.

寃곌낵 諛 怨좎같

솉쁺 뿉깂삱 異붿텧臾쇱쓽 닔뱷瑜 諛 珥 뤃由ы럹 븿웾

100 g쓽 솉쁺 떆猷뚮 80% 뿉깂삱쓣 씠슜븯뿬 異붿텧븯쓣 븣쓽 닔뱷瑜좎 3.73%씠떎. 蹂 뿰援ъ뿉꽌뒗 gallic acid瑜 몴以臾쇱쭏濡 솉쁺 뿉깂삱 異붿텧臾쇱쓽 珥 뤃由ы럹 븿웾쓣 痢≪젙븯쑝硫, 洹 寃곌낵 292.43짹8.42 mg gallic acid equivalent (GAE)/100 g dry extract쓽 뤃由ы럹쓣 븿쑀븿쓣 솗씤븯떎(Table 2). 씠뒗 쑀깋媛먯옄쓽 珥 뤃由ы럹 븿웾쓣 蹂닿퀬븳 湲곗〈쓽 뿰援ъ 쑀궗븳 寃곌낵씠떎(Jang and Yoon, 2012; Alam et al., 2016).

Yield and total polyphenol contents of Hongyoung extract

Extract Yield (%) Total polyphenol (mg GAE/100 g)
Hongyoung 3.73 292.43짹8.42


SNU-80 꽭룷뿉꽌 솉쁺 뿉깂삱 異붿텧臾 泥섎━뿉 쓽븳 깮議댁쑉 遺꾩꽍

SNU-80 뿭삎꽦 媛묒긽꽑븫꽭룷뿉꽌 솉쁺 뿉깂삱 異붿텧臾쇱쓣 0, 500, 750, 1,000, 1,500, 2,000 關g/mL쓽 냽룄濡 泥섎━븯뿬 24, 48, 72떆媛 룞븞 諛곗뼇븳 썑, MTS 諛⑸쾿쓣 씠슜븯뿬 꽭룷쓽 깮議댁쑉쓣 遺꾩꽍븯떎. 議곌뎔怨 鍮꾧탳븳 寃곌낵, SNU-80 꽭룷쓽 깮議댁쑉씠 냽룄 쓽議댁쟻쑝濡 媛먯냼븯怨, 1,000 關g /mL쓽 냽룄뿉꽌뒗 꽭룷 깮議댁쑉씠 50% 誘몃쭔쑝濡 媛먯냼븿쓣 솗씤븷 닔 엳뿀떎(Fig. 1). 씠뒗 솉쁺 異붿텧臾쇱씠 쟾由쎌꽑븫, 寃곗옣븫, 諛깊삁蹂, 援ш컯븫 벑쓽 븫꽭룷뿉 빐 꽭룷룆꽦 슚怨쇨 엳떎뒗 湲곗〈쓽 뿰援ъ 쑀궗븳 寃곌낵씠떎(Kim et al., 2023; Park et al., 2008).

Fig. 1. Anti-proliferation effect of Hongyoung on SNU-80 cells.
The cells were treated with different concentrations of Hongyoung for 24, 48 and 72 hr. Data represent the mean 짹 S.D. through three independent experiments (**; P<0.01 compared with untreated control, ***; P<0.001 compared with untreated control).

솉쁺 뿉깂삱 異붿텧臾쇱쓽 꽭룷二쇨린 議곗젅 遺꾩꽍

솉쁺씠 SNU-80 뿭삎꽦 媛묒긽꽑븫꽭룷쓽 꽭룷遺꾩뿴 二쇨린 긽뿉 誘몄튂뒗 쁺뼢쓣 flow cytometry濡 議곗궗븯떎. G1, S 洹몃━怨 G2/M湲곗뿉 빐떦븯뒗 議곌뎔꽭룷뿉꽌뒗 媛곴컖 77.31%, 5.38% 洹몃━怨 14.33%쓽 遺꾪룷瑜 蹂댁떎. 諛섎㈃뿉 1,000 關g/mL 냽룄쓽 솉쁺 異붿텧臾쇱쓣 泥섎━뻽쓣 븣뒗 G1湲곌 51.73%濡 媛먯냼븯怨, S湲곕뒗 9.30% 洹몃━怨 G2/M湲곕뒗 24.25%濡 利앷븯뒗 寃껋쓣 愿李고븯떎(Fig. 2). 씠긽쓽 寃곌낵濡 蹂댁븘 S 諛 G2/M湲 arrest媛 諛쒖깮뻽쓬쓣 솗씤븯쑝硫 삉븳 議곌뎔꽭룷뿉꽌 sub-G1湲곕뒗 1.99%吏留, 500 關g/mL뿉꽑 2.31%, 750 關g/mL뿉꽌뒗 3.81%, 1,000 關g/mL뿉꽌뒗 6.90%濡 솗뿰엳 利앷븿쓣 愿李고븷 닔 엳뿀떎. 씠뒗 泥쒖뿰臾 異붿텧臾 泥섎━뿉 쓽븳 뿭삎꽦 媛묒긽꽑븫꽭룷뿉꽌 굹궃 꽭룷二쇨린뿉 븳 湲곗〈 뿰援щ 뮮諛쏆묠븯怨 엳떎(Kim et al., 2023).

