鱼藤素抑制人胰腺癌细胞株Panc-1的增殖并诱导细胞自噬的发生
어등소억제인이선암세포주Panc-1적증식병유도세포자서적발생
Deguelin inhibited the proliferation and induced autophage in human pancreatic cancer lines Panc-1
  • 万方数据
    中华实验外科杂志 중화실험외과잡지
    CHINESE JOURNAL OF EXPERIMENTAL SURGERY
    2014年 1期 23-27 ,共5页

    龙官保%李旭%郑建伟%粱盟%秦仁义%申铭

    룡관보%리욱%정건위%량맹%진인의%신명

    胰腺癌%鱼藤素%细胞周期%细胞凋亡%自噬 胰腺癌%魚籐素%細胞週期%細胞凋亡%自噬
    이선암%어등소%세포주기%세포조망%자서
    Pancreatic cancer%Deguelin%Cell cycle%Cell apoptosis%Autophagy
    目的 观察鱼藤素在人胰腺癌细胞株Panc-1中的作用.方法 细胞计数试剂盒(CCK-8)法检测鱼藤素对人胰腺癌细胞株Panc-1增殖的影响；碘化丙锭(PI)染色法检测鱼藤素对人胰腺癌细胞株Panc-1周期的影响；膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)法检测鱼藤素对人胰腺癌细胞株Panc-1凋亡的影响；单丹(磺)酰戊二胺(MDC)和吖啶橙/溴乙啶(AO/EO)染色法检测鱼藤素对人胰腺癌细胞株Panc-1自噬的影响；Western blot技术检测鱼藤素对人胰腺癌细胞株Panc-1自噬蛋白及蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)信号通路蛋白表达的调节作用.结果 Panc-1细胞经不同浓度(0、10、25、50、100、200和500 μmol/L)的鱼藤素处理24 h后,细胞存活率分别为(96.21±0.30)％、(95.40±1.78)％、(94.21 ±2.43)％、(91.27±4.24)％、(86.14±3.10)％、(80.30±3.21)％、(69.54±3.36)％；Panc-1细胞经不同浓度(0、10、25、50、100、200和500μmol/L)的鱼藤素处理48h后,细胞存活率分别为(95.94±0.20)％、(93.46±1.66)％、(88.62±0.72)％、(80.16±2.64)％、(63.64±2.94)％、(49.87±3.50)％、(34.99±2.09)％；Panc-1细胞经不同浓度(0、10、25、50、100、200和500 μmol/L)的鱼藤素处理72 h后,细胞存活率分别为(94.50±0.14)％、(86.98±1.26)％、(70.87±2.87)％、(51.85±6.73)％、(33.16±6.33)％、(16.29±0.83)％、(6.01±0.45)％；经不同浓度的鱼藤素处理后,Panc-1细胞的存活率明显下降,呈时间-剂量依赖性(P＜0.05)；Panc-1细胞经不同浓度(0、10、25、50、100和200μmol/L)的鱼藤素处理24h后,G2/M期细胞比例分别为(13.98±2.31)％、(16.94±2.31)％、(22.61±1.79)％、(34.15±0.09)％、(43.01±1.45)％、(45.46±0.94)％；Panc-1细胞经不同浓度(0、10、25、50、100和200 μmol/L)的鱼藤素处理48 h后,G2/M期细胞比例分别为(12.32±0.80)％、(14.33±0.78)％、(25.72±3.27)％、(36.91±1.50)％、(44.35 ±0.82)％、(48.94±1.63)％；不同浓度的鱼藤素作用24、48 h后,Panc-1细胞G2/M期比例明显增加(P＜0.05).Panc-1细胞经不同浓度(0、10、25、50、100和200 μmol/L)的鱼藤素处理48 h后,对照组(0μmol/L)的凋亡率为(10.82±2.99)％,低浓度(10、25、50 μmol/L)鱼藤素处理组的凋亡率分别(11.88±2.40)％、(13.91±2.03)％、(17.28±3.67)％,与对照组比较,其促凋亡作用并不明显(P＞0.05)；高剂量(100、200 μnol/L)鱼藤素处理组的凋亡率分别为(24.80±2.92)％、(30.16±3.08)％,与对照组比较,有部分促凋亡作用(P＜0.05).MDC和AO/EO染色结果显示Panc-1细胞内的点状阳性结构和橘红色荧光明显增加；Western blot结果显示,鱼藤素作用后自噬关键蛋白Atg5、Atg7和LC3Ⅱ表达增加,而磷酸化Akt (p-Akt)、磷酸化mTOR (p-mTOR)表达减少；使用自噬抑制剂3-甲基腺嘌呤(3-MA)和氯喹(CQ)抑制自噬后,对照组的凋亡率为(11.26±2.89)％,3-MA和CQ处理组的凋亡率分别为(14.98±2.77)％和(18.10±3.03)％,100 μmol/L的鱼藤素引起的凋亡率为(22.37±2.45)％；分别联用3-MA和CQ后,100 μmol/L鱼藤素处理组的促凋亡率分别为(39.24±2.78)％和(55.53±3.25)％,与未加自噬抑制剂前比较,差异有统计学意义(P＜0.01).结论 鱼藤素可以明显抑制人胰腺癌细胞株Panc-1的增殖,并通过抑制Akt/mTOR信号通路和激活相关自噬蛋白的表达,进而诱导Panc-1细胞发生保护性自噬.
