异硫氰酸苯已酯（PHI）诱导前列腺癌PC3细胞凋亡的分子机制研究
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摘要
[目的]观察异硫氰酸苯己酯(PHI)对前列腺癌PC3细胞株增殖、凋亡的作用,研究PHI对前列腺癌细胞凋亡相关蛋白、组蛋白乙酰化、甲基化、Akt信号转导通路的影响,阐述诱导凋亡的分子机制。
[方法]采用MTT方法绘制PHI作用后PC3细胞的生长曲线;采用TUNNEL方法检测PHI对PC3细胞凋亡的影响;用蛋白免疫印迹法(Western Blot)观察PC3细胞凋亡相关蛋白BcL-2,caspase-9、caspase-3、McL-1、Cyt-C、XIAP的变化及组蛋白乙酰化H3、H4,组蛋白甲基化H3K9、H3K4水平的变化;同时研究Akt信号转导通路蛋白表达水平的变化。
[结果](1)PHI能抑制PC3细胞增殖,IC_(50)约为20μmol/L,并能诱导PC3细胞凋亡,随作用时间延长及浓度的增加,凋亡率渐上升,呈时效及量效关系;(2)PHI下调PC3细胞BcL-2,caspase-9、caspase-3、McL-1、Cyt-C、XIAP的表达,呈时效及量效变化;(3)PHI处理3小时就可见增加PC3细胞组蛋白乙酰化H3、H4,组蛋白甲基化H3K4水平,降低甲基化H3K9水平;(4)PHI处理后3小时PC3细胞Akt信号转导通路Akt及p-Akt均未见明显变化,而作用7小时后,总蛋白Akt、mTOR、p70S6K的表达变化不明显,而磷酸化蛋白p-Akt、p-mTOR、p-p70S6K均表达下降。
[结论](1)PHI可抑制PC3细胞的增殖并诱导其凋亡,可能与激活细胞内源性凋亡通路有关。(2)PHI可使组蛋白H3、H4高乙酰化,并调控组蛋白甲基化水平。(3)PHI可抑制Akt通路,从而参与了PHI抑制PC3细胞的增殖、诱导凋亡的过程。PHI是一种潜在抗前列腺癌的新药。
Ojects To investigate PHI(phenyhexyle isothiocyanate) effect on cell apoptosis,histone acetylation and histone methylation,and Akt signaling pathway in prostate cancer cell line(PC3) in vitro.We further study its potential mechanism.
Methods The viability of PC3 cells after treated with PHI was checked by MTT method.Apoptotic cells were measured by TUNNEL assay.The proteins of bcL-2,caspase-9,caspase-3,McL-1,Cyt-C,XIAP,histone acetylated H3 and H4,histone methylated H3K9 and H3K4 and the protein of Akt signaling pathway(Akt,p-Akt,mTOR,p-mTOR,p70S6K,p-p70S6K) were detected by Western Blot.
Results(1) PHI induced a dose-dependent decrease of cells density and viability.(2) Apoptotic cells were increased after exposure to PHI with time and concentration dependent.With the augmenting of time and dose, the expression of bcL-2,caspase-9,caspase-3,McL-1,Cyt-C,XIAP were decreased.(3) PHI significantly induced an accumulation of histone acetylated H3;H4,and histone methylated H3k4 and decreased methylated H3K9.(4) The change of proteins of Akt、mT0R、p70S6K was not seen and phosphorylation of Akt(p-Akt)、p70S6K(p-p70S6K)、mTOR(p-mTOR) was decreased after exposure to PHI for 7 hours.The protein of Akt、p-Akt was no change after exposure to PHI for 3 hours.
Conclusions(1) PHI was able to inhibit the cell growth and induce apoptosis in PC3 cells through mitochondria pathway.(2) Acetylation of histone H3,H4 was enhanced significantly,coupled to selective methylation of histone H3 lysine 4,followed with demethylation on lysine 9、of H3,which are marks of chromatin unfolding and gene transcription. (3) PHI inhibited Akt signaling pathway after 7 hours exposure,which inhibited cells growth and induced apoptosis. PHI might be a potential novel anti-prostate cancer agent.
