樟芝皿培菌丝提取物体外对SPCA-1肺癌细胞增殖的抑制作用
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摘要
利用细胞克隆实验、划痕实验和alamarBlue~(TM)细胞活力测定实验评价樟芝皿培菌丝体醇提物石油醚萃取相和氯仿萃取相对SPCA-1非小细胞肺癌的体外抑制作用,并进一步利用流式细胞术Annexin VFITC(fluorescein isothiocyanate)/PI(propidium iodide)双染测定细胞早期凋亡率、流式细胞术2′,7′-二氯荧光素二乙酸酯(2′,7′-dichlorofluorescindiacetate,H2DCFDA)单染测定ROS(reactive oxygen species)释放量、流式细胞术PI单染测定细胞周期变化和ELISA(enzyme-linked immunosorbent assay)检测细胞凋亡相关蛋白Caspase-3、p53和PARP的表达,探讨樟芝皿培菌丝体醇提物石油醚萃取相和氯仿萃取相抑制SPCA-1增殖的作用机理。研究结果表明:樟芝皿培菌丝体醇提物石油醚萃取相和氯仿萃取相均能抑制SPCA-1细胞克隆的形成、迁移和增殖,石油醚萃取相的活性明显好于氯仿萃取相;石油醚萃取相通过促进SPCA-1释放ROS来诱发线粒体依赖性早期凋亡的发生,并使促凋亡蛋白Caspase-3和p53表达上升,同时抑制蛋白PARP表达,从而发挥其抑制SPCA-1肺癌细胞增殖的作用;而氯仿萃取相主要通过将SPCA-1细胞阻滞在S期抑制其增殖。
Colony formation method,wound-healing method and alamarBlue~(TM)cell viability assay were used to evaluate in vitro anti-tumor effects of petroleum ether extract and chloroform extract from dish-cultivated T.camphoratus mycelia on SPCA-1 non-small cell lung cancer cells.Flow cytometry Annexin V-FITC/PI double staining method was used to determine early apoptotic rate; flow cytometry 2 ',7 '-dichlorofluorescindiacetate(H2 DCFDA)single staining method was used to determine ROS(reactive oxygen species)release; flow cytometry PI single staining method was applied to determine cell cycle changes; and ELISA method was used to determine expression of apoptosis-related proteins Caspase-3,p53 and PARP,hence to explore the anti-proliferation mechanism of extracts from dish-cultivated T.camphoratus mycelia on SPCA-1 lung cancer cells.The results showed that both petroleum ether extract and chloroform extract inhibited the colony formation,migration and proliferation of SPCA-1 cells.The activity of petroleum ether extract was significantly better than that of chloroform extract.The petroleum ether extract induced mitochondria-dependent early apoptosis by promoting the release of ROS from SPCA-1,which led to an increase in the activity of the pro-apoptotic proteins Caspase-3 and p53 and inhibition of PARP protein,thereby exerting its anti-tumor activity.The chloroform extract might exert its anti-tumor activity mainly by arresting SPCA-1 cells in S phase.
Colony formation method,wound-healing method and alamarBlue~(TM)cell viability assay were used to evaluate in vitro anti-tumor effects of petroleum ether extract and chloroform extract from dish-cultivated T.camphoratus mycelia on SPCA-1 non-small cell lung cancer cells.Flow cytometry Annexin V-FITC/PI double staining method was used to determine early apoptotic rate; flow cytometry 2 ',7 '-dichlorofluorescindiacetate(H2 DCFDA)single staining method was used to determine ROS(reactive oxygen species)release; flow cytometry PI single staining method was applied to determine cell cycle changes; and ELISA method was used to determine expression of apoptosis-related proteins Caspase-3,p53 and PARP,hence to explore the anti-proliferation mechanism of extracts from dish-cultivated T.camphoratus mycelia on SPCA-1 lung cancer cells.The results showed that both petroleum ether extract and chloroform extract inhibited the colony formation,migration and proliferation of SPCA-1 cells.The activity of petroleum ether extract was significantly better than that of chloroform extract.The petroleum ether extract induced mitochondria-dependent early apoptosis by promoting the release of ROS from SPCA-1,which led to an increase in the activity of the pro-apoptotic proteins Caspase-3 and p53 and inhibition of PARP protein,thereby exerting its anti-tumor activity.The chloroform extract might exert its anti-tumor activity mainly by arresting SPCA-1 cells in S phase.