    목적 관찰어등소재인이선암세포주Panc-1중적작용.방법 세포계수시제합(CCK-8)법검측어등소대인이선암세포주Panc-1증식적영향；전화병정(PI)염색법검측어등소대인이선암세포주Panc-1주기적영향；막련단백V-이류청산형광소(Annexin V-FITC)법검측어등소대인이선암세포주Panc-1조망적영향；단단(광)선무이알(MDC)화아정등/추을정(AO/EO)염색법검측어등소대인이선암세포주Panc-1자서적영향；Western blot기술검측어등소대인이선암세포주Panc-1자서단백급단백격매B(Akt)/뢰파매소파단백(mTOR)신호통로단백표체적조절작용.결과 Panc-1세포경불동농도(0、10、25、50、100、200화500 μmol/L)적어등소처리24 h후,세포존활솔분별위(96.21±0.30)％、(95.40±1.78)％、(94.21 ±2.43)％、(91.27±4.24)％、(86.14±3.10)％、(80.30±3.21)％、(69.54±3.36)％；Panc-1세포경불동농도(0、10、25、50、100、200화500μmol/L)적어등소처리48h후,세포존활솔분별위(95.94±0.20)％、(93.46±1.66)％、(88.62±0.72)％、(80.16±2.64)％、(63.64±2.94)％、(49.87±3.50)％、(34.99±2.09)％；Panc-1세포경불동농도(0、10、25、50、100、200화500 μmol/L)적어등소처리72 h후,세포존활솔분별위(94.50±0.14)％、(86.98±1.26)％、(70.87±2.87)％、(51.85±6.73)％、(33.16±6.33)％、(16.29±0.83)％、(6.01±0.45)％；경불동농도적어등소처리후,Panc-1세포적존활솔명현하강,정시간-제량의뢰성(P＜0.05)；Panc-1세포경불동농도(0、10、25、50、100화200μmol/L)적어등소처리24h후,G2/M기세포비례분별위(13.98±2.31)％、(16.94±2.31)％、(22.61±1.79)％、(34.15±0.09)％、(43.01±1.45)％、(45.46±0.94)％；Panc-1세포경불동농도(0、10、25、50、100화200 μmol/L)적어등소처리48 h후,G2/M기세포비례분별위(12.32±0.80)％、(14.33±0.78)％、(25.72±3.27)％、(36.91±1.50)％、(44.35 ±0.82)％、(48.94±1.63)％；불동농도적어등소작용24、48 h후,Panc-1세포G2/M기비례명현증가(P＜0.05).Panc-1세포경불동농도(0、10、25、50、100화200 μmol/L)적어등소처리48 h후,대조조(0μmol/L)적조망솔위(10.82±2.99)％,저농도(10、25、50 μmol/L)어등소처리조적조망솔분별(11.88±2.40)％、(13.91±2.03)％、(17.28±3.67)％,여대조조비교,기촉조망작용병불명현(P＞0.05)；고제량(100、200 μnol/L)어등소처리조적조망솔분별위(24.80±2.92)％、(30.16±3.08)％,여대조조비교,유부분촉조망작용(P＜0.05).MDC화AO/EO염색결과현시Panc-1세포내적점상양성결구화귤홍색형광명현증가；Western blot결과현시,어등소작용후자서관건단백Atg5、Atg7화LC3Ⅱ표체증가,이린산화Akt (p-Akt)、린산화mTOR (p-mTOR)표체감소；사용자서억제제3-갑기선표령(3-MA)화록규(CQ)억제자서후,대조조적조망솔위(11.26±2.89)％,3-MA화CQ처리조적조망솔분별위(14.98±2.77)％화(18.10±3.03)％,100 μmol/L적어등소인기적조망솔위(22.37±2.45)％；분별련용3-MA화CQ후,100 μmol/L어등소처리조적촉조망솔분별위(39.24±2.78)％화(55.53±3.25)％,여미가자서억제제전비교,차이유통계학의의(P＜0.01).결론 어등소가이명현억제인이선암세포주Panc-1적증식,병통과억제Akt/mTOR신호통로화격활상관자서단백적표체,진이유도Panc-1세포발생보호성자서.
