吴茱萸碱抑制人骨肉瘤HOS细胞的增殖并促进其凋亡
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摘要
目的探讨吴茱萸碱对骨肉瘤HOS细胞株增殖凋亡的影响及其可能的机制。方法通过利用0、1、2、4、8μmol/L浓度吴茱萸碱处理HOS细胞24、48 h后,利用CCK-8法检测HOS细胞活性。用3μmol/L的吴茱萸碱处理HOS细0、24、48 h后,利用细胞凋亡-Hoechst染色试剂盒染色观察HOS细胞细胞核染色质的形态。用3μmol/L吴茱萸碱处理0、24、48 h后,利用流式细胞仪检测HOS细胞的凋亡率。3μmol/L吴茱萸碱处理0、24、48 h后,Westernblot检测HOS细胞内Caspase 3、Bcl-2蛋白的表达变化情况。结果 2~8μmol/L的吴茱萸碱可抑制HOS细胞的细胞活性,抑制其增殖,呈剂量-时间依赖性。8μmol/L处理48 h后细胞存活率为0.453±0.071,与对照组比较,差异有统计学意义(P<0.01)。Hoechst-33258染色观察可见凋亡细胞染色质颜色发白,呈固缩状或者碎裂状染色质,染色质着色不均匀,核形态各异。流式细胞仪检测3μmol/L吴茱萸碱处理HOS细胞0、24、48 h后的凋亡率分别为(5.32±1.62)%、(10.85±1.49)%和(12.47±0.59)%,与对照组比较,差异有统计学意义(P<0.01)。吴茱萸碱可上调HOS细胞内的Caspase 3蛋白的表达,同时下调Bcl-2蛋白的表达,与对照组相比较,差异有统计学意义(P<0.05)。结论吴茱萸碱可降低人骨肉瘤HOS细胞的细胞活性,抑制其体外增殖,诱导其发生凋亡,其机制可能与其上调Caspase 3蛋白的表达,下调Bcl-2蛋白的表达有关。
Objective To study the effects of Evodiamine on proliferation and apoptosis of osteosarcoma HOS cell line and its possible mechanisms,several cell experiments in vitro were performed. Methods HOS cells were treated with0,1,2,4,and 8 μmol/L concentrations of Evodiamine for 24 and 48 h,and HOS cell activity was measured by CCK-8 method. HOS cells were treated with 3μmol/L Evodiamine for 0,24,and 48 h,and the morphology of nuclear chromatin in HOS cells was observed by apoptosis-Hoechst staining kit. After treatment with 3 μmol/L Evodiamine for 0,24,and 48 h,the apoptosis rate of HOS cells was detected by flow cytometry. After treatment with 3 μmol/L Evodiamine for 0,24 and48 h,the expression of Caspase 3 and Bcl-2 protein in HOS cells was detected by Western-blot. Results 2~8 μmol/L of Evodiamine inhibited the cell viability of HOS cells and inhibited their proliferation in a dose-time dependent manner. The cell viability was only(0.453±0.071)after 48 h of treatment at 8 μmol/L. Compared with the control group,the difference was statistically significant(P<0.01). Hoechst-33258 staