藤黄酸诱导恶性血液病细胞株凋亡及机理研究
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摘要
[目的]
探讨藤黄酸对多种恶性血液病细胞株的作用和机制,以及藤黄酸对正常免疫细胞及造血干细胞功能的影响。
[方法]
以不同浓度(0、0.065、0.125、0.25、0.5、1.0、2.0、4.0μg/ml)藤黄酸处理Jurkat、Raji、RPMI8226、U266、HL-60、K562、K562/A02细胞株24h-72h,四甲基偶氮唑盐(MTT)法检测藤黄酸对7种细胞株的增殖抑制作用；碘化丙啶(PI)染色分析检测细胞周期改变；采用Annexin V/PI双染法观察细胞形态并检测细胞的凋亡,JC-1染色法检测细胞线粒体跨膜电位(△Ψm)水平；流式细胞仪检测荧光素激活的细胞半胱氨酸蛋白酶Caspase3、Caspase 8、Caspase 9阳性细胞水平；流式微球技术检测慢性白血病BCR-ABL融合蛋白变化。采用MTT法检测藤黄酸对PHA及LPS诱导的健康人外周血中淋巴细胞增殖活性的影响,中性红染色法检测对健康人外周血中单核细胞吞噬功能的影响,CFU-GM半固体集落培养法检测对造血干细胞的集落形成能力的影响。
[结果]
1.藤黄酸体外对Jurkat、Raji、RPMI8226、U266、HL-60、K562、K562/A02等7种细胞株均具有不同程度的增殖抑制作用。K562／A02细胞(GA>2μg/ml)比k562细胞(GA>0.5μg/ml)需要更高的藤黄酸浓度才显示增殖抑制作用。藤黄酸对各种恶性血液病细胞株24h的IC50依次为：K562 2.23μg/ml; Raji 1.86μg/ml; Jurkat 1.35μg/ml; HL-60 0.68μg/ml; RPMI82260.52μg/ml; U2660.44μg/ml。藤黄酸0、0.5、1.0、2.0、4.0μg/ml浓度对其中K562、Jurkat、Raji细胞周期动态随访3天统计学未见明显差异。
2.藤黄酸作用于Jurkat、Raji、RPMI8226、U266、HL-60、K562等6种细胞株呈典型凋亡形态学改变。藤黄酸呈浓度依赖性增高上述所有细胞株的损伤线粒体跨膜电位(△Ψm)水平。藤黄酸2μg／ml作用24h、48h后,藤黄酸处理标本较对照标本激活的caspase 3、caspase 8、caspase 9阳性细胞比例均有不同程度幅度的升高。
3.2.0μg/ml藤黄酸处理K562细胞24h后细胞中的BCR-ABL融合蛋白水平下降。
4.藤黄酸体外对PHA诱导的正常淋巴细胞和LPS诱导的正常淋巴细胞的增殖有抑制作用且呈浓度依赖性。藤黄酸对正常单核细胞吞噬中性红染液的能力有抑制作用。藤黄酸对正常骨髓／外周血造血干细胞CFU-GM集落形成能力有抑制作用,低浓度范围时(<1μg/ml)对正常骨髓造血干细胞CFU-GM集落形成能力无影响。
[结论]
1.藤黄酸体外对多种恶性血液病细胞株具有不同程度的增殖抑制作用,其增殖抑制作用在部分细胞株并非通过特异的细胞周期阻滞来实现。
2.藤黄酸体外对淋巴瘤、骨髓瘤、白血病细胞株均有不同程度的诱导凋亡作用,机理是通过线粒体跨膜电位途径和胞浆激活途径诱导肿瘤细胞凋亡。
3.藤黄酸体外对免疫细胞功能有一定程度的抑制效应,藤黄酸对正常骨髓／外周血造血干细胞功能有不同程度的抑制作用,低浓度时对骨髓干细胞功能无影响。
[Objective]To investigate the apoptosis-inducing effect of Gambogic acid (GA) on malignant hematologic cell lines and its mechanisms, and the function-inhibiting effect of GA on normal human immunocytes and hematopoietic stem cells (HSCs).
[Methods] The Jurkat、Raji、RPMI8226、U266、HL-60、K562、K562/A02 cell lines were treated with GA at different concentrations for 24h-72h, the inhibition rates were detected by MTT assay. Cell morphology and apoptosis were measured by Annexin V/PI staining assay. Mitochondrial membrane potential (ΔΨm) was measured by JC assay. Activated Caspase 3、Caspase 8 and Caspase 9 level in above living cells were measured by Caspase-GloTM fluorescein staining kit. The GA effect on lymphocyte proliferation function was measured by MTT assay when lymphocytes were induced by PHA or LPS. Phagocytotic function of normal monocytes was measured by the intake dose of Neutral Red Dye. CFU-GM was assessed by semi-solid culture assay for determination of colony formation capacity of HSCs.