Fig. 2. Cell cycle analysis of Hongyoung on SNU-80 cells.
The cells were treated at different concentrations of Hongyoung for 48 hr. (A) The cell cycle profiles were analyzed by FACS using CytExpert Software. (B) The percentage of cells in each phase of the cell cycle is represented by G0/G1, S. and G2/M phase. (C) The percent of sub-G1 phase (apoptotic cells) were plotted. Data represent the mean 짹 S.D. through three independent experiments (*; P<0.05 compared with untreated control, ***; P<0.001 compared with untreated control).

솉쁺 異붿텧臾쇱쓽 MMP-2 諛 MMP-9쓽 븫쟾씠 뼲젣 슚怨

븫꽭룷쓽 移⑥뒿 諛 쟾씠뿉 二쇰맂 뿭븷쓣 븯뒗 꽭룷쇅 湲곗쭏 湲덉냽븿쑀 떒諛깅텇빐슚냼(matrix metalloproteinase, MMP)뒗 꽭룷쇅 떒諛깆쭏쓣 몴쟻쑝濡 젅떒븯뒗 븘뿰 쓽議댁꽦 궡씤꽦 peptidase씠떎. MMP뒗 湲곗留됱쓣 援ъ꽦븯뒗 떒諛깆쭏쓣 遺꾪빐빐 삁愿 諛 由쇳봽愿쑝濡 븫꽭룷쓽 씠룞쓣 珥됱쭊븯硫, 醫낆뼇쓽 珥덇린 꽦옣 諛 쟾씠뿉룄 愿뿬븿씠 諛앺議뚮떎(Ann and Lynn, 1997; Jacob and Prekeris, 2015). MMP뒗 湲곗쭏 꽑샇룄뿉 뵲씪 type IV collagenase, stromelysin, interstitial collagenase 벑쑝濡 援щ텇븯硫, MMP-2(gelatinase A, 遺꾩옄웾 72 kDa) MMP-9(gelatinase B, 遺꾩옄웾 92 kDa) 湲곗留됱쓣 援ъ꽦븯뒗 type IV collagen쓣 遺꾪빐빐 移⑥뒿쓣 쑀룄븯뒗 type IV collagenase뿉 빐떦맂떎(Bassiouni et al., 2021; Liotta and Stetler-Stevenson, 1991).

