黄癸素（α-羟基-δ-癸酰乙基磺酸小檗碱）的抗肿瘤效应及其诱导细胞凋亡等作用的研究
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摘要
目的:探讨黄癸素的体内外抗肿瘤作用及其诱导细胞凋亡、分化等抗肿瘤作用机制。
方法:⑴应用MTT法检测黄癸素对体外培养的HepG2、MGC80-3、SW480等13株肿瘤细胞增殖的抑制作用。⑵应用小鼠移植性实体瘤H22、S180和裸鼠移植瘤Colon26的治疗试验观察黄癸素的体内抗肿瘤活性。⑶采用吸光度法观察黄癸素对B16细胞黑色素含量的影响,探讨其诱导细胞分化作用。⑷通过Rhodamine 123染色、琼脂糖凝胶电泳、Hoechst 33258染色、caspase3和caspase 8活性检测,观察黄癸素诱导细胞凋亡作用。⑸采用免疫组化法检测裸鼠Colon26移植瘤CD44V6的表达,并观察黄癸素的影响。
结果:黄癸素显著抑制体外培养HepG2、MGC80-3、SW480等肿瘤细胞的增殖;显著抑制小鼠H22、S180实体瘤的在体生长,对裸鼠移植瘤Colon26也有明显抑制作用;经黄癸素处理后的B16细胞黑色素生成能力增强,细胞趋向分化成熟;经黄癸素处理后,Rhodamine 123的荧光强度明显降低,琼脂糖凝胶电泳观察到DNA Ladder的出现,Hoechst 33258染色可观察到较高比例凋亡细胞,caspase3、caspase 8活性增强,显示黄癸素有促凋亡作用;黄癸素给药组Colon26移植瘤CD44V6表达较对照组降低,提示有抗肿瘤转移的可能。
结论:黄癸素有较强的体内外抗肿瘤活性,并能诱导B16细胞分化、凋亡,降低CD44V6表达。
Objective: To investigate the antitumor effects of berberineα-hydroxyβ-decanoylethyl sulfonate (HB), and its mechanism such as induction of cell apoptosis and differentiation.
Methods: 1) MTT assay was employed to determine the cytotoxicity of HB in HepG2,MGC80-3, SW480 and other cells. 2) HB was administrated to treat mouse transplanted solid tumors H22 and S180,Colon26(a human cancer)in nude mice, and the tumor growth was observed. 3) The difference of melanin content in B16 cells between HB treatment group and control group was determined by using absorption photometry. 4)Through Rhodamine 123 dyeing, agarose gel electrophoresis, Hoechst 33258 dyeing, active detection of caspase 3 and caspase 8, observe the cell apoptosis induced by HB. 5)CD44V6 expression of Colon26 tumors which was transplanted in nude mice was detected by immunohistochemical technigue, and the influence of HB was observed on it.
RESULT: HB significantly inhibits the proliferation of HepG2, MGC80-3, SW480 and other cells in vitro;inhibits the growth of H22 and S180 in mice, Colon26 tumors in nude mice. Melanin content of B16 cells increase after treatment with HB. The evidences of B16 cells apoptosis induced by HB were provided, such as Rhodamine 123 fluorescence intensity obviously decreased, DNA Ladder detected, nuclei pyknosed, caspase3 and caspase 8 activity increased. CD44V6 expression was decreased in Colon26 tumors which was treated with HB.
Conclusion: HB has significant antitumor activity in vitro & in vivo, and can induce differentiation and apoptosis of B16 cells, reduce CD44V6 expression in Colon26 tumors.