[方法]采用MTT方法绘制PHI作用后PC3细胞的生长曲线;采用TUNNEL方法检测PHI对PC3细胞凋亡的影响;用蛋白免疫印迹法(Western Blot)观察PC3细胞凋亡相关蛋白BcL-2,caspase-9、caspase-3、McL-1、Cyt-C、XIAP的变化及组蛋白乙酰化H3、H4,组蛋白甲基化H3K9、H3K4水平的变化;同时研究Akt信号转导通路蛋白表达水平的变化。
[结果](1)PHI能抑制PC3细胞增殖,IC_(50)约为20μmol/L,并能诱导PC3细胞凋亡,随作用时间延长及浓度的增加,凋亡率渐上升,呈时效及量效关系;(2)PHI下调PC3细胞BcL-2,caspase-9、caspase-3、McL-1、Cyt-C、XIAP的表达,呈时效及量效变化;(3)PHI处理3小时就可见增加PC3细胞组蛋白乙酰化H3、H4,组蛋白甲基化H3K4水平,降低甲基化H3K9水平;(4)PHI处理后3小时PC3细胞Akt信号转导通路Akt及p-Akt均未见明显变化,而作用7小时后,总蛋白Akt、mTOR、p70S6K的表达变化不明显,而磷酸化蛋白p-Akt、p-mTOR、p-p70S6K均表达下降。
[结论](1)PHI可抑制PC3细胞的增殖并诱导其凋亡,可能与激活细胞内源性凋亡通路有关。(2)PHI可使组蛋白H3、H4高乙酰化,并调控组蛋白甲基化水平。(3)PHI可抑制Akt通路,从而参与了PHI抑制PC3细胞的增殖、诱导凋亡的过程。PHI是一种潜在抗前列腺癌的新药。
Ojects To investigate PHI(phenyhexyle isothiocyanate) effect on cell apoptosis,histone acetylation and histone methylation,and Akt signaling pathway in prostate cancer cell line(PC3) in vitro.We further study its potential mechanism.
Methods The viability of PC3 cells after treated with PHI was checked by MTT method.Apoptotic cells were measured by TUNNEL assay.The proteins of bcL-2,caspase-9,caspase-3,McL-1,Cyt-C,XIAP,histone acetylated H3 and H4,histone methylated H3K9 and H3K4 and the protein of Akt signaling pathway(Akt,p-Akt,mTOR,p-mTOR,p70S6K,p-p70S6K) were detected by Western Blot.
Results(1) PHI induced a dose-dependent decrease of cells density and viability.(2) Apoptotic cells were increased after exposure to PHI with time and concentration dependent.With the augmenting of time and dose, the expression of bcL-2,caspase-9,caspase-3,McL-1,Cyt-C,XIAP were decreased.(3) PHI significantly induced an accumulation of histone acetylated H3;H4,and histone methylated H3k4 and decreased methylated H3K9.(4) The change of proteins of Akt、mT0R、p70S6K was not seen and phosphorylation of Akt(p-Akt)、p70S6K(p-p70S6K)、mTOR(p-mTOR) was decreased after exposure to PHI for 7 hours.The protein of Akt、p-Akt was no change after exposure to PHI for 3 hours.
Conclusions(1) PHI was able to inhibit the cell growth and induce apoptosis in PC3 cells through mitochondria pathway.(2) Acetylation of histone H3,H4 was enhanced significantly,coupled to selective methylation of histone H3 lysine 4,followed with demethylation on lysine 9、of H3,which are marks of chromatin unfolding and gene transcription. (3) PHI inhibited Akt signaling pathway after 7 hours exposure,which inhibited cells growth and induced apoptosis. PHI might be a potential novel anti-prostate cancer agent.
引文
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