SNU-80 꽭룷뿉 솉쁺 異붿텧臾쇱쓣 500, 750, 1,000 關g/mL쓽 냽룄濡 泥섎━븯뿬 MMP-2 MMP-9쓽 쑀쟾옄 諛쒗쁽 뼲젣 슚怨쇰 議곌뎔怨 鍮꾧탳븳 寃곌낵, 냽룄 쓽議댁쟻쑝濡 諛쒗쁽씠 뼲젣맖쓣 솗씤븯떎(Fig. 3A). 삉븳 SNU-80 꽭룷뿉 븳 솉쁺 異붿텧臾쇱쓽 MMP-2/-9 떒諛깆쭏 솢꽦 뼲젣瑜 넻빐 븫쟾씠 뼲젣뿉 誘몄튂뒗 슚怨쇰 議곗궗븯湲 쐞빐, 500, 750, 1,000 關g/mL 냽룄쓽 솉쁺 異붿텧臾쇱쓣 泥섎━븯뿬 MMP-2쓽 솢꽦 뼲젣 슚怨쇰 議곌뎔怨 鍮꾧탳븯떎. 洹 寃곌낵, 500 關g/mL뿉꽌뒗 1.25諛, 750 關g/mL뿉꽌뒗 1.55諛, 1,000 關g/mL뿉꽌뒗 2.35諛 媛먯냼瑜 蹂댁떎. 洹몃━怨 MMP-9쓽 솢꽦 뼲젣 슚怨쇰 議곌뎔怨 鍮꾧탳븯쓣 븣, 500 關g/mL뿉꽌뒗 2.08諛, 750 關g/mL뿉꽌뒗 4.05諛, 1,000 關g/mL뿉꽌뒗 12.49諛 媛먯냼瑜 蹂댁떎(Fig. 3B). 씠윭븳 寃곌낵瑜 넻븯뿬 議곌뎔뿉 鍮꾪빐, 솉쁺 異붿텧臾쇱쓣 泥섎━븳 꽭룷뿉꽌뒗 MMP-2 MMP-9쓽 쑀쟾옄 諛 떒諛깆쭏 솢꽦씠 쑀쓽븯寃 媛먯냼븯쑝硫 臾댁뾿蹂대떎 MMP-9쓽 쑀쟾옄 諛 떒諛깆쭏 솢꽦뿉 븳 뼲젣 슚怨쇨 넂쓬쓣 솗씤븷 닔 엳뿀떎. 씠뒗 솉쁺 異붿텧臾 泥섎━뿉 쓽븳 븫꽭룷뿉꽌 굹궦 MMP-2/-9뿉 븳 뿰援ъ 쑀궗븳 寃곌낵씠떎(Kim et al., 2023).

Fig. 3. Effect of Hongyoung on the expression of MMP-2/9 in PMA-treated SNU-80 cells.
(A) Effect of Hongyoung on the activities of MMP-2/9 mRNA in PMA-treated SNU-80 cells. (B) The activity of MMP-2/-9 protein were determined by gelatin zymography in conditioned media. (C) The relative expression of MMP-2/-9 were analyzed by band intensity using ImageJ program. Data represent similar results from three independent experiments (*; P<0.05 compared with only PMA-treated cells, **; P<0.01 compared with only PMA-treated cells, ***; P<0.001 compared with only PMA-treated cells).

솉쁺 異붿텧臾쇱쓽 apoptosis 遺꾩꽍

Annexin V 떒諛깆쭏 젙긽쟻쑝濡쒕뒗 꽭룷留 궡痢≪뿉 쐞移섑븯떎媛 apoptosis媛 吏꾪뻾릺硫 꽭룷留 쇅痢≪쑝濡 쟾쐞릺뒗 phosphatidylserine뿉 寃고빀븯뒗 꽦吏덉쓣 媛吏怨 엳뼱 apoptosis瑜 솗씤븯뒗뜲 궗슜맂떎. 蹂 뿰援ъ뿉꽌뒗 솉쁺 異붿텧臾쇱뿉 쓽븳 SNU-80 꽭룷쓽 apoptosis 쑀룄瑜 븣븘蹂닿린 쐞븯뿬 솉쁺 異붿텧臾쇱쓣 500, 750, 1,000 關g/mL쓽 냽룄濡 48떆媛 룞븞 泥섎━븳 썑 Annexin V Propidium iodide 뿼깋쑝濡 議곗궗븯떎. 議곌뎔뿉 鍮꾪빐 1,000 關g/mL쓽 냽룄뿉꽌 Annexin V뿉 뿼깋맂 apoptosis 珥덇린 떒怨꾩쓽 꽭룷媛 빟 6諛 利앷븯떎(Fig. 4).

Fig. 4. Effect of Hongyoung on apoptosis in SNU-80 cells.
(A) Annexin V/Propidium iodide (PI) double staining assay detected by flow cytometry. The cells were treated with Hongyoung (500~1,000 關g/mL) for 48 hr. (B) Relative amount of FITC-Annexin V-positive (apoptotic) cells. Data represent similar results from three independent experiments (*, P<0.05 compared with untreated control). (C) Western Blot analysis of (cleaved) caspase-3 and (fragmented) PARP proteins in SNU-80 cells. The cells were treated with Hongyoung at the indicated concentrations for 48 hr. (D) Relative protein expression of cleaved caspase-3 and fragmented PARP. Data represent similar results from three independent experiments (**; P<0.01 compared with untreated control).