方法:⑴应用MTT法检测黄癸素对体外培养的HepG2、MGC80-3、SW480等13株肿瘤细胞增殖的抑制作用。⑵应用小鼠移植性实体瘤H22、S180和裸鼠移植瘤Colon26的治疗试验观察黄癸素的体内抗肿瘤活性。⑶采用吸光度法观察黄癸素对B16细胞黑色素含量的影响,探讨其诱导细胞分化作用。⑷通过Rhodamine 123染色、琼脂糖凝胶电泳、Hoechst 33258染色、caspase3和caspase 8活性检测,观察黄癸素诱导细胞凋亡作用。⑸采用免疫组化法检测裸鼠Colon26移植瘤CD44V6的表达,并观察黄癸素的影响。
结果:黄癸素显著抑制体外培养HepG2、MGC80-3、SW480等肿瘤细胞的增殖;显著抑制小鼠H22、S180实体瘤的在体生长,对裸鼠移植瘤Colon26也有明显抑制作用;经黄癸素处理后的B16细胞黑色素生成能力增强,细胞趋向分化成熟;经黄癸素处理后,Rhodamine 123的荧光强度明显降低,琼脂糖凝胶电泳观察到DNA Ladder的出现,Hoechst 33258染色可观察到较高比例凋亡细胞,caspase3、caspase 8活性增强,显示黄癸素有促凋亡作用;黄癸素给药组Colon26移植瘤CD44V6表达较对照组降低,提示有抗肿瘤转移的可能。
结论:黄癸素有较强的体内外抗肿瘤活性,并能诱导B16细胞分化、凋亡,降低CD44V6表达。
Objective: To investigate the antitumor effects of berberineα-hydroxyβ-decanoylethyl sulfonate (HB), and its mechanism such as induction of cell apoptosis and differentiation.
Methods: 1) MTT assay was employed to determine the cytotoxicity of HB in HepG2,MGC80-3, SW480 and other cells. 2) HB was administrated to treat mouse transplanted solid tumors H22 and S180,Colon26(a human cancer)in nude mice, and the tumor growth was observed. 3) The difference of melanin content in B16 cells between HB treatment group and control group was determined by using absorption photometry. 4)Through Rhodamine 123 dyeing, agarose gel electrophoresis, Hoechst 33258 dyeing, active detection of caspase 3 and caspase 8, observe the cell apoptosis induced by HB. 5)CD44V6 expression of Colon26 tumors which was transplanted in nude mice was detected by immunohistochemical technigue, and the influence of HB was observed on it.
RESULT: HB significantly inhibits the proliferation of HepG2, MGC80-3, SW480 and other cells in vitro;inhibits the growth of H22 and S180 in mice, Colon26 tumors in nude mice. Melanin content of B16 cells increase after treatment with HB. The evidences of B16 cells apoptosis induced by HB were provided, such as Rhodamine 123 fluorescence intensity obviously decreased, DNA Ladder detected, nuclei pyknosed, caspase3 and caspase 8 activity increased. CD44V6 expression was decreased in Colon26 tumors which was treated with HB.
Conclusion: HB has significant antitumor activity in vitro & in vivo, and can induce differentiation and apoptosis of B16 cells, reduce CD44V6 expression in Colon26 tumors.
引文
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[4]顾瑶华,朱悦,郝建华.鱼腥草研究进展[J].中国药业,2008,17(8):58-59.
[5]叶翩,张淑玲,揭盛华等.人肝癌裸鼠移植模型的研究进展[J].世界华人消化杂志,2006,14(36):3500-3503.
[6]李娜,高俊岩,刘敏.细胞凋亡和肿瘤的关系研究进展[J].当代医学,2009,15(16):13-14.
[7]李娜,胡玉红,孙盛梅等.小檗碱诱导肿瘤细胞凋亡的研究进展[J].黑龙江医药科学,2007,30(1):102-103.
[8]金鸣,董宁宁,吴伟等.羟基红花黄色素A缓解大鼠心肌细胞凋亡作用的研究[J].中草药,2009,40(6):924-930.
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[10]韩阳,官大威,侯震寰等.Caspase-8及其研究进展[J].中国法医学杂志,2006,21(2):94-96.
[11]王盛兰,钟延丰,管增伟等.电镜和荧光显微技术在细胞凋亡研究中的应用[J].北京大学学报(医学版),2003,35(1):91-93.
[12]牛建昭,魏育林,曹炜.细胞坏死和凋亡(Apoptosis)DNA状态的比较[J].解剖学报,1995,26(2):142-145.