引文
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[34]周彦娇,张勇,姚智.活性氧在线粒体凋亡信号传导中的调节作用[J].中国运动医学杂志,2005,24(6):746-750.
[35]马淇,刘垒,陈佺.活性氧、线粒体通透性转换与细胞凋亡[J].生物物理学报,2012,28(7):523-536.
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[37]ALMAHLI H,HADCHITY E,JABALLAH MY,et al.Development of novel synthesized phthalazinone-based PARP-1 inhibitors with apoptosis inducing mechanism in lung cancer[J].Bioorganic Chemistry,2018,77:443-456.
[38]RAJAVEL T,PACKIYARAJ P,SURYANARAYANAN V,et al.β-Sitosterol targets Trx/Trxl reductase to induce apoptosis in A549 cells via ROS mediated mitochondrial dysregulation and p53 activation[J].Scientific Reports,2018,8(1):2071.
[2]SONG TY,YEN GC.Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CC14-induced hepatic toxicity in rats[J].Journal of Agricultural and Food Chemistry,2003,51(6):1571-1577.
[3]LEE YP,TSAI WC,KO CJ,et al.Anticancer effects of eleven triterpenoids derived from Antrodia camphorata[J].Anticancer Research,2012,32(7):2727-2734.
[4]SUN SY,HAIL NJ,LOTAN R.Apoptosis as a novel target for cancer chemoprevention[J].Journal of theNational Cancer Institute,2004,96(9):662-672.
[5]KIM BC,KIM HG,LEE SA,et al. Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway[J].Biochemical Pharmacology,2005,70(9):1398-1407.
[6]LIN LT,TAI CJ,SU CH,et al.The ethanolic extract of Taiwanofungus camphoratus(Antrodia camphorata)induces cell cycle arrest and enhances cytotoxicity of cisplatin and doxorubicin on human hepatocellular carcinoma cells[J].BioMed Research International,2015,2015:415269.
[7]CHEN LY,SHEU MT,LIU DZ,et al. Pretreatment with an ethanolic extract of Taiwanofungus camphoratus(Antrodia camphorata)enhances the cytotoxic effects of amphotericin B[J].Journal of Agricultural and Food Chemistry,2011,59(20):112 55-112 63.
[8]ZHANG F,CAI H,LIU X,et al·Simultaneous determination of 19 fatty acids in Antrodia camphorata by derivatized GC-MS and evaluation of antioxidant activity of Antrodia camphorata crude oil[J].Archives of Pharmacal Research,2015:1-8.
[9]TSAI TC,TUNG YT,KUO YH,et al.Anti-inflammatory effects of Antrodia camphorata,a herbal medicine,in a mouse skin ischemia model[J].Journal of Ethnopharmacology,2015,159:113-121.
[10]LIN SY,SHEEN LY,CHIANG BH,et al.Dietary effect of Antrodia camphorate extracts on immune responses in WEHI-3 leukemia BALB/c mice[J].Nutrition and Cancer,2010,62(5):593-600.
[11]赵春艳,吴素蕊,高观世,等.牛樟芝药用菌功能成分作用及应用进展[J].食品工业科技,2016,37(23):391-395.
[12]邢宏博,胡永丹,吕俊,等.樟芝活性代谢产物的研究进展[J].生物加工过程,2017,15(4):1-8.
[13]王冲,徐琳,华晓东,等.牛樟芝人工培养技术及药理作用的研究进展[J].天津药学,2018,30(5):48-53.
[14]张东柱.台湾特有珍贵药用真菌牛樟芝[J].食药用菌,2011,19(1):33-34.
[15]徐泰浩,林芳仪.台湾地区樟芝研究概况与发展趋势[J].食药用菌,2011,19(6):24-29.
[16]赖敏男.台湾牛樟芝发展史及人工栽培现况[J].食药用菌,2017,25(2):84-89.
[17]QIAO X,SONG W,WANG Q,et al. Comprehensive chemical analysis of triterpenoids and polysaccharides in the medicinal mushroom Antrodia cinnamomea[J].RSC Advances,2015,5(58):47040-47052.
[18]YANG HR,ZHANG JS,ZHANG HN,et al.Compound of stout camphor medicinal mushroom Taiwanofungus camphoratus(Higher Basidiomycetes)induces protective autophagy in SPCA-1 cells through AMPK inhibitionindependent blockade of Akt/mTOR pathway.International Journal of Medicine Mushrooms,2018,20(8):727-738.
