新牛蛙抗肿瘤肽RGD-T-La(FS)嵌合体的设计及其抗肿瘤作用
|
摘要
为了研究新牛蛙抗肿瘤肽RGD-嵌合体的抗肿瘤作用及其对肿瘤细胞的作用机制。本研究以新牛蛙抗菌肽Temporin-La(T-La)为基序,通过生物信息学分析,改变特定氨基酸残基设计合成新的抗肿瘤肽Temporin-La(S)(TLa(S))和Temporin-La(FS)(T-La(FS)),氨基端偶联RGD肽成RGD-T-La、RGD-T-La(S)和RGD-T-La(FS)抗肿瘤肽,通过圆二色谱检测多肽二级结构,MTT法体外筛选抗肿瘤细胞活性多肽。使用流式细胞仪测定不同肿瘤细胞对不同多肽的吸收量,筛选对抗肿瘤肽敏感的肿瘤细胞。利用激光共聚焦显微镜实时观察抗肿瘤活性强的抗肿瘤肽对敏感肿瘤细胞的杀伤作用,并用扫描电镜观察抗肿瘤肽及其RGD嵌合体肽对肿瘤细胞的作用机制。在新牛蛙抗菌肽Temporin-La基序上设计T-La(S)和T-La(FS)2种抗肿瘤活性肽,圆二色谱仪测定其二级结构均呈α螺旋型,MTT结果显示黑色素瘤细胞(B16)对几种多肽的敏感性最强,10 mg/L质量浓度时,RGD-T-La(FS)对B16的毒性作用最强,细胞存活率为24.65%。激光共聚焦显微镜实时观察到,RGD-La(FS)和RGD-La(S)对肿瘤细胞都有较强的杀伤作用。扫描电镜结果显示,RGD嵌合体多肽对肿瘤细胞的杀伤作用具有位点靶向性。流式细胞仪检测HepG2对FITC-RGD-T-La(FS)的吸收量最大,检测到荧光强度为800 950.70。经改造的多肽T-La(FS)可以增加其对肿瘤细胞的杀伤作用,且偶联RGD的嵌合体RGD-T-La(FS)对肿瘤细胞的杀伤作用更具有靶向专一性,为抗肿瘤肽作为抗肿瘤药物的临床应用提供科学依据。
In order to study the antitumor effect and the mechanism of the new synthetic bullfrog skin antibacterial peptide.With natural antibacterial peptide Temporin-La(T-La)as the motif,amino acid residues were changed to design and synthetize of new antitumor peptide Temporin-Ls(TLa(S))and Temporin-LS(T-La(FS)),then formed a coupling of RGD antitumor peptide RGD-TLa,RGD-T-La(S)and RGD-T-La(FS).The use of circular dichroism spectra chromatograph predicted the two level structure of them,and drawing 3 Dconstructional detail of the peptide by the PyMoL molecular graphics system.The cytotoxicity test(MTT)was used to select the polypeptide which has strong effect on the tumor cells.Flow cytometry was used to determine the absorption of different peptides different tumor cells.Then antineoplasmic activity of antitumor peptide was detected by confocal laser scanning microscopy,and action mechanism of killing tumor cells was observed by scanning electron microscope.Through circular dichroism spectra chromatograph determining,the secondary structure of T-La(S)and T-La(FS)was alpha helix.MTT results showed that melanoma cells(B16)were most sensitive to the peptides.At 10μg/mL concentration,RGD-T-La(FS)had the strongest toxicity to B16 cells,the survival rate was 24.65%.The killing effect of FITC-RGD-L(FS)on cells was larger and faster than that of FITC-RGD-L(S).The RGD chimeric peptide was more targeted against tumor cells and acted rapidly.HepG2 has a maximum absorption of FITC-RGD-T-La(FS)and the fluorescence intensity was 800950.70.Reformed peptide T-La(FS)increased its cytotoxic effect on tumor cells,and the coupling of RGD chimeric RGD-T-La(FS)on tumor cell killing effect had a more targeted specificity.It provided a basis for clinical application of antitumor peptide as antitumor drugs.
In order to study the antitumor effect and the mechanism of the new synthetic bullfrog skin antibacterial peptide.With natural antibacterial peptide Temporin-La(T-La)as the motif,amino acid residues were changed to design and synthetize of new antitumor peptide Temporin-Ls(TLa(S))and Temporin-LS(T-La(FS)),then formed a coupling of RGD antitumor peptide RGD-TLa,RGD-T-La(S)and RGD-T-La(FS).The use of circular dichroism spectra chromatograph predicted the two level structure of them,and drawing 3 Dconstructional detail of the peptide by the PyMoL molecular graphics system.The cytotoxicity test(MTT)was used to select the polypeptide which has strong effect on the tumor cells.Flow cytometry was used to determine the absorption of different peptides different tumor cells.Then antineoplasmic activity of antitumor peptide was detected by confocal laser scanning microscopy,and action mechanism of killing tumor cells was observed by scanning electron microscope.Through circular dichroism spectra chromatograph determining,the secondary structure of T-La(S)and T-La(FS)was alpha helix.MTT results showed that melanoma cells(B16)were most sensitive to the peptides.At 10μg/mL concentration,RGD-T-La(FS)had the strongest toxicity to B16 cells,the survival rate was 24.65%.The killing effect of FITC-RGD-L(FS)on cells was larger and faster than that of FITC-RGD-L(S).The RGD chimeric peptide was more targeted against tumor cells and acted rapidly.HepG2 has a maximum absorption of FITC-RGD-T-La(FS)and the fluorescence intensity was 800950.70.Reformed peptide T-La(FS)increased its cytotoxic effect on tumor cells,and the coupling of RGD chimeric RGD-T-La(FS)on tumor cell killing effect had a more targeted specificity.It provided a basis for clinical application of antitumor peptide as antitumor drugs.
引文
[1]BROWN K L,POON G F T,DARLENE B,et al.Host defense peptide LL-37selectively reduces proinflammatory macrophage responses[J].J Immunol,2011,186(9):5497-5505.
[2]徐鹏,张昊龙,刘芳宁,等.PRRSV N类特异性肽对CSF、PRRS免疫猪肺门淋巴结细胞因子分泌的影响[J].中国兽医学报,2017,37(6):1007-1011.
[3]俞玉萍.含RGD序列多肽靶向结合整合素αvβ3的分子基础研究[D].浙江杭州:浙江大学,2014.
[4]KEMP J A,SHIM M S,HEO C Y,et al.Combo nanomedicine:co-delivery of multi-modal therapeutics for efficient,targeted,and safe cancer therapy[J].Adv Drug Deliv Rev,2016,98:3.
[5]YANG Z Z,LI J Q,WANG Z Z,et al.Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas[J].Biomaterials,2014,35,5226-5239.
[6]刁昱文.肿瘤细胞靶向抗菌肽嵌合体的设计及其抗肿瘤活性机制研究[D].吉林长春:吉林大学,2012.
[7]HANCOCK R K,SAHL H G.Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies[J].Nat Biotechnol,2006,24(12):1551-1557.
[8]赵瑞利,韩俊友,韩文瑜,等.新牛蛙活性肽Catesbeianin-1a的抗菌抗肿瘤作用[J].中国兽医学报,2013,33(9):1407-1411.
[9]DANHIER F,BRETON A L,PRE’V.RGD-based strategies to target avb3integrin in cancer therapy and diagnosis[J].Mol Pharm,2012,9:2961-2973.
[10]ROBERTS P J,DER C J.Targeting the Raf-MEKERK mitogen-activated protein kinase cascade for the treatment of cancer[J].Oncogene,2007,26:3291-3310.
[11]HU G,ZHANG H,ZHANG L,et al.Integrin-mediated active tumor targeting and tumor microenvironment response dendrimer-gelatin nanoparticles for drug delivery and tumor treatment[J].Int J Pharm,2015,496:1057-1068.
[12]GAO H,YANG Z,CAO S,et al.Tumor cells and neovasculature dual targeting delivery for glioblastoma treatment[J].Biomaterials,2014,35:2374-2382.
[13]HOU J,DIAO Y,LI W,et al.,RGD peptide conjugation results in enhanced antitumor activity of PD0325901against glioblastoma by both tumor-targeting delivery and combination therapy[J].Int J Pharm,2016.505:329-340.