[13]王涛,金戈,李江滨等.肿瘤细胞诱导分化研究进展[J].河南肿瘤学杂志,2002,15(2):154-156.
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[2]Li B,Zhu WL,Chen KX.Advances in the study of berberine and its derivatives[J].Acta Pharmaceutica Sinica 2008,43(8):773-787.
[3]杨菁,林菁.小檗碱抗肿瘤作用机制的研究进展[J].中国中药杂志,2002,68:277-281.
[4]顾瑶华,朱悦,郝建华.鱼腥草研究进展[J].中国药业,2008,17(8):58-59.
[5]叶翩,张淑玲,揭盛华等.人肝癌裸鼠移植模型的研究进展[J].世界华人消化杂志,2006,14(36):3500-3503.
[6]李娜,高俊岩,刘敏.细胞凋亡和肿瘤的关系研究进展[J].当代医学,2009,15(16):13-14.
[7]李娜,胡玉红,孙盛梅等.小檗碱诱导肿瘤细胞凋亡的研究进展[J].黑龙江医药科学,2007,30(1):102-103.
[8]金鸣,董宁宁,吴伟等.羟基红花黄色素A缓解大鼠心肌细胞凋亡作用的研究[J].中草药,2009,40(6):924-930.
[9]陈剑,宫兆华,王季石.Caspase-3在凋亡中的作用及在恶性血液病中的研究进展[J].国外医学输血及血液学分册,2001,24(3):207-209.
[10]韩阳,官大威,侯震寰等.Caspase-8及其研究进展[J].中国法医学杂志,2006,21(2):94-96.
[11]王盛兰,钟延丰,管增伟等.电镜和荧光显微技术在细胞凋亡研究中的应用[J].北京大学学报(医学版),2003,35(1):91-93.
[12]牛建昭,魏育林,曹炜.细胞坏死和凋亡(Apoptosis)DNA状态的比较[J].解剖学报,1995,26(2):142-145.
[13]王涛,金戈,李江滨等.肿瘤细胞诱导分化研究进展[J].河南肿瘤学杂志,2002,15(2):154-156.
[14]王志红,林菁.盐酸小檗碱对HL-60细胞的诱导分化及其作用机制[J].中国药科大学学报,2006,37(2):165-168.
[15]张秀亮,朱志图,哈敏文. CD44V6与肿瘤侵袭和转移研究进展[J].医学综述, 2008,14(8):1162-1165.
[1]Iwasa Kinuko,Nanba H,Lee DU,et al. Structure activity relationships of protoberberines having antimicrobial activity[J].Planta Med,1998,64: 748-751.
[2]Hong SW,Kim SH,Jeun JA,et al. Antimicrobial activity of 9-O-acyl- and 9-O-benzyl-substituted berberrubines[J].Planta Med,2000,66:361-363.
[3]Kim SH,Lee SJ,Lee JH,et al. Anti microbial activity of 9-O-acyl- and 9-O-alkylberberrubine derivatives[J].Planta Med,2002,68:277-281.
[4]Zheng HY. Experimental and Theoretical Studies of Protoberberine Alkaloids on the Mechanism of Multiple Activities[D].Tianjing:Tianjing Medical College,2004.
[5]Yang Y,Ye XL,Li XG,et al. Synthesis and antimicrobial activity of 8-alkylberberine derivatives with a long aliphatic chain[J].Planta Med, 2007,73(6):602-604.
[6]李耐三,汪海峰.13-已基小檗碱盐的制备及其抗病毒和抗菌作用:中国,200310112632.9[P]. 2005-06-22.
[7]李学刚,叶小利,袁吕江.抗结核杆菌药物3-烷氧基-8-烷基-12-R3-药根碱盐的合成方法及应用:中国,200810232837.3[P].2009-02-18.
[8]张立新,孙诺.小檗碱及其结构类似物在逆转多药耐药泵中的应用:中国,200810112350.1[P]. 2008-11-12.
[9]Iwasa K,Nishiyama Y,Ichimaru M,et al.Structure activity relationships of quaternary protoberberine alkaloids having antimalarial activity[J]. Eur J MedChem,1999,34:1077-1083.
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