[19]YANG HR,SUN WB,ZHANG JS,et al.Autophagy inhibition enhances SPCA-1 cell proliferation inhibition induced by By-1 from the stout camphor medicinal mushroom,Taiwanofungus camphoratus(Agaricomycetes).International Journal of Medicine Mushrooms,2018,20(4):321-335.
[20]YANG HR,BAI X,ZHANG HN,et al.Antrodin C,an NADPH dependent metabolism,encourages crosstalk between autophagy and apoptosis in lung carcinoma cells by use of an AMPK inhibition-independent blockade of the Akt/mTOR pathway[J].Molecules,2019,24(5):993.
[21]WANG WH,WANG LY,YANG HR,et al.Antitumor effect of by-1 from spent broth from submerged cultures of stout camphor medicinal mushroom,Taiwanofungus camphoratus(Higher basidiomycetes),on A549adenocarcinoma cells[J].International Journal of Medicinal Mushrooms.2017,19(3):225-232.
[22]LEE YP,TSAI WC,KO CJ,et al.Anticancer effects of eleven triterpenoids derived from Antrodia camphorata[J].Anticancer Research.2012,32(7):2727-2734.
[23]吴建胜.皿培式牛樟芝子实体对小鼠发炎性肠病及肠癌恶病质减缓作用[D].基隆:国立台湾海洋大学,2012.
[24]李传华,贾薇,杨海芮,等.人工栽培黑柄炭角菌子实体的生物活性[J].食用菌学报,2016,23(2):59-64.
[25]汪雯翰,朱丽娜,张赫男,等.抑制前列腺癌的食药用菌醇提物的筛选及灵芝醇提物对癌细胞的抑制机理[J].菌物学报,2018,37(6):782-793.
[26]BRAY F,FERLAY J,SOERJOMATARAM I,et al.Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA:A Cancer Journal for Clinicians,2018,68(6):394-424.
[27]杨露璐.非小细胞肺癌新的治疗性靶点的探索[D].广州:南方医科大学,2014.
[28]ANGAMUTHU V,SHANMUGAVADIVU M,NAGARAJAN G,et al.Pharmacological activities of antroquinonol-mini review[J].Chemico-Biological Interactions,2019,297:8-15.
[29]姚晓军,刘伦旭.肺癌的流行病学及治疗现状[J].现代肿瘤医学,2014,22(8):1982-1986.
[30]白玉梅,卢保华.非小细胞肺癌治疗进展[J].山西医药杂志,2013,42(14):767-768.
[31]高成彬.浅谈肺癌的临床治疗方法与体会[J].中国卫生标准管理,2015,6(7):102-103.
[32]LEE CI,WU CC,HSIEH SL,et al.Anticancer effects on human pancreatic cancer cells of triterpenoids,polysaccharides and 4,3-β-D-glucan derived from the fruiting body of Antrodia camphorata.Food and Function.2014,5(12):3224-3232.
[33]LEE YP,TSAI WC,KO CJ,et al.Anticancer effects of eleven triterpenoids derived from Antrodia camphorata.Anticancer Research.2012,32(7):2727-2734.
[34]周彦娇,张勇,姚智.活性氧在线粒体凋亡信号传导中的调节作用[J].中国运动医学杂志,2005,24(6):746-750.
[35]马淇,刘垒,陈佺.活性氧、线粒体通透性转换与细胞凋亡[J].生物物理学报,2012,28(7):523-536.
[36]CAI Y,SHENG ZY,CHEN Y,et al.Effect of withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer[J].Asian Pacific Journal of Cancer Prevention:APJCP,2014,15(4):1711-1714.
[37]ALMAHLI H,HADCHITY E,JABALLAH MY,et al.Development of novel synthesized phthalazinone-based PARP-1 inhibitors with apoptosis inducing mechanism in lung cancer[J].Bioorganic Chemistry,2018,77:443-456.
[38]RAJAVEL T,PACKIYARAJ P,SURYANARAYANAN V,et al.β-Sitosterol targets Trx/Trxl reductase to induce apoptosis in A549 cells via ROS mediated mitochondrial dysregulation and p53 activation[J].Scientific Reports,2018,8(1):2071.