灵芝免疫调节蛋白LZ-8及云芝免疫调节蛋白FIP-tvc对几种癌细胞系的抑制活性检测
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摘要
在体外培养的人非小细胞肺癌A549细胞、人胃腺癌BGC-823细胞、人结肠癌HCT116细胞、人肝癌Hep 3B细胞、人宫颈癌Hela细胞中加入不同浓度的酵母异源表达的LZ-8或FIP-tvc,培养24 h后显微镜下观察细胞形态变化,并用CCK8试剂盒检测细胞存活率,以用来检测灵芝免疫调节蛋白LZ-8及云芝免疫调节蛋白FIP-tvc对几种癌细胞生长抑制能力.结果显示,LZ-8和FIP-tvc在5～10μg/mL浓度时使A549、BGC-823、Hela和Hep 3B细胞贴壁性变差,且CCK8结果显示前3种细胞的存活率有不同程度的降低(80%～95%存活率),而在20μg/mL浓度时FIP-tvc对Hep 3B细胞的存活率开始有明显抑制效果(89%存活率),但对HCT 116细胞的形态无影响,对其存活率也无抑制作用.
In this paper,the morphological changes of human non-small cell lung cancer cell line A549,human gastric adenocarcinoma cell line BGC-823,human colon cancer cell line HCT 116,human liver cancer cell line Hep 3 B and human cervical carcinoma cell line Hela were observed after culturing with LZ-8 or FIP-tvc for 24 hours,and the viability of these cell lines was further analyzed with CCK8 kit to analyze the inhibitory effect of LZ-8 from Ganoderma lucidum and FIP-tvc from Trametes versicolor to these cancer cell lines. The results showed that the cell adherence of A549,BGC-823,Hela and Hep 3 B was significantly inhibited by LZ-8 and FIP-tvc at 5-10 μg/mL,and the viability of the first three cell lines was simultaneously inhibited and analyzed with CCK8 kit(80%-95%),but the inhibitory effect of FIP-tvc to the viability of Hep 3 B was appeared when the concentration was reached to20 μg/mL(89%). While morphology of HCT 116 was not changed and its viability was also not inhibited by LZ-8 or FIP-tvc at any test concentrations.
In this paper,the morphological changes of human non-small cell lung cancer cell line A549,human gastric adenocarcinoma cell line BGC-823,human colon cancer cell line HCT 116,human liver cancer cell line Hep 3 B and human cervical carcinoma cell line Hela were observed after culturing with LZ-8 or FIP-tvc for 24 hours,and the viability of these cell lines was further analyzed with CCK8 kit to analyze the inhibitory effect of LZ-8 from Ganoderma lucidum and FIP-tvc from Trametes versicolor to these cancer cell lines. The results showed that the cell adherence of A549,BGC-823,Hela and Hep 3 B was significantly inhibited by LZ-8 and FIP-tvc at 5-10 μg/mL,and the viability of the first three cell lines was simultaneously inhibited and analyzed with CCK8 kit(80%-95%),but the inhibitory effect of FIP-tvc to the viability of Hep 3 B was appeared when the concentration was reached to20 μg/mL(89%). While morphology of HCT 116 was not changed and its viability was also not inhibited by LZ-8 or FIP-tvc at any test concentrations.
引文
[1] TANAKA S,KO K,KINO K,et al. Complete amino acid sequence of an immunomodulatory protein,Ling Zhi-8(LZ-8). An immunomodulator from a fungus,Ganoderma lucidium,having similarity to immunoglobulin variable regions[J]. J Biol Chem,1989,264(28):16372-16377.
[2] KINO K,YAMASHITA A,YAMAOKA K,et al. Isolation and characterization of a new immunomodulatory protein,Ling Zhi-8(LZ-8),from Ganoderma lucidium[J]. J Biol Chem,1989,264(1):472-478.
[3] KO J L,HSU C I,LIN R H,et al. A new fungal immunomodulatory protein,FIP-fve isolated from the edible mushroom,Flammulina velutipes and its complete amino acid sequence[J]. Eur J Biochem,1995,228(2):244-249.
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[5] LINWH,HUNGCH,HSUCI,etal.DimerizationoftheN-terminalamphipathicalpha-helixdomainofthefungalimmunomodulatory protein from Ganoderma tsugae(FIP-gts)defined by a yeast two-hybrid system and site-directed mutagenesis[J]. J Biol Chem,1997,272(32):20044-20048.