Apoptosis뒗 꽭룷 궡 愿젴 떒諛깆쭏뿉 쓽빐 議곗젅릺뒗뜲, 몴쟻쑝濡 Bcl-2 family, caspase-3, PARP 벑씠 媛옣 옒 븣젮졇 엳떎. 꽭룷 궡 옄洹 벑쓽 씠쑀濡 apoptosis 湲곗쟾씠 쑀諛쒕릺硫 誘명넗肄섎뱶由ъ븘 궡쓽 cytochrome c媛 諛⑹텧릺怨, caspase cascade媛 솢꽦솕릺뼱 沅곴레쟻쑝濡쒕뒗 caspase-3媛 솢꽦솕맂떎. 씠븣 DNA 蹂듦뎄뿉 愿뿬븯뒗 DNA-binding zinc finger protein씤 PARP뒗 솢꽦솕맂 caspase-3뿉 쓽빐 fragmentation씠 諛쒖깮릺怨, 씠濡 씤빐 apoptosis媛 씪뼱굹寃 맂떎. 뵲씪꽌 SNU-80 꽭룷뿉 꽭룷궗硫 쑀룄 寃쎈줈瑜 솗씤븯湲 쐞빐 愿젴 떒諛깆쭏씤 caspase-3, PARP쓽 諛쒗쁽뼇쓣 솗씤븯떎. 洹 寃곌낵 pro-caspase-3 PARP쓽 諛쒗쁽씠 냽룄 쓽議댁쟻쑝濡 媛먯냼븯怨, cleaved caspase-3 fragmented PARP媛 利앷븯뒗 寃껋쓣 솗씤븯떎(Fig. 4). 씠 媛숈 寃곌낵瑜 넻빐 솉쁺 異붿텧臾쇱씠 SNU-80 꽭룷뿉꽌 apoptosis媛 쑀룄맖쓣 솗씤븷 닔 엳뿀떎. 씠뒗 泥쒖뿰臾 異붿텧臾 泥섎━뿉 쓽븳 뿭삎꽦 媛묒긽꽑븫꽭룷뿉꽌 굹궃 apoptosis뿉 븳 뿰援ш껐怨쇰뱾쓣 뮮諛쏆묠 븯怨 엳떎(Al-Mohanna et al., 2023; Allegri et al., 2018; Hong et al., 2014; Jung and Hwang, 2021; Yeo et al., 2014; Yeo et al., 2014).

蹂 뿰援щ뒗 SNU-80 뿭삎꽦 媛묒긽꽑븫꽭룷뿉꽌쓽 솉쁺 異붿텧臾 泥섎━瑜 넻븳 븫꽭룷 꽦옣 뼲젣, 꽭룷궗硫 諛 븫쟾씠 뼲젣 뒫젰쓣 솗씤븯떎. 솉쁺 異붿텧臾 泥섎━뒗 50% 씠긽쓽 넂 븫꽭룷 꽦옣 뼲젣 슚怨쇰 굹깉쑝硫, S湲곗 G2/M湲곕뒗 利앷븯怨 G1湲곕뒗 媛먯냼븯怨, 꽭룷궗硫 뒫젰쓣 굹궡뒗 sub-G1湲곗쓽 鍮꾩쑉 2諛 씠긽 利앷븿쓣 愿李고븯떎. 삉븳, MMP-2 諛 MMP-9쓽 쑀쟾옄 諛쒗쁽 諛 떒諛깆쭏 솢꽦 媛먯냼瑜 넻븳 넂 븫쟾씠 뼲젣 솢꽦쓣 솗씤븯떎. 솉쁺 異붿텧臾쇱쓣 泥섎━븯쓣 븣 apoptosis 珥덇린 떒怨꾩쓽 꽭룷媛 쑀쓽誘명븯寃 利앷븯쑝硫, apoptosis 愿젴맂 떒諛깆쭏씤 caspase-3, PARP쓽 諛쒗쁽뼇쓣 솗씤븿쑝濡쒖뜥 솉쁺 泥섎━濡 씤빐 apoptosis媛 吏꾪뻾맖쓣 솗씤븯떎. 醫낇빀쟻쑝濡, 씠 媛숈 뿰援ш껐怨쇰뒗 SNU-80 뿭삎꽦 媛묒긽꽑븫꽭룷뿉꽌 솉쁺 異붿텧臾쇱쓽 빆븫 슚怨쇰 솗씤븯쑝硫 뼢썑 씠 媛숈 泥섎━뿉 븳 異붽쟻씤 슚뒫 諛 湲곗쟾 뿰援ш 븘슂븷 寃껋쑝濡 궗猷뚮맂떎.

ACKNOWLEDGEMENT

None.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest with the contents of this article.

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