[14]朱琳,赵瑞利,刘珊珊,等.牛蛙凝集素生物学活性及其小鼠体内组织靶向性分布[J].中国兽医学报,2016,36(2):185-190.
[15]ROSENFELD Y,N LEV,SHAI Y.Effect of the hydrophobicity to net positive charge ratio on antibacterial and anti-endotoxin activities of structurally similar antimicrobial peptides[J].Biochemistry,2010,49(5):853-861.
[16]JIANG Z,A I VASIL,J HALE,et al.,Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides[J].Adv Exp Med Biol,2009,611:561-562.
[17]LAI Y,GALLO R L.AMPed up immunity:how antimicrobial peptides have multiple roles in immune defense[J].Trends Immunol,2009,30(3):131-141.
[18]REDDY K V,YEDERY R D,ARANHA C.Antimicrobial peptides:premises and promises[J].Int J Antimicrob Agents,2004,24(6):536-547.
[2]徐鹏,张昊龙,刘芳宁,等.PRRSV N类特异性肽对CSF、PRRS免疫猪肺门淋巴结细胞因子分泌的影响[J].中国兽医学报,2017,37(6):1007-1011.
[3]俞玉萍.含RGD序列多肽靶向结合整合素αvβ3的分子基础研究[D].浙江杭州:浙江大学,2014.
[4]KEMP J A,SHIM M S,HEO C Y,et al.Combo nanomedicine:co-delivery of multi-modal therapeutics for efficient,targeted,and safe cancer therapy[J].Adv Drug Deliv Rev,2016,98:3.
[5]YANG Z Z,LI J Q,WANG Z Z,et al.Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas[J].Biomaterials,2014,35,5226-5239.
[6]刁昱文.肿瘤细胞靶向抗菌肽嵌合体的设计及其抗肿瘤活性机制研究[D].吉林长春:吉林大学,2012.
[7]HANCOCK R K,SAHL H G.Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies[J].Nat Biotechnol,2006,24(12):1551-1557.
[8]赵瑞利,韩俊友,韩文瑜,等.新牛蛙活性肽Catesbeianin-1a的抗菌抗肿瘤作用[J].中国兽医学报,2013,33(9):1407-1411.
[9]DANHIER F,BRETON A L,PRE’V.RGD-based strategies to target avb3integrin in cancer therapy and diagnosis[J].Mol Pharm,2012,9:2961-2973.
[10]ROBERTS P J,DER C J.Targeting the Raf-MEKERK mitogen-activated protein kinase cascade for the treatment of cancer[J].Oncogene,2007,26:3291-3310.
[11]HU G,ZHANG H,ZHANG L,et al.Integrin-mediated active tumor targeting and tumor microenvironment response dendrimer-gelatin nanoparticles for drug delivery and tumor treatment[J].Int J Pharm,2015,496:1057-1068.
[12]GAO H,YANG Z,CAO S,et al.Tumor cells and neovasculature dual targeting delivery for glioblastoma treatment[J].Biomaterials,2014,35:2374-2382.
[13]HOU J,DIAO Y,LI W,et al.,RGD peptide conjugation results in enhanced antitumor activity of PD0325901against glioblastoma by both tumor-targeting delivery and combination therapy[J].Int J Pharm,2016.505:329-340.
[14]朱琳,赵瑞利,刘珊珊,等.牛蛙凝集素生物学活性及其小鼠体内组织靶向性分布[J].中国兽医学报,2016,36(2):185-190.
[15]ROSENFELD Y,N LEV,SHAI Y.Effect of the hydrophobicity to net positive charge ratio on antibacterial and anti-endotoxin activities of structurally similar antimicrobial peptides[J].Biochemistry,2010,49(5):853-861.
[16]JIANG Z,A I VASIL,J HALE,et al.,Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides[J].Adv Exp Med Biol,2009,611:561-562.
[17]LAI Y,GALLO R L.AMPed up immunity:how antimicrobial peptides have multiple roles in immune defense[J].Trends Immunol,2009,30(3):131-141.
[18]REDDY K V,YEDERY R D,ARANHA C.Antimicrobial peptides:premises and promises[J].Int J Antimicrob Agents,2004,24(6):536-547.