摘要
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.
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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第1期23-27,共5页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目
关键词
胰腺癌
鱼藤素
细胞周期
细胞凋亡
自噬
Pancreatic cancer
Deguelin
Cell cycle
Cell apoptosis
Autophagy