    Objective To observe the influences and mechanisms of deguelin,a rotenoid from Mundulea sericea,on Panc-1 cell.Methods Cell counting kit-8 (CCK-8) assay detected the cell proliferation; propidium iodide (PI) staining was used to observe the change of cell cycle; Annexin V-FITC assay was performed to test apoptosis ; In order to examine the influence on autophage,monodansylcadaverine (MDC) and acridine orange/ethidium bromide (AO/EO) stain was adopted; Western blotting explored the expression of proteins include autophage and protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signal.Results After the 24 hours intervention of different concentration (0,10,25,50,100,200 and 500 μmol/L) of deguelin,the survival rate of Panc-1 cell were (96.21 ± 0.30) ％,(95.40 ±1.78)％,(94.21 ±2.43)％,(91.27 ±4.24)％,(86.14 ±3.10)％,(80.30 ±3.21)％,(69.54 ±3.36) ％ respectively; 48 hours were (95.94 ± 0.20) ％,(93.46 ± 1.66) ％,(88.62 ± 0.72) ％,(80.16 ±2.64)％,(63.64 ±2.94)％,(49.87 ±3.50)％,(34.99 ±2.09)％ respectively; 72 hours were (94.50 ±0.14)％,(86.98± 1.26)％,(70.87 ±2.87)％,(51.85 ±6.73)％,(33.16 ±6.33) ％,(16.29 ± 0.83) ％,(6.01 ± 0.45) ％ respectively.After the intervention of different concentration of deguelin,the survival rate of Panc-1 cell significantly decreased with a drug concentration-and time-dependent manner (P ＜ 0.05) ; After the 24 hours intervention of different concentration (0,10,25,50,100 and 200 μmol/L) of deguelin,the rate of G2/M phase of Panc-1 cell were (13.98 ±2.31)％,(16.94±2.31)％,(22.61±1.79)％,(34.15 ±0.09)％,(43.01 ±1.45)％,(45.46±0.94)％,respectively; 48 hours were (12.32 ± 0.80) ％,(14.33 ± O.78) ％,(25.72 ± 3.27) ％,(36.91 ±1.50)％,(44.35 ± 0.82)％,(48.94 ± 1.63)％,respectively; G2/M phase cell was obviously increased after the intervention of 24 and 48 h ; After the 48 hours intervention of different concentration (0,10,25,50,100 and 200 μmol/L) of deguelin,the apoptosis rate of Panc-1 cell were (10.82 ±2.99)％,(11.88 ±2.40)％,(13.91 ±2.03)％,(17.28 ±3.67)％,(24.80±2.92)％,(30.16±3.08) ％,respectively; After the 48 hours of deguelin intervention,the low concentration (10,25 and 50 μmol/L) of deguelin did not promote Panc-1 cell apoptosis (P ＞ 0.05),the depth of deguelin(100 and 200 μmol/L) induced part of apoptosis (P ＜ 0.05) ; The staining of MDC within the point-like positive structure and AO/EO within the orange-red fluorescence significantly increased ; Atg5,Atg7 and LC3 Ⅱ expression increased; p-Akt and p-mTOR' expression reduced; Use 3-methyladenine (3-MA) and CQ inhibit autophage,the apoptosis rate of control group,3-MA group and CQ group was (11.26 ± 2.89) ％,(14.98 ± 2.77)％,(18.10 ± 3.03)％,respectively; Combination 3-MA and degulin (100μmol/L)gourp' s apoptosis was (39.24 ± 2.78) ％ ; Combination CQ and degulin (100μmol/L) gourp' s apoptosis was (55.53 ± 3.25) ％.The apoptosis was increased rapidly when autophage was inhibited by 3-MA or CQ.Conclusion Deguelin inhibited proliferation of Panc-1 cell obviously; Deguelin induced autophage with the activation of autopahge proteins by down-regulation of Akt/mTOR signal proteins in Panc-1 cell that protected the Panc-1 cell to survival.
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