摘要
目的探讨阿魏菇总三萜(PFTP-E)、齐墩果酸(OA)和紫杉醇(PTX)对结肠癌细胞HCT116和CT26的生长抑制作用及可能机制。方法采用紫外和红外光谱分析PFTP-E成分;噻唑蓝实验检测PFTP-E、OA、PTX体外抑制HCT116、CT26细胞的增殖活性;Hoechst 33258染色观察细胞凋亡;流式细胞术检测PFTP-E、OA、PTX对细胞凋亡、周期、线粒体膜电位及胞内活性氧的影响;蛋白质印迹法(Western Blot)检测PFTP-E、OA、PTX对细胞凋亡相关蛋白B细胞淋巴瘤因子2(Bcl-2)、Bcl-2相关X蛋白(Bax)、多聚二磷酸腺苷核糖聚合酶(PARP)、半胱氨酸蛋白酶-3(Caspase-3)、Caspase-9,细胞周期蛋白B1(Cyclin B1),内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、真核起始因子2α(eIF2α)、促凋亡因子C/EBP同源蛋白(CHOP)和自噬微管相关蛋白轻链3A/B(LC3A/B)表达的影响。结果PFTP-E主要成分为三萜类、甾体。PFTP-E、OA、PTX以时间和浓度依赖性抑制HCT116和CT26细胞的增殖,诱导细胞凋亡,并将细胞周期阻滞在G0/G1和G2/M期。PFTP-E可引起HCT116和CT26细胞线粒体膜电势崩溃并导致胞内活性氧升高,与PTX作用类似。PFTP-E、PTX可上调细胞色素C(CytC)、Bax、GRP78、磷酸化PERK、磷酸化eIF2α和CHOP表达,下调Bcl-2表达,明显增加剪切型PARP、Caspase-3及Caspase-9含量,诱导内质网应激;而OA可明显上调HCT116细胞LC3A/B的表达,倾向于诱导自噬凋亡。结论PFTP-E与PTX诱导结肠癌细胞凋亡效果及机制相似,均与线粒体损伤途径、周期阻滞、内质网应激等有关,且PFTP-E副作用明显低于PTX,为PFTP-E作为抗癌药物的筛选与研究提供了有力的参考依据。
Objective To investigate the effects of ethyl acetate phase of Pleurotus ferulatus triterpenoid component(PFTP-E),oleanolic acid(OA)and paclitaxel(PTX)on growth inhibition and possible regulation mechanisms of colon cancer cells HCT116 and CT26.Methods Ultraviolet and infrared spectroscopy was used to analyze the component of PFTP-E.MTT assay was used to detect the proliferation of HCT116 and CT26 cells by PFTP-E,OA and PTX in vitro.Apoptosis was observed by Hoechst 33258 staining.Flow cytometry was used to detect apoptosis,cell cycle,mitochondrial membrane potential and intracellular ROS.Western Blot were used to detect the expressions of apoptosis-related proteins B cell lymphoma-2(Bcl-2),BCL2 associated X protein(Bax),poly(adenosine diphosphate-ribose)polymerase(PARP),Caspase-3,Caspase-9,cell cycle related proteinB1(Cyclin B1),endoplasmic reticulum stress-related proteins glucose-regulated protein 78(GRP78),protein kinase R-like ER kinase(PERK),eukaryotic initiation factor 2α(eIF2α),C/EBP-homologous protein(CHOP),and autophagy microtubule associated protein light chain 3A/B(LC3A/B).Results PFTP-E is mainly composed of steroids and triterpenoids.PFTP-E,OA and PTX can inhibit the proliferation of HCT116 and CT26 cells in a time-and dose-dependent manner,induce apoptosis,and block the cell cycle at G0/G1 and G2/M phases.PFTP-E can collapse mitochondrial membrane potential of HCT116 and CT26 cells and increase the content of intracellular reactive oxygen species,which is similar to the effect of PTX.PFTP-E and PTX can up-regulate CytC,Bax,GRP78,p-PERK,p-eIF2αand CHOP,down-regulate Bcl-2,significantly increase the content of cleaved PARP,Caspase3 and Caspase9,and induce endoplasmic reticulum stress response.OA can significantly increase the expression of LC3A/B in HCT116 cells and tend to induce autophagy and apoptosis.Conclusions PFTP-E and PTX have a similar effect on apoptosis,and that is related to mitochondrial injury pathway,cycle arrest,and endoplasmic reticulum stress response.The side effects of PFTP-E are significantly lower than PTX,which provides a meaningful reference for the screening and research of PFTP-E as an anticancer drug.
作者
王磊
张富春
刘军
Wang Lei;Zhang Fuchun;Liu Jun(College of Life Science and Technology,Xinjiang University,Xinjiang Key Laboratory of Biological Resources Genetic Engineering,Urumqi 830046,China)
出处
《国际生物医学工程杂志》
CAS
2019年第6期451-462,483,共13页
International Journal of Biomedical Engineering
关键词
三萜类
结肠肿瘤
细胞凋亡
阿魏菇
内质网应激
Triterpenoid
Colonic neoplasms
Apoptosis
Pleurotus ferulatus
Endoplasmic reticulum stress
