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Effects of Amifostine on Apoptosis, Cell Cycle and Cytoprotection of Human Colon Cancer Cell Lines
Biomed Sci Letters 2023;29:287-295
Published online December 31, 2023;  https://doi.org/10.15616/BSL.2023.29.4.287
© 2023 The Korean Society For Biomedical Laboratory Sciences.

Eun Ju Lee†,*

Department of Clinical Laboratory Science, Daejeon Health Institute of Technology, Daejeon 34504, Korea
Correspondence to: Eun Ju Lee. Department of Clinical Laboratory Science, Daejeon Health Institute of Technology, 21 Chungjeong St., Dong-Gu, Daejeon 34504, Korea.
Tel: +82-42-670-9163, Fax: +82-42-670-9160, e-mail: e16lee@hit.ac.kr
*Professor.
Received September 25, 2023; Revised November 8, 2023; Accepted November 9, 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
Amifostine was developed to protect cells, but it is known to induce cytotoxicity and apoptosis, and the exact mechanism is unknown. In this study, we investigated how the DNA mismatch repair (MMR) system interacts with p53 to prevent apoptosis, cell cycle arrest, and cytoprotective effects induced by amifostine. HCT116 colon cancer cells sublines HCT116/p53+, HCT116/p53-, HCT116/E6 and HCT116+ch3/E6 cells were used for evaluation. Amifostine induced G1 arrest and increased toxicity two-fold in p53- cells regardless of MMR expression. Both G1 cell cycle arrest and induction of p53 protein peaked at 24 h after the start of amifostine exposure. Both G1 cell cycle arrest and induction of p53 protein peaked at 24 h after the start of amifostine exposure. Amifostine induced the expression of p21 protein in both p53+ and p53- cells. As for apoptosis, compared to p53- cells, p53+ cells showed 3.5~4.2 times resistance to amifostine-induced apoptosis. HCT116+E6 with both p53 and MMR loss showed maximum apoptosis at 48 h, and HCT116+ch3/E6 with p53 loss showed maximum apoptosis at 24 h. As a result, it was confirmed through in vitro experiments that amifostine-induced G1 cell cycle arrest and apoptosis are mediated through a pathway dependent on MMR and p53 protein.
Keywords : Amifostine, MMR, p53, p21, Cell cycle, Apoptosis, G1 cell cycle arrest
꽌 濡

쑀쟾꽦 鍮꾩슜醫낆꽦 寃곗옣븫(hereditary nonpolyposis colorectal cancer, HNPCC)怨 떎뼇븳 醫낅쪟쓽 븫뿉꽌 DNA mismatch repair(遺덉씪移 蹂듦뎄, MMR) 寃고븿씠 썝씤쑝濡 諛앺議뚮떎(Fink et al., 1998; Fishel and Kolodner, 1995; Jin et al., 2017; Salem et al., 2020). MMR DNA 以묓빀슚냼뿉 쓽빐 옒紐 吏앹뼱吏 돱겢젅삤떚뱶 蹂듭젣 삉뒗 옱議고빀 룞븞 諛섎났릺뒗 뿼湲곗꽌뿴쓽 slippage뿉 쓽븳 遺덉씪移섎 蹂댁젙븳떎(Fishel and Kolodner, 1995; Sunitha and Mats, 2003). MMR 떆뒪뀥쓽 寃고븿 룎뿰蹂씠 諛쒖깮瑜좎쓣 湲됯꺽엳 利앷떆耳 諛쒖븫怨쇱젙쓣 珥됱쭊떆궎怨(Sunitha and Mats, 2003; Jin et al., 2017; Salem et al., 2020), hMLH1怨 hMSH2쓽 깮떇 꽭룷 룎뿰蹂씠뒗 쟾泥 HNPCC 솚옄쓽 빟 젅諛섏쓣 李⑥븳떎(Fishel and Kolodner, 1995; Salem et al., 2020)怨 븣젮졇 엳떎. DNA 遺덉씪移 蹂듦뎄(MMR) 떒諛깆쭏 DNA 以묓빀슚냼 삤瑜섎줈 씤븳 遺덉씪移 蹂댁젙 쇅뿉룄 쑀궗遺꾩뿴怨 媛먯닔遺꾩뿴 옱議고빀(Fishel and Kolodner, 1995; Sunitha and Mats, 2003) 諛 쟾궗 寃고빀 蹂듦뎄쓽 議곗젅뿉 李몄뿬븯뒗 寃껋쑝濡 븣젮졇 엳떎(Hickman and Samson, 1999; Lin et al., 2001; Chakraborty et al., 2016). DNA 遺덉씪移섎 닔젙븯吏 紐삵븯硫 룎뿰蹂씠 鍮꾩쑉씠 利앷븯硫, MSH2 삉뒗 MLH1쓽 룎뿰蹂씠濡 씤븳 MMR 넀떎 遺遺꾩쓽 쑀쟾꽦 鍮꾩슜醫낆꽦 寃곗옣븫쓽 썝씤씠 릺硫(Fishel et al., 1993; Papadopoulos et al., 1994; Salem et al., 2020) 옄沅곷궡留됱븫, 궃냼븫, 쑀諛⑹븫, 쟾由쎌꽑븫, 룓븫 諛 痍뚯옣븫 벑쓽 썝씤씠 릺湲곕룄 븳떎(Fishel and Kolodner, 1995; Munakata et al., 2023). 룎뿰蹂씠 鍮꾩쑉쓣 利앷떆궎뒗 寃 쇅뿉룄, MMR쓽 넀떎 빆븫빟臾 궡꽦쓣 利앷떆궓떎. 솚옄뿉寃 궗슜븯뒗 빆븫 냽룄뿉꽌, 빆븫젣뒗 꽭룷궗硫몄쓣 쑀諛쒗븳떎. 꽭룷궗硫몄쓣 珥됱쭊븯젮硫 꽭룷媛 DNA 넀긽쓽 議댁옱瑜 씤떇븷 닔 엳뼱빞 븯굹, MMR쓽 넀떎濡 씤빐 媛먯 뒫젰씠 넀긽릺嫄곕굹 蹂듦뎄 떆룄媛 遺議깊븯뿬 꽭룷궗硫 珥됱쭊 떊샇媛 媛먯냼븯寃 맂떎(Kat et al., 1995; Jiang et al., 2021). p53쓽 寃쎌슦 꽭룷 DNA뿉 넀긽씠 諛쒖깮븯쓣 븣 넀긽씠 떎 닔꽑릺湲 쟾뿉 꽭룷遺꾩뿴씠 吏꾪뻾릺吏 紐삵븯룄濡 븯硫 닔꽑씠 뼱젮슱 寃쎌슦 꽭룷媛 뒪뒪濡 궗硫명븯룄濡 븳떎. p53 湲곕뒫씠 寃곗뿬맂 꽭룷뒗 쑀쟾쟻쑝濡 遺덉븞젙븯怨 珥앹껜쟻씤 쑀쟾옄 蹂寃쎌뿉 痍⑥빟븯寃 맂떎(Jiang et al., 2021). p53 湲곕뒫 옣븷뒗 p53 끃븘썐 留덉슦뒪(knockout mice)뿉꽌 利앷맂 醫낆뼇 삎꽦怨 愿젴씠 엳쑝硫(Carder et al., 1993), p53 씤媛 븫쓽 留ㅼ슦 留롮 遺遺꾩뿉꽌 룎뿰蹂씠 맂떎(Bhattacharyya et al., 1994; Fink et al., 1998; Hollstein et al., 1991; Jiang et al., 2021)怨 븣젮졇 엳떎.