[Results]
1. Jurkat、Raji、RPMI8226、U266、HL-60、K562、562/A02 cells'growth were inhibited in a concentration dependent manner. K562/A02 cells needed higher GA concentration (>2μg/ml) to show antiproliferative effect compared with k562 cells (>0.5μg/ml). The IC50 of GA on K562 was 2.23μg/ml, Rajil.86μg/ml, Jurkatl.35μg/ml, HL-60 0.68μg/ml, RPMI8226 0.52μg/ml, U266 0.44μg/ml respectively。GA did not significantly influence the cell cycle of K562、urkat、Raji cell lines at the concentration of 0、0.5、1.0、2.0、4.0μg/ml for 24h-72h.
2. The typical apoptosis morphology change could be visualized after Jurkat、Raji、RPMI8226. U266. HL-60、K562 cell lines were incubated with GA. GA decreased the Mitochondrial membrane potential(ΔΨm) of all above cell lines in a concentration dependent manner. GA increased the apoptosis rate of all above cell lines and increased activated caspase 3, caspase 8, caspase 9 positive cell number for different degree in 24h and 48h.
3. After treatment with GA, the BCR-ABL protein level in K562 cells was decreased by Cytometric beads array.
4. GA could inhibit the proliferation of normal lymphocytes treated with PHA or LPS in concentration dependent manner. GA had negative effect on monocytes to ingest Neutral Red Dye, GA could inhibit the CFU-GM colony formation capacity of normal bone marrow/peripheral HSCs but at low concentration (<1μg/ml) the effect on that of normal bone marrow HSCs was not significant.
[Conclusion]
1. GA has inhibitive effect on various malignant hematologic cell lines'growth in vitro. The cell cycles are not disturbed obviously in some malignant hematologic cell lines.
2. GA induces apoptosis on some lymphoma, leukemia and multiple myeloma cell lines through both intrinsic and extrinsic pathways.
3. GA has negative effect on the proliferation of immunocytes and HSCs in vitro. The cytotoxic effect on the function of normal bone marrow HSCs is not found at low GA concentration.
探讨藤黄酸对多种恶性血液病细胞株的作用和机制,以及藤黄酸对正常免疫细胞及造血干细胞功能的影响。
[方法]
以不同浓度(0、0.065、0.125、0.25、0.5、1.0、2.0、4.0μg/ml)藤黄酸处理Jurkat、Raji、RPMI8226、U266、HL-60、K562、K562/A02细胞株24h-72h,四甲基偶氮唑盐(MTT)法检测藤黄酸对7种细胞株的增殖抑制作用；碘化丙啶(PI)染色分析检测细胞周期改变；采用Annexin V/PI双染法观察细胞形态并检测细胞的凋亡,JC-1染色法检测细胞线粒体跨膜电位(△Ψm)水平；流式细胞仪检测荧光素激活的细胞半胱氨酸蛋白酶Caspase3、Caspase 8、Caspase 9阳性细胞水平；流式微球技术检测慢性白血病BCR-ABL融合蛋白变化。采用MTT法检测藤黄酸对PHA及LPS诱导的健康人外周血中淋巴细胞增殖活性的影响,中性红染色法检测对健康人外周血中单核细胞吞噬功能的影响,CFU-GM半固体集落培养法检测对造血干细胞的集落形成能力的影响。
[结果]
1.藤黄酸体外对Jurkat、Raji、RPMI8226、U266、HL-60、K562、K562/A02等7种细胞株均具有不同程度的增殖抑制作用。K562／A02细胞(GA>2μg/ml)比k562细胞(GA>0.5μg/ml)需要更高的藤黄酸浓度才显示增殖抑制作用。藤黄酸对各种恶性血液病细胞株24h的IC50依次为：K562 2.23μg/ml; Raji 1.86μg/ml; Jurkat 1.35μg/ml; HL-60 0.68μg/ml; RPMI82260.52μg/ml; U2660.44μg/ml。藤黄酸0、0.5、1.0、2.0、4.0μg/ml浓度对其中K562、Jurkat、Raji细胞周期动态随访3天统计学未见明显差异。
2.藤黄酸作用于Jurkat、Raji、RPMI8226、U266、HL-60、K562等6种细胞株呈典型凋亡形态学改变。藤黄酸呈浓度依赖性增高上述所有细胞株的损伤线粒体跨膜电位(△Ψm)水平。藤黄酸2μg／ml作用24h、48h后,藤黄酸处理标本较对照标本激活的caspase 3、caspase 8、caspase 9阳性细胞比例均有不同程度幅度的升高。
3.2.0μg/ml藤黄酸处理K562细胞24h后细胞中的BCR-ABL融合蛋白水平下降。
4.藤黄酸体外对PHA诱导的正常淋巴细胞和LPS诱导的正常淋巴细胞的增殖有抑制作用且呈浓度依赖性。藤黄酸对正常单核细胞吞噬中性红染液的能力有抑制作用。藤黄酸对正常骨髓／外周血造血干细胞CFU-GM集落形成能力有抑制作用,低浓度范围时(<1μg/ml)对正常骨髓造血干细胞CFU-GM集落形成能力无影响。
[结论]
1.藤黄酸体外对多种恶性血液病细胞株具有不同程度的增殖抑制作用,其增殖抑制作用在部分细胞株并非通过特异的细胞周期阻滞来实现。
2.藤黄酸体外对淋巴瘤、骨髓瘤、白血病细胞株均有不同程度的诱导凋亡作用,机理是通过线粒体跨膜电位途径和胞浆激活途径诱导肿瘤细胞凋亡。
3.藤黄酸体外对免疫细胞功能有一定程度的抑制效应,藤黄酸对正常骨髓／外周血造血干细胞功能有不同程度的抑制作用,低浓度时对骨髓干细胞功能无影响。
[Objective]To investigate the apoptosis-inducing effect of Gambogic acid (GA) on malignant hematologic cell lines and its mechanisms, and the function-inhibiting effect of GA on normal human immunocytes and hematopoietic stem cells (HSCs).
[Methods] The Jurkat、Raji、RPMI8226、U266、HL-60、K562、K562/A02 cell lines were treated with GA at different concentrations for 24h-72h, the inhibition rates were detected by MTT assay. Cell morphology and apoptosis were measured by Annexin V/PI staining assay. Mitochondrial membrane potential (ΔΨm) was measured by JC assay. Activated Caspase 3、Caspase 8 and Caspase 9 level in above living cells were measured by Caspase-GloTM fluorescein staining kit. The GA effect on lymphocyte proliferation function was measured by MTT assay when lymphocytes were induced by PHA or LPS. Phagocytotic function of normal monocytes was measured by the intake dose of Neutral Red Dye. CFU-GM was assessed by semi-solid culture assay for determination of colony formation capacity of HSCs.
[Results]
1. Jurkat、Raji、RPMI8226、U266、HL-60、K562、562/A02 cells'growth were inhibited in a concentration dependent manner. K562/A02 cells needed higher GA concentration (>2μg/ml) to show antiproliferative effect compared with k562 cells (>0.5μg/ml). The IC50 of GA on K562 was 2.23μg/ml, Rajil.86μg/ml, Jurkatl.35μg/ml, HL-60 0.68μg/ml, RPMI8226 0.52μg/ml, U266 0.44μg/ml respectively。GA did not significantly influence the cell cycle of K562、urkat、Raji cell lines at the concentration of 0、0.5、1.0、2.0、4.0μg/ml for 24h-72h.
2. The typical apoptosis morphology change could be visualized after Jurkat、Raji、RPMI8226. U266. HL-60、K562 cell lines were incubated with GA. GA decreased the Mitochondrial membrane potential(ΔΨm) of all above cell lines in a concentration dependent manner. GA increased the apoptosis rate of all above cell lines and increased activated caspase 3, caspase 8, caspase 9 positive cell number for different degree in 24h and 48h.
3. After treatment with GA, the BCR-ABL protein level in K562 cells was decreased by Cytometric beads array.
4. GA could inhibit the proliferation of normal lymphocytes treated with PHA or LPS in concentration dependent manner. GA had negative effect on monocytes to ingest Neutral Red Dye, GA could inhibit the CFU-GM colony formation capacity of normal bone marrow/peripheral HSCs but at low concentration (<1μg/ml) the effect on that of normal bone marrow HSCs was not significant.
[Conclusion]
1. GA has inhibitive effect on various malignant hematologic cell lines'growth in vitro. The cell cycles are not disturbed obviously in some malignant hematologic cell lines.
2. GA induces apoptosis on some lymphoma, leukemia and multiple myeloma cell lines through both intrinsic and extrinsic pathways.
3. GA has negative effect on the proliferation of immunocytes and HSCs in vitro. The cytotoxic effect on the function of normal bone marrow HSCs is not found at low GA concentration.
引文
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14.崔国惠,舒文秀,吴青,等.藤黄酸对K562细胞Herg钾通道蛋白的调控作用[J].中草药,2009,40(6)：915-919.
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22. Han QB, Yan GL, Wang YL, et al. A Pair of Novel Cytotoxic Polyprenylated Xanthone Epimers from Gamboges[J]. Chem Biodiversity,2006,3(1):101-105.
23. Weber MM, Fottuer C, Liu SB, et al. Overexpression of the insulin-like growth factor I receptor in human colon carcinomas[J]. Cancer,2002,95(10):2086-2095.
24. Zhang HZ,Kasibhatla S,Wang Y, et al. Discovery, characterization and SAR of gambogic acid as a potent apoptosis inducer by a HTS assay[J]. Bioorg Med Chem,2004,12(2):309-317.
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2.刘宇,季宇彬.肿瘤细胞的凋亡及其分子机制研究[J].哈尔滨商业大学学报(自然科学版),2008,24(5)：524-527.
3.周艺群,谷志远.线粒体与细胞凋亡[J].解剖科学进展,2006,12(1)：60-62.
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5.李东,欧阳建,李翠萍,等.通关藤对U937细胞凋亡相关基因表达谱的影响[J].中国生化药物杂志,2008,27(4)：240-243.
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