[6] CHANG H H,YEH C H,SHEU F. A novel immunomodulatory protein from Poria cocos induces Toll-like receptor 4-dependent activation within mouse peritoneal macrophages[J]. J Agric Food Chem,2009,57(14):6129-6139.
[7] SHEU F,CHIEN P J,CHIEN A L,et al. Isolation and characterization of an immunomodulatory protein(APP)from the Jew’s Ear mushroom Auricularia polytricha[J]. Food Chemistry,2004,87(4):593-600.
[8] LIN C H,SHEU G T,LIN Y W,et al. A new immunomodulatory protein from Ganoderma microsporum inhibits epidermal growth factor mediated migration and invasion in A549 lung cancer cells[J]. Process Biochemistry,2010,45(9):1537-1542.
[9] ZHOU X W,XIE M Q,FAN H,et al. Genomic cloning and characterization of a FIP-gsi gene encoding a fungal immunomodulatory protein from Ganoderma sinense[J]. International Journal of Medicinal Mushrooms,2009,11(1):77-86.
[10] LI F,WEN H,ZHANG Y,et al. Purification and characterization of a novel immunomodulatory protein from the medicinal mushroom Trametes versicolor[J]. Sci China Life Sci,2011,54(4):379-385.
[11] LIN J W,GUAN S Y,DUAN Z W,et al. Gene cloning of a novel fungal immunomodulatory protein from Chroogomphis rutilus and its expression in Pichia pastoris[J]. Journal of Chemical Technology&Biotechnology,2016,91(11):2761-2768.
[12] XU H,KONG Y Y,CHEN X,et al. Recombinant FIP-gat,a fungal immunomodulatory protein from Ganoderma atrum induces growth inhibition and cell death in breast cancer cells[J]. Journal of Agricultural&Food Chemistry,2016,64(13):2690.
[13] LI S,JIANG Z,XU W,et al. FIP-sch2,a new fungal immunomodulatory protein from Stachybotrys chlorohalonata,suppresses proliferation and migration in lung cancer cells[J]. Appl Microbiol Biotechnol,2017,101(8):3227-3235.
[14] LI S,JIANG Z,SUN L,et al. Characterization of a new fungal immunomodulatory protein,FIP-dsq2 from Dichomitus squalens[J].J Biotechnol,2017,246(3):45-51.
[15] LI Q Z,WANG X F,ZHOU X W. Recent status and prospects of the fungal immunomodulatory protein family[J]. Crit Rev Biotechnol,2011,31(4):365-375.
[16] PAAVENTHAN P,JOSEPH J S,SEOW S V,et al. A 1.7A structure of Fve,a member of the new fungal immunomodulatory protein family[J]. J Mol Biol,2003,332(2):461-470.
[17] HUANG L,SUN F,LIANG C,et al. Crystal structure of LZ-8 from the medicinal fungus Ganoderma lucidium[J]. Proteins,2009,75(2):524-527.
[18] XUE Q,DING Y,SHANG C,et al. Functional expression of LZ-8,a fungal immunomodulatory protein from Ganoderma lucidium in Pichia pastoris[J]. J Gen Appl Microbiol,2008,54(6):393-398.
[19] CONG W R,XU H,LIU Y,et al. Production and functional characterization of a novel fungal immunomodulatory protein FIP-SN15 shuffled from two genes of Ganoderma species[J]. Appl Microbiol Biotechnol,2014,98(13):5967-5975.
[20] WU C T,LIN T Y,HSU H Y,et al. Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway[J]. Carcinogenesis,2011,32(12):1890-1896.
[21] HAAK-FRENDSCHO M,KINO K,SONE T,et al. Ling Zhi-8:a novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression[J]. Cellular Immunology,1993,150(1):101-113.
[22]许燕,胡明慧,高蓓蓉,等.灵芝真菌免疫调节蛋白在体外对不同胃癌细胞的抑制作用[J].药学实践杂志,2016,34(3):223-226,277.
[2] KINO K,YAMASHITA A,YAMAOKA K,et al. Isolation and characterization of a new immunomodulatory protein,Ling Zhi-8(LZ-8),from Ganoderma lucidium[J]. J Biol Chem,1989,264(1):472-478.
[3] KO J L,HSU C I,LIN R H,et al. A new fungal immunomodulatory protein,FIP-fve isolated from the edible mushroom,Flammulina velutipes and its complete amino acid sequence[J]. Eur J Biochem,1995,228(2):244-249.
[4] HSU H C,HSU C I,LIN R H,et al. Fip-vvo,a new fungal immunomodulatory protein isolated from Volvariella volvacea[J].Biochem J,1997,323(Pt 2):557-565.
[5] LINWH,HUNGCH,HSUCI,etal.DimerizationoftheN-terminalamphipathicalpha-helixdomainofthefungalimmunomodulatory protein from Ganoderma tsugae(FIP-gts)defined by a yeast two-hybrid system and site-directed mutagenesis[J]. J Biol Chem,1997,272(32):20044-20048.
[6] CHANG H H,YEH C H,SHEU F. A novel immunomodulatory protein from Poria cocos induces Toll-like receptor 4-dependent activation within mouse peritoneal macrophages[J]. J Agric Food Chem,2009,57(14):6129-6139.
[7] SHEU F,CHIEN P J,CHIEN A L,et al. Isolation and characterization of an immunomodulatory protein(APP)from the Jew’s Ear mushroom Auricularia polytricha[J]. Food Chemistry,2004,87(4):593-600.
[8] LIN C H,SHEU G T,LIN Y W,et al. A new immunomodulatory protein from Ganoderma microsporum inhibits epidermal growth factor mediated migration and invasion in A549 lung cancer cells[J]. Process Biochemistry,2010,45(9):1537-1542.
[9] ZHOU X W,XIE M Q,FAN H,et al. Genomic cloning and characterization of a FIP-gsi gene encoding a fungal immunomodulatory protein from Ganoderma sinense[J]. International Journal of Medicinal Mushrooms,2009,11(1):77-86.
[10] LI F,WEN H,ZHANG Y,et al. Purification and characterization of a novel immunomodulatory protein from the medicinal mushroom Trametes versicolor[J]. Sci China Life Sci,2011,54(4):379-385.
[11] LIN J W,GUAN S Y,DUAN Z W,et al. Gene cloning of a novel fungal immunomodulatory protein from Chroogomphis rutilus and its expression in Pichia pastoris[J]. Journal of Chemical Technology&Biotechnology,2016,91(11):2761-2768.
[12] XU H,KONG Y Y,CHEN X,et al. Recombinant FIP-gat,a fungal immunomodulatory protein from Ganoderma atrum induces growth inhibition and cell death in breast cancer cells[J]. Journal of Agricultural&Food Chemistry,2016,64(13):2690.
[13] LI S,JIANG Z,XU W,et al. FIP-sch2,a new fungal immunomodulatory protein from Stachybotrys chlorohalonata,suppresses proliferation and migration in lung cancer cells[J]. Appl Microbiol Biotechnol,2017,101(8):3227-3235.
[14] LI S,JIANG Z,SUN L,et al. Characterization of a new fungal immunomodulatory protein,FIP-dsq2 from Dichomitus squalens[J].J Biotechnol,2017,246(3):45-51.
[15] LI Q Z,WANG X F,ZHOU X W. Recent status and prospects of the fungal immunomodulatory protein family[J]. Crit Rev Biotechnol,2011,31(4):365-375.
[16] PAAVENTHAN P,JOSEPH J S,SEOW S V,et al. A 1.7A structure of Fve,a member of the new fungal immunomodulatory protein family[J]. J Mol Biol,2003,332(2):461-470.
[17] HUANG L,SUN F,LIANG C,et al. Crystal structure of LZ-8 from the medicinal fungus Ganoderma lucidium[J]. Proteins,2009,75(2):524-527.
[18] XUE Q,DING Y,SHANG C,et al. Functional expression of LZ-8,a fungal immunomodulatory protein from Ganoderma lucidium in Pichia pastoris[J]. J Gen Appl Microbiol,2008,54(6):393-398.
[19] CONG W R,XU H,LIU Y,et al. Production and functional characterization of a novel fungal immunomodulatory protein FIP-SN15 shuffled from two genes of Ganoderma species[J]. Appl Microbiol Biotechnol,2014,98(13):5967-5975.
[20] WU C T,LIN T Y,HSU H Y,et al. Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway[J]. Carcinogenesis,2011,32(12):1890-1896.
[21] HAAK-FRENDSCHO M,KINO K,SONE T,et al. Ling Zhi-8:a novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression[J]. Cellular Immunology,1993,150(1):101-113.
[22]许燕,胡明慧,高蓓蓉,等.灵芝真菌免疫调节蛋白在体外对不同胃癌细胞的抑制作用[J].药学实践杂志,2016,34(3):223-226,277.