WR-2721濡 紐낅챸맂 amifostine 1950뀈 썡꽣 由щ뱶 쑁援 뿰援ъ냼뿉꽌 빑 諛⑹궗꽑쑝濡쒕꽣 援곗씤쓣 蹂댄샇븯湲 쐞빐 빀꽦릺뿀떎(Shaw et al., 1988; Capizzi and Oster, 2000). 쟾援ъ빟臾쇱씤 amifostine怨 솢꽦 궗궛臾쇱씤 WR-1065뒗 븙꽦 議곗쭅怨 鍮꾩븙꽦 議곗쭅뿉 李⑤벑쟻씤 쁺뼢쓣 誘몄튂뒗 寃껋쑝濡 蹂닿퀬릺뿀떎(Capizzi, 1999; Taylor et al., 1997). 삁瑜 뱾뼱, amifostine 씠삩솕 諛⑹궗꽑 諛 솕븰슂踰뺤젣쓽 룆꽦 슚怨쇰줈遺꽣 젙긽 議곗쭅쓣 蹂댄샇븯吏留, 醫낆뼇뿉뒗 슚怨쇨 뾾嫄곕굹 빆醫낆뼇 슚怨쇰 뼢긽떆궓떎. Amifostine씠 젙긽 꽭룷瑜 蹂댄샇븯뒗 硫붿빱땲利섏 옒 븣젮졇 엳吏 븡怨, amifostine뿉 쓽븳 젙긽 議곗쭅쓽 꽑깮쟻 蹂댄샇뒗 p53 쓽議댁꽦 寃쎈줈쓽 솢꽦솕 愿젴씠 엳쑝硫, amifostine p53 떒諛깆쭏뿉 寃고빀븯怨 븯뼢 쑀쟾옄쓽 쟾궗솢꽦솕瑜 뼢긽떆궎뒗 寃껋쑝濡 蹂닿퀬릺뿀떎(Falcicchio et al., 2020; Lee et al., 2003).

蹂 뿰援ъ뿉꽌뒗 amifostine뿉 쓽빐 쑀諛쒕맂 꽭룷궗硫 諛 꽭룷 二쇨린 吏꾪뻾뿉 븳 p53 떒諛깆쭏 諛 p21 떒諛깆쭏쓽 뿭븷쓣 議곗궗븯湲 쐞빐 MMR+, MMR-, p53+ 諛 p53꽭룷뿉꽌 amifostine씠 p53 떒諛깆쭏 諛 p21 떒諛깆쭏쓽 諛쒗쁽뿉 誘몄튂뒗 쁺뼢쓣 븣븘蹂닿퀬옄 븳떎.

옱猷 諛 諛⑸쾿

Cell lines

寃곗옣븫 꽭룷二쇱씤 HCT116 꽭룷뒗 American Type Culture Collection (ATCC, CCL 247)뿉꽌 援щℓ븯뿬 궗슜븯떎. HCT116/p53꽭룷二쇰뒗 몴쟻 긽룞 옱議고빀(homologous recombination)뿉 쓽빐 몢 p53 由 쑀쟾옄媛 紐⑤몢 寃곗떎맂 꽭룷씠떎(Boyer et al., 1995). HCT116+ch3쑝濡 紐낅챸맂 꽭룷二쇰뒗 3踰 뿼깋泥닿 蹂댁셿맂 꽭룷濡 DNA 遺덉씪移 蹂듦뎄 뒫젰씠 엳뒗 꽭룷씠떎(Koi et al., 1994). HCT116/E6 HCT116+ch3/E6 꽭룷二쇰뒗 쑀鍮꾪댄떞 쓽議 寃쎈줈瑜 넻빐 p53 떒諛깆쭏쓽 遺꾪빐瑜 옄洹뱁븯뒗 넂 닔以쓽 씤媛 쑀몢醫 諛붿씠윭뒪 쑀삎-16 E6 떒諛깆쭏쓣 諛쒗쁽븯뒗 꽭룷二쇱씠떎(Davis et al., 1998; Bunz et al., 1999). 紐⑤뱺 꽭룷二쇰뒗 100 mM L-湲猷⑦誘 諛 10% 뿴 遺덊솢꽦솕 깭븘 냼 삁泥(FBS, Fetal Bovine Serum)씠 蹂댁땐맂 Iscove쓽 蹂삎맂 몮踰좎퐫 諛곗(DMEM, Dulbecco Modified Eagle Medium, Irvine Scientific, Irvine, CA, USA)뿉꽌 쑀吏릺뿀떎. 뿼깋泥 蹂댁셿 씪씤 400 關gmL-1 젣꽕떚떊(geneticin, GIBCO BRL, Gaithersburg, MD, USA)씠 蹂댁땐맂 諛곗뿉꽌 꽦옣떆耳곕떎. HCT116/p53+ 諛 HCT116/p53꽭룷뒗 100 mM L-湲猷⑦誘 諛 10% 뿴 遺덊솢꽦솕 깭븘 냼 삁泥씠 蹂댁땐맂 McCoy's 諛곗(Irvine Scientific, Irvine, CA, USA)뿉 꽔뼱 37꼦쓽 삩룄瑜 쑀吏븯硫댁꽌 5% CO2 諛곗뼇湲곗뿉꽌 諛곗뼇븯떎. 꽭룷뒗 70~80% 옄씪硫 遺꾩<븯뿬 怨꾨 諛곗뼇븳 꽭룷瑜 떎뿕뿉 씠슜븯떎. 紐⑤뱺 꽭룷二쇰뒗 UCSD Cancer Center쓽 Dr Howell濡쒕꽣 뼸뼱 궗슜븯떎.

Reagents

Amifostine US Bioscience Corporation뿉꽌 援ъ엯뻽떎. 50 mg/mL쓽 썝븸쓣 0.9% NaCl뿉 슜빐븯怨 4꼦뿉 蹂닿븯떎.

Clonogenic assay

肄쒕줈땲 삎꽦쓣 蹂닿린 쐞븯뿬, 떎뼇븳 냽룄쓽 amifostine쓣 24떆媛 룞븞 泥섎━븳 썑 꽭룷瑜 PBS濡 꽭泥숉븯떎. 깉濡쒖슫 諛곗瑜 泥④븳 썑 37꼦쓽 삩룄瑜 쑀吏븯硫댁꽌 5% CO2 諛곗뼇湲곗뿉꽌 10~14씪 룞븞 諛곗뼇븳 썑 꽭룷瑜 PBS濡 꽭泥숉븯怨 硫뷀깂삱濡 怨좎젙븯뿬 0.1% Crystal Violet쑝濡 뿼깋븯떎. 50媛 씠긽쓽 꽭룷媛 紐⑥씤 꽭룷 겢윭뒪꽣뒗 肄쒕줈땲濡 젏닔瑜 留ㅺ꼈떎. 떎뿕 媛 빟臾 냽룄뿉 빐 理쒖냼 3쉶 씠긽 닔뻾릺뿀떎. IC50 媛믪 닔 꽑삎 蹂닿컙踰(log-linear interpolation)쓣 궗슜븯뿬 怨꾩궛븯떎.

꽭룷二쇨린 痢≪젙(cell cycle analysis)

Amifostine쓣 24떆媛 룞븞 1 횞 106 ~ 2 횞 106 꽭룷뿉 泥섎━븳 썑, amifostine 泥섎━ 떆옉 썑 0, 1, 2, 3, 4, 5, 6씪吏몄뿉 꽭룷瑜 梨꾩랬븯뿬 鍮숇깋 PBS濡 2쉶 꽭泥숉븯怨, 鍮숇깋 100% 뿉깂삱뿉 怨좎젙븯떎. 106媛 꽭룷瑜 怨꾩닔븯뿬 썝떖遺꾨━븯怨, 李④슫 PBS 500 關L뿉 옱쁽긽븯뿬 0.1 mg/mL RNAse A (Sigma Chemical Co.)濡 37꼦뿉꽌 30遺 룞븞 泥섎━븯떎. 50 mg/mL 냽룄쓽 Propidium iodide (Molecular Probes, Eugene, OR)瑜 꽭룷 쁽긽븸뿉 泥④뻽떎. 뼹쓬 쐞뿉꽌 30遺 룞븞 蹂닿븳 썑, FACScan 쑀꽭룷 遺꾩꽍湲(Becton-Dickinson, San Jose, CA)瑜 궗슜븯뿬 꽭룷瑜 遺꾩꽍뻽떎. Multicycle AV Cell Cycle 냼봽듃썾뼱(Phoenix Flow Systems, San Diego, CA)瑜 궗슜븯뿬 꽭룷 二쇨린쓽 媛 떒怨꾩뿉꽌 꽭룷 鍮꾩쑉쓣 怨꾩궛뻽떎(Lee et al., 2003).

꽭룷궗硫 痢≪젙(apoptosis analysis)

Amifostine 泥섎━ 떆옉 썑 24떆媛, 48떆媛, 72떆媛꾩뿉 遺쑀 꽭룷 遺李⑸맂 꽭룷瑜 梨꾩랬븯뿬 썝떖遺꾨━븯怨 PBS 100 關L뿉 옱쁽긽븯怨 acridine orange ethidium bromide 濡 뿼깋븯떎. 洹 썑, 꽭룷뒗 삎愿 쁽誘멸꼍뿉 쓽빐 꽭룷궗硫 삎깭뿉 빐 룊媛릺뿀떎. 꽭룷뒗 삎깭븰쟻 湲곗(McGahon et al., 1995)뿉 뵲씪 꽭룷궗硫몃줈 젏닔瑜 留ㅺ꼈떎.

Western blot analysis

떎뼇븳 냽룄쓽 amifostine쓣 泥섎━븳 썑 떎뼇븳 떆젏뿉꽌 0.15 M NaCl, 5 mM EDTA 1%, Triton X-100, 10 mM TRIS (pH 7.4), 5 mM DTT, 0.1 mM phenylmethylsulphonyl fluoride 諛 5 mM epsilon-aminocaproic acid瑜 븿쑀븯뒗 셿異⑹븸뿉꽌 떒諛깆쭏쓣 異붿텧븯떎. 떒諛깆쭏 7.5% 諛 15% 뤃由ъ븘겕由댁븘誘몃뱶 寃(Precast Acrylamide Gels, Bio-Rad, Hercules, CA, USA)쓣 궗슜븯뿬 쟾湲곗쁺룞쑝濡 젙웾 諛 遺꾪쉷솕 븯떎. 떒諛깆쭏쓣 polyvinylidene difluoride 留(Immobilon, Millipore, Bedford, MA, USA)쑝濡 삷寃쇰떎. 留됱쓣 5% 臾댁諛 슦쑀, 0.05% Tween쑝濡 1떆媛 룞븞 李⑤떒븯怨 0.05% Tween쑝濡 꽭泥숉븳 떎쓬 4꼦뿉꽌 諛ㅼ깉 1李 빆泥댁뿉 끂異쒖떆耳곕떎. 留덉슦뒪 떒씪 겢濡 anti-p21 (sc-817) 諛 anti-p53 (sc-126) 빆泥대뒗 誘멸뎅 罹섎━룷땲븘二 궛겕猷⑥쫰쓽 Santa Cruz Biotechnology뿉꽌 援ъ엯뻽떎. 1李 빆泥대뒗 5~10% 臾댁諛 슦쑀뿉 1:400쑝濡 씗꽍븯떎. 꽭泥 썑, 1:3,000쓽 씗꽍 鍮꾩쑉濡 horseradish peroxidase-conjugated anti-mouse antibodies (Amersham Life Science, Inc., Arlington Heights, IL, USA)뿉 끂異쒖떆궎怨, 蹂듯빀泥대 솕븰諛쒓킅쑝濡 媛떆솕븯떎.

넻怨꾩쿂由

紐⑤뱺 떎뿕뱾 3쉶뵫 諛섎났 吏꾪뻾븯쑝硫, 씠뿉 븳 寃곌낵뒗 룊洹 짹 몴以삤李(mean 짹 sd)濡 怨꾩궛븯떎. Amifostine 誘몄쿂由ш뎔(議곌뎔)怨 amifostine 泥섎━援 媛꾩쓽 李⑥씠뿉 뵲瑜 쑀쓽꽦 two sided t-test 諛⑸쾿쓣 씠슜븯떎. 媛 遺꾩꽍쓽 넻怨꾩쟻 쑀쓽닔以 95% 떊猶곗닔以(쑀쓽룄 0.05, P<0.05)濡 寃利앺븯떎.

寃 怨

Amifostine쓽 룆꽦뿉 븳 p53怨 MMR쓽 슚怨

Amifostine뿉 븳 HCT116 寃곗옣븫 꽭룷쓽 誘쇨컧룄뿉 븳 p53, MM쓽 쁺뼢쓣 寃곗젙븯湲 쐞빐 clonogenic assay瑜 궗슜뻽떎(Fig. 1). 꽭룷瑜 遺李⑹떆궓 썑, 떎뼇븳 냽룄쓽 amifostine뿉 24떆媛 룞븞 끂異쒖떆궎怨, 10~14씪 썑뿉 肄쒕줈땲닔瑜 룊媛븯떎. HCT116 꽭룷뿉꽌 p53 쑀쟾옄쓽 寃곗떎 IC50 媛믪쓽 鍮꾩쑉濡 젙웾솕(Table 1)븳 諛붿 媛숈씠 amifostine 誘쇨컧룄瑜 2.1짹0.2諛 利앷떆耳곕떎(룊洹 짹 sd, n=3, P<0.05 -뼇硫 t-寃젙). 쑀궗븯寃, E6뿉 쓽븳 p53 떒諛깆쭏쓽 遺꾪빐뒗 amifostine 誘쇨컧룄瑜 1.9짹0.2諛 利앷떆耳곕떎(룊洹 짹 sd, n=3, P<0.05, 뼇痢 t-寃젙). 遺덉씪移 蹂듦뎄 뒫젰씠 엳뒗 HCT116+ ch3 꽭룷뿉꽌 E6뿉 쓽븳 p53 떒諛깆쭏쓽 遺꾪빐뒗 amifostine뿉 븳 誘쇨컧룄瑜 1.89짹0.2諛 利앷떆耳곕떎(룊洹 짹 sd, n=3, 뼇硫 t-뀒뒪듃뿉 쓽븳 P<0.05). Amifostine뿉 븳 HCT116쓣 寃곗옣븫 꽭룷쓽 誘쇨컧룄뿉 븳 p53, MMR 떒諛깆쭏쓽 쁺뼢 p53 떒諛깆쭏씠 긽떎맂 寃쎌슦 뜑 겕寃 쁺뼢쓣 二쇱뿀떎(Table 2).

Fig. 1. Effect of p53 and MMR on amifostine sensitivity. HCT116 cells were exposed for 24뎗, and dose-response curves were generated by clonogenic assay. Each data point represents the mean of at least 3 independent experiments performed with triplicate culture.

IC50 Values for HCT116 sublines*

Cell lines Phenotype Amifostine IC50 (mM)
MMR p53
HCT116+ch2 + 4.87짹0.5
HCT116+ch3 + + 4.3짹0.3
HCT116/E6 2.57짹0.7
HCT116+ch3/E6 + 2.25짹0.5

*Each values represents mean 짹 sd of 3 independent experiments each performed with triplicate cultures for each drug concentraion



Magnitude change in amifostine sensitivity expressed as IC50 ratios

Cell lines Effect of loss p53 Effect of loss MMR
In MMR+ cells In MMRcells In p53+ cells In p53cells
Fold 1.91 1.89 0.88 0.86
P-value 0.081 0.0012 > 0.05 > 0.05


Amifostine씠 쑀룄븳 꽭룷궗硫몄뿉 븳 p53怨 MMR 떒諛깆쭏쓽 슚怨

삎愿 쁽誘멸꼍 寃궗踰뺤쓣 궗슜븯뿬 꽭룷궗硫 꽭룷瑜 젙웾솕뻽떎(Fig. 2). HCT116/p53+, HCT116/p53-, HCT116/E6 諛 HCT116+ch3/E6 꽭룷瑜 HCT116/p53+ 꽭룷쓽 IC50뿉 빐떦븯뒗 4.8 mM amifostine뿉 24떆媛 룞븞 끂異쒖떆耳곕떎. apoptotic 꽭룷쓽 遺꾪쉷 amifostine뿉 끂異쒕릺湲 떆옉븳 썑 24, 48 洹몃━怨 72떆媛꾩뿉 痢≪젙븯떎. HCT116 꽭룷뿉꽌 몴쟻 옱議고빀뿉 쓽븳 p53 쑀쟾옄쓽 寃곗떎 amifostine뿉 끂異쒕맂 썑 24떆媛꾩뿉 媛곴컖 3.5짹1.2諛 諛 4.2짹0.7諛곕줈(Table 3) amifostine 쑀룄맂 꽭룷궗硫몄쓣 利앷떆耳곕떎(룊洹 짹 s.d., n=3, P<0.05, MMR+ MMR꽭룷쓽 鍮꾧탳瑜 쐞븳 뼇痢 t-寃젙). 쑀궗븯寃, E6뿉 쓽븳 p53 떒諛깆쭏쓽 遺꾪빐뒗 amifostine뿉 끂異쒕릺湲 떆옉븳 썑 24, 48 諛 72떆媛꾩뿉 媛곴컖 2.1짹0.2諛 諛 4.3짹0.8諛곌퉴吏 amifostine뿉 쓽븳 꽭룷궗硫몄쓣 利앷떆耳곕떎(룊洹 짹 s.d., n=3, p53+ p53꽭룷쓽 鍮꾧탳瑜 쐞븳 뼇硫 t-寃젙뿉 쓽븳 P<0.05). 遺덉씪移 蹂듦뎄 뒫젰씠 엳뒗 HCT116+ch3 꽭룷뿉꽌 p53 떒諛깆쭏쓽 E6 留ㅺ컻 遺꾪빐뒗 amifostine 쑀룄 꽭룷궗硫몄쓣 72떆媛 諛 96떆媛꾩뿉 媛곴컖 4.2짹0.4 諛 2.0짹0.2濡 利앷떆耳곕떎(룊洹 짹 s.d., n=3, p53+ P53꽭룷쓽 鍮꾧탳瑜 쐞븳 뼇硫 t-寃젙뿉 쓽븳 P<0.05).

Fig. 2. Effect of p53 and MMR on amifostine-induced apoptosis. HCT 116 sublines were exposed to amifostine for 24 h. The percent of cells in apoposis was determined by supravital fluorescence microscopy at 24, 48 and 72 h. Data points represent mean 짹 sd of 3 different experiment each performed with triplicate cultures.

Magnitude of change in amifostine-induced apoptosis as a result of p53, MMR, or both functions expressed as apoptosis ratios*

Effect of loss of p53 Effect of loss of MMR
in MMR+ cells in MMRcells in p53+ cells in p53cells
3.5짹1.2(a) 4.2짹0.7(b) 1.3짹0.2(c) 2.1짹0.9(d)

*Ratio of the apoptosis value in the functionally deficient to proficient cells determined from acridine orange and ethidium bromide and this value was compared untreated with amifostine treated cells. Each value is mean 짹 sd.

  • P=0.02: comparison between the MMR+/p53- and the MMR+ /p53+ cells

  • P=0.002: comparison between the MMR-/p53- and MMR-/p53+ cells

  • P>0.05: comparison between the MMR-/p53+ and the MMR+ /p53+ cells

  • P=0.02: comparison between the MMR-/p53- and the MMR+ /p53cells



Amifostine뿉 쓽빐 쑀룄맂 꽭룷 二쇨린 젙吏 p53怨 MMR 떒諛깆쭏쓽 愿젴꽦

HCT116/p53+, HCT116/p53-, HCT116/E6 諛 HCT116+ch3/E6 꽭룷瑜 HCT116/p53+ 꽭룷쓽 IC50뿉 빐떦븯뒗 4.8 mM amifostine뿉 24떆媛 룞븞 끂異쒖떆耳곕떎. p53씠 諛쒗쁽릺뒗 HCT116/p53+ 諛 HCT116+ch3 꽭룷뿉꽌 amifostin쓣 泥섎━븯硫 G1 젙吏媛 諛쒖깮븯뿬 24 h뿉 理쒓퀬議곗뿉 떖뻽떎(Fig. 3). 洹몃윭굹, p53諛 MMR 寃고븤맂 HCT116+ch2 HCT116+ ch3/E6 꽭룷뿉꽌 amifostine G1 젙吏瑜 씪쑝궎吏 紐삵뻽쑝硫, 씠뒗 amifostine뿉 쓽빐 쑀룄맂 G1 젙吏媛 p53 떒諛깆쭏뿉 쓽議댁쟻엫쓣 굹궦떎. Amifostine 삉븳 p53- MMR 寃고븤맂 HCT116+ch3/E6 꽭룷瑜 젣쇅븳 꽭룷 紐⑤몢뿉꽌 諛쒖깮븯뒗 G2/M 젙吏瑜 쑀諛쒗븯뿬 G2/M 젙吏媛 p53怨 MMR 떒諛깆쭏怨 룆由쎌쟻씤 寃쎈줈瑜 넻빐 留ㅺ컻맖쓣 굹궦떎(Fig. 4).

Fig. 3. Effect of p53 and MMR on amifostine-induced cell cycle arrest. HCT116 sublines were exposed to 4.8 mM amifostine for 24 h. Cells were harvested at 1, 2, 3, 4, and 5 days after the beginning of amifostine exposure. The percent of cells in G2/M, S and G1 phase was determines by flowcytometery. Data points represent mean 짹 SD of 3 different experiments. d0 mean amifostine untreated cells. D1 mean 24 h after the beginning of amifostine exposure.

Fig. 4. Effect of p53 and MMR on amifostine-induced G2/M cell cycle arrest. Magnitude of change in a amifostine-induced G2/M arrest as a result of p53, MMR or both functions expressed as the under the curve of fraction of cells. HCT116 sublines were exposed to 4.8 mM amifostine for 24 h. The percent o cells in G2/M phase was determines by flowcytometery. Columns represent mean 짹 SD of 3 different experiments. (a) P=0.0035: comparison the amifostine untreated MMR+/p53+ and the amifostine treated MMR+/p53+ cells (b) P=0.0038: comparison between the amifostine untreated MMR-/p53+ and the amifostine treated MMR-/p53+ cells (c) P= 0.01: comparison between the amifostine untreated MMR-/p53- and the amifostine treated MMR-/p53cells (d) P=0.045: comparison between the amifostine untreated MMR+/p53- and the amifostine treated MMR+/p53cells (e) P=0.008: The G2/M phase was 2.5 fold larger in amifostine-treated HCT116/E6 cells relative to amifostine-treated HCT116+ch3/E6 cells.

Effect of amifostine on the expression of p53 protein and p21 protein

Amifostine뿉 쓽빐 쑀諛쒕맂 꽭룷궗硫 諛 꽭룷 二쇨린 吏꾪뻾뿉 븳 p53 떒諛깆쭏 諛 p21 떒諛깆쭏쓽 뿭븷쓣 異붽濡 議곗궗븯湲 쐞빐 MMR+, MMR-, p53+ 諛 p53꽭룷뿉꽌 amifostine씠 p53 떒諛깆쭏 諛 p21 떒諛깆쭏쓽 諛쒗쁽뿉 誘몄튂뒗 쁺뼢쓣 議곗궗븯떎(Fig. 5). HCT116/p53+, HCT116/p53-, HCT116/E6 諛 HCT116+ch3/E6 꽭룷瑜 HCT116/p53+ 꽭룷쓽 IC50뿉 빐떦븯뒗 4.8 mM amifostine뿉 24떆媛 룞븞 끂異쒖떆耳곕떎. p53+ 꽭룷뿉꽌 amifostine amifostine 쑀룄 G1 젙吏 留덉갔媛吏濡 p53 떒諛깆쭏쓽 鍮좊Ⅸ 쑀룄瑜 쑀諛쒗뻽쑝硫, 씠뒗 24떆媛꾩뿉 理쒓퀬議곗뿉 떖뻽떎. 삉븳, amifostine p21 떒諛깆쭏쓽 鍮좊Ⅸ 쑀룄瑜 씪쑝耳곕떎. p21 떒諛깆쭏쓽 쑀룄뒗 HCT116/p53+, HCT116/p53-, HCT116/E6 諛 HCT116+ ch3/E6 꽭룷 紐⑤몢뿉꽌 諛쒖깮뻽쑝硫, 씠뒗 amifostine뿉 븳 諛섏쓳쑝濡 p21 떒諛깆쭏쓽 쑀룄媛 p53 諛 MMR 떒諛깆쭏怨 룆由쎌쟻엫쓣 굹궦떎. MMR-/p53꽭룷뿉꽌 amifostine G1 젙吏瑜 씪쑝궎吏 븡怨 p21 떒諛깆쭏쓽 諛쒗쁽쓣 쑀룄뻽뒗뜲, 씠뒗 amifostine뿉 븳 諛섏쓳쑝濡 p21 떒諛깆쭏쓽 怨쇰컻쁽씠 꽭룷 二쇨린쓽 G1 떒怨꾩뿉꽌 꽭룷瑜 젙吏떆궎湲곗뿉 異⑸텇븯吏 븡떎뒗 寃껋쓣 굹궦떎.

Fig. 5. Effect of amifostine on the expression of p53 protein and p21 protein. Cells were exposed to amifostine for 24뎗 and cellular proteins were analysed by Western blotting at 24, 48, 72 h after the beginning of exposure to amifostine. Amifostine induced the expression of p53 protein in p53-proficient cells and the expression of p21 protein in p53-proficient and -deficient cells.
怨 李

븫씠 媛뽯뒗 怨듯넻맂 듅꽦 쑀쟾쟻 遺덉븞젙꽦씠떎. 遺遺꾩쓽 븫뿉꽌 DNA 넀긽 諛섏쓳쓽 뼱뼡 寃고븿뿉 쓽빐 븫꽭룷媛 삎꽦릺怨 諛쒕떖븯뒗吏뒗 젙솗엳 븣젮 吏吏 븡븯吏留, DNA 넀긽 諛섏쓳쓽 寃고븿怨 븫 궗씠뿉뒗 遺꾨챸븳 뿰愿 愿怨꾧 엳떎. 삁瑜 뱾뼱, 15%쓽 궛諛쒖꽦 옣 醫낆뼇(sporadic colorectal tumour) 鍮꾩젙긽쟻쑝濡 吏㏐굅굹 湲 dinucleotide 諛섎났꽌뿴쓣 媛吏怨 엳떎. 씠윭븳 DNA 룎뿰蹂씠뒗 microsatellite instability씪怨 븯뒗뜲, 遺덉씪移 蹂듦뎄(MMR)媛 젣 湲곕뒫쓣 紐삵븯뿬 異뺤쟻맂 DNA 蹂듭젣 삤瑜(replication error) 븣臾몄뿉 깮湲곕뒗 寃껋쑝濡 異붿륫맂떎(Fink et al., 1998; Fishel and Kolodner, 1995). Microsatellite instability뒗 궛諛쒖꽦 吏곸옣 醫낆뼇肉먮쭔 븘땲씪 쑀쟾꽦 鍮꾩슜醫낆꽦 옣븫(hereditary non polypasis colorectal cancer)뿉꽌룄 굹궃떎. 쑀쟾꽦 鍮꾩슜醫낆꽦 옣븫 MSH2 MLH1怨 媛숈 遺덉씪移섎났援(MMR) 쑀쟾옄쓽 湲곕뒫긽떎蹂씠(loss of function mutation) 뿰愿 릺뼱엳뒗 寃껋쑝濡 蹂닿퀬릺뿀떎(Lord and Ashworth, 2012). 遺덉씪移 蹂댁젙 쇅뿉룄 MMR 떒諛깆쭏 듅젙 DNA 쁺뿭뿉 寃고빀븷 닔 엳쑝硫 씠 寃고빀 p53 諛 꽭룷궗硫 쑀룄 愿젴씠 엳뒗 寃껋쑝濡 蹂닿퀬릺뿀吏留(Fishel et al., 1993; Kat et al., 1993; Papadopoulos et al., 1994; Parsons et al., 1993) DNA 넀긽 쑀룄 썑 p53 쑀룄 諛 꽭룷궗硫몄뿉꽌 MMR 떒諛깆쭏쓽 뿭븷 븘吏 씠빐릺吏 븡븯떎. 蹂 뿰援ъ뿉꽌뒗 p53 諛 MMR 諛쒗쁽 긽깭媛 떎瑜 HCT116 꽭룷二쇰 씠슜븯뿬 amifostine쓽 꽭룷 빟由ы븰뿉 븳 p53怨 MMR 떒諛깆쭏쓽 슚怨쇱뿉 빐 븣븘蹂댁븯떎. Amifostine paclitaxel쓽 꽭룷룆꽦쑝濡쒕꽣 HCT116/p53+ 꽭룷瑜 꽑깮쟻쑝濡 蹂댄샇븯뒗 寃곌낵뿉꽌 젙긽 議곗쭅 p53씠 뭾遺븯湲 븣臾몄뿉 amifostine씠 씠삩솕 諛⑹궗꽑 諛 솕븰슂踰뺤젣쓽 룆꽦 슚怨쇰줈遺꽣 젙긽 議곗쭅쓣 蹂댄샇븯吏留, p53씠 寃고븤맂 븙꽦 議곗쭅쓣 蹂댄샇븯吏 紐삵븿쓣 꽕紐낇븷 닔 엳뿀떎. 利 留롮 怨좏삎 醫낆뼇 p53 떒諛깆쭏 옄泥 삉뒗 p53 떒諛깆쭏쓽 븯瑜 寃쎈줈뿉 寃고븿 삎깭媛 諛쒗쁽맖씠 留롮 뿰援ъ뿉꽌 諛쒗몴릺뿀떎(Falcicchio et al., 2020; Roser et al., 2021; Jiang et al., 2021; Munakata et al., 2023). 삉븳, amifostine paclitaxel 쑀룄 꽭룷룆꽦쑝濡쒕꽣 MRC-5 씤媛 룓 꽟쑀븘꽭룷瑜 蹂댄샇븯怨 A427 룓븫 꽭룷뿉꽌 paclitaxel 꽭룷룆꽦씠 利앷릺뒗 寃껋쑝濡 굹궗떎(Taylor et al., 1997). A427 꽭룷뒗 p53 떒諛깆쭏쓣 諛쒗쁽븯吏留(Li et al., 2021), p53 떒諛깆쭏쓽 寃고븿쑝濡 씤빐 p53 寃쎈줈媛 鍮꾪솢꽦솕릺뼱 DNA 넀긽 諛섏쓳쑝濡(Strobeck et al., 2001; John et al., 2007) 吏꾪뻾맂떎. Amifostine 泥섎━맂 A427 꽭룷뿉꽌 paclitaxel쓽 誘쇨컧꽦 利앷뒗 DNA 뙆넀쓽 利앷 愿젴씠 엳떎怨(Taylor et al., 1997) 諛쒗몴릺뿀떎. 移섎즺瑜 쐞븳 솕븰슂踰뺤젣 궗슜怨 愿젴븯뿬 MMR 솢꽦씠 긽떎릺硫 寃뚮냸(genome) 쟾泥댁쓽 룎뿰蹂씠 鍮꾩쑉쓣 利앷떆耳, 빟臾 궡꽦씠 諛쒖깮븯뿬 DNA瑜 寃異쒗븯뒗 꽭룷쓽 뒫젰쓣 吏곸젒쟻쑝濡 넀긽떆궡쑝濡쒖뜥 꽭룷궗硫몄쓣 利앷떆궓떎. MMR 떒諛깆쭏 꽭룷 二쇨린쓽 솢꽦솕 以묒옱뿉 愿뿬븯뿬 DNA 넀긽뿉 븳 諛섏쓳쑝濡 꽭룷 二쇨린 泥댄겕룷씤듃 꽭룷궗硫몄씠 씪뼱굹寃 릺怨, MMR씠 寃고븤맂 꽭룷뒗 궡꽦쓣 媛뽯뒗 寃껋쑝濡 蹂닿퀬릺뿀떎(Davis et al., 1998). MMR씠 꽭룷룆꽦쓣 뼱뼸寃 留ㅺ컻븯뒗吏뒗 젙솗엳 꽕紐낇븷 닔 뾾吏留, 6-thioguanine怨 cisplatin 벑뿉 쓽빐 깮꽦맂 DNA 蹂묐 紐⑤몢 룎뿰蹂씠瑜 쑀諛쒗븯誘濡 MMR 븣臾몄뿉 꽭룷媛 二쎌 븡쓣 寃껋씠씪怨 삁긽릺怨, MMR씠 鍮꾪솢꽦솕릺硫 DNA 以묓빀슚냼 삤瑜섎 蹂듦뎄븯吏 紐삵븯湲 븣臾몄뿉 룎뿰蹂씠쑉씠 留ㅼ슦 넂쓣 肉먮쭔 븘땲씪 DNA뿉 遺媛臾쇱씠 吏냽릺湲 븣臾몄씠떎. 媛앷쟻씤 빟臾 궡꽦 諛쒕떖 냽룄 愿젴븯뿬 醫낆뼇뿉꽌 MMR 寃고븤 꽭룷쓽 以묒슂꽦뿉 븳 뿰援ш 쁽옱 떆湲됲엳 븘슂븯떎怨 궗猷뚮맂떎.

Amifostine씠 泥섎━맂 꽭룷뿉꽌 p53 떒諛깆쭏 꽭룷 二쇨린瑜 議곗젅븯뒗뜲 以묒슂븳 뿭븷쓣 븳떎. Amifostine p53씠 엳뒗 꽭룷 寃고븤맂 꽭룷 紐⑤몢뿉꽌 G2 젙吏瑜 씪쑝耳곗쑝硫, 씠뒗 amifostine뿉 쓽빐 쑀룄맂 G2 젙吏媛 p53怨 臾닿븿쓣 굹궦떎. Amifostine 삉븳 p53-룆由쎌쟻 寃쎈줈瑜 넻빐 p21 떒諛깆쭏쓽 怨쇰컻쁽쓣 쑀룄뻽뒗뜲, amifostine뿉 쓽빐 쑀룄맂 G2 젙吏媛 p21 떒諛깆쭏怨 愿젴 엳뒗 寃껋쑝濡쒖꽌, 떎젣濡 p21 떒諛깆쭏 Cdc2쓽 씤궛솕瑜 뼲젣븯怨 G2 젙吏瑜 떆뻾븯뒗 寃껋쑝濡 蹂닿퀬릺뿀떎(Shapiro and Harper, 1999; Smits et al., 2000). MMR 諛쒗쁽 꽭룷뿉꽌 MNNG 泥섎━ 썑 G2 꽭룷 二쇨린 젙吏 諛 꽭룷궗硫몄쓣 珥됱쭊븯怨(Davis et al., 1998), 쑀궗븯寃, 6-thioguanine 泥섎━ 썑 MMR 諛쒗쁽 꽭룷뿉꽌 G2 젙吏媛 蹂닿퀬릺뿀떎(Davis et al., 1998). 씠뿉 諛섑빐, 룞씪븳 끂異쒖뿉 6-thioguanine G2 젙吏瑜 쑀룄븯吏 븡怨 삤엳젮 MMR 遺議 꽭룷뿉꽌 G1 젙吏瑜 쑀룄뻽떎. 꽭룷 二쇨린쓽 G2 젙吏 泥댄겕룷씤듃뒗 꽭룷媛 DNA 遺덉씪移섏쓽 蹂듦뎄瑜 떆룄븯怨 룎뿰蹂씠 맂 DNA쓽 蹂듭젣瑜 諛⑹븯룄濡 뿀슜븷 닔 엳떎. DNA 넀긽뿉 븳 諛섏쓳쑝濡 p53怨 p21뿉 쓽빐 以묒옱맂 G1-S 떒怨꾩뿉 癒몃Т瑜닿쾶 맂떎. 理쒓렐뿉뒗 MLH1씠 寃고븤맂 씤媛 醫낆뼇 꽭룷二쇰룄 cisplatin 泥섎━媛 G2 꽭룷 二쇨린 젙吏뿉 愿뿬븯吏 紐삵븯뒗 寃껋쑝濡 蹂닿퀬릺뿀떎(Davis et al., 1998). MMR씠 DNA 遺덉씪移섎퓧留 븘땲씪 DNA 넀긽씠 媛먯맆 븣 꽭룷 蹂듭젣瑜 젣븳븯뒗 怨쇱젙뿉룄 愿뿬븿쓣 쓽誘명븳떎.

蹂 뿰援 寃곌낵 amifostine p53+ 꽭룷뿉꽌 G1 젙吏瑜 씪쑝耳곗쑝硫, 씠뒗 G1 젙吏媛 p53 떒諛깆쭏怨 愿젴 엳쓬쓣 굹궦떎. p53+ 꽭룷뿉꽌, G1 젙吏 p53 떒諛깆쭏쓽 쑀룄뒗 紐⑤몢 amifostine 끂異 떆옉 썑 24떆媛꾩뿉 理쒓퀬議곗뿉 떖뻽쑝硫, 씠뒗 amifostine뿉 쓽빐 쑀룄맂 G1 젙吏媛 p53 쓽議댁꽦 寃쎈줈 愿젴꽦뿉 븳 利앷굅씠떎. p53-씤 MMR+ MMR꽭룷쓽 寃쎌슦 HCT116 E6쓽 寃쎌슦 빟媛꾩쓽 G1 젙吏 G2/M쓣 쑀룄븳 諛섎㈃ HCT16+ch3/E6쓽 寃쎌슦뒗 꽭룷 二쇨린 젙吏媛 愿李곕릺吏 븡븯떎.

Amifostine MMR怨 긽愿뾾씠 p53+ 꽭룷 p53꽭룷 紐⑤몢뿉꽌 p21 떒諛깆쭏쓽 諛쒗쁽쓣 쑀룄뻽吏留 p53+ 꽭룷뿉꽌留 G1 젙吏瑜 씪쑝耳곌린 븣臾몄뿉, p21 떒諛깆쭏쓽 諛쒗쁽留뚯쑝濡쒕뒗 amifostine뿉 쓽븳 G1 젙吏瑜 꽕紐낇븯湲곗뿉 異⑸텇븯吏 븡븯떎. p21 쑀쟾옄쓽 쟾궗瑜 議곗젅븯뒗 寃 쇅뿉룄, p53 떒諛깆쭏 p27Kip1 諛 p57Kip2 媛숈 Cip/Kip 뙣諛由ъ쓽 븯뼢 떒諛깆쭏쓣 肄붾뵫 븯뒗 쑀쟾옄쓽 쟾궗瑜 議곗젅븯뒗 寃껋쑝濡 諛쒗몴릺뿀떎(Feng et al., 2012; Shapiro and Harper, 1999; Smits et al., 2000). p21 떒諛깆쭏怨 留덉갔媛吏濡 p27Kip1 떒諛깆쭏怨 p57Kip2 떒諛깆쭏 궗씠겢由 E-CDK2 蹂듯빀泥대 삎꽦븯怨 CDK2 뼲젣 諛 G1 젙吏瑜 珥됱쭊븯뒗 寃껋쑝濡 꽕紐낅릺뿀떎(Feng et al., 2012; Shen et al., 2001). 뵲씪꽌, p21 떒諛깆쭏쓽 怨쇰컻쁽씠 amifostine 泥섎━맂 꽭룷뿉꽌 G1 젙泥대 쑀吏븯湲곗뿉 異⑸텇븯吏 븡떎뒗 寃껋 amifostine씠 p53 떒諛깆쭏쓽 븯瑜섏뿉꽌 븯굹 씠긽쓽 떎瑜 꽭룷 二쇨린 뼲젣젣쓽 諛쒗쁽쓣 쑀룄뻽쓬쓣 떆궗븳떎.

꽭룷 蹂댄샇젣씤 amifostine HCT116 꽭룷뿉꽌 꽭룷궗硫몄쓣 쑀룄븷 媛뒫꽦씠 엳떎. p53 떒諛깆쭏쓽 議댁옱 븯뿉꽌 HCT116 꽭룷뒗 amifostine뿉 빐 궙 닔以쓽 궡꽦쓣 굹궡뼱, amifostine뿉 쓽빐 쑀룄맂 꽭룷궗硫몃줈 遺꽣 꽭룷瑜 蹂댄샇븳떎. Amifostine뿉 븳 궡꽦 1.5~2諛곗쓽 踰붿쐞濡 셿留뚰뻽怨, p53 떒諛깆쭏뿉 쓽빐 遺뿬맂 긽쟻쑝濡 옉 궡꽦 젙룄쓽 깮臾쇳븰쟻 愿젴꽦 쁽옱 紐낇솗븯吏 븡吏留, 엫긽 뿰援(Feng et al., 2012; Smits et al., 2000)뿉꽌 떖꽦맂 삁옣 냽룄蹂대떎 빟媛 넂 냽룄뿉꽌 꽭룷궗硫몄쓣 쑀諛쒗븯뿬 엫긽쟻 愿젴꽦씠 엳쓬쓣 굹궦떎.

醫낇빀븯硫, 蹂 뿰援 寃곌낵뒗 p53 떒諛깆쭏씠 amifostine뿉 븳 꽭룷 諛섏쓳쓣 議곗젅븯뒗 뜲 以묒슂븳 뿭븷쓣 븯怨, p53 떒諛깆쭏씠 amifostine뿉 쓽빐 쑀룄맂 꽭룷궗硫몄뿉 븳 빆꽦 湲곗쟾怨 amifostine뿉 쓽빐 쑀룄맂 G1 젙吏 諛 꽭룷 蹂댄샇쓽 湲곗쟾쑝濡 솗씤맂떎뒗 寃껋쓣 굹궦떎.

ACKNOWLEDGEMENT

This paper was supported by Daejeon Health Institute of Technology in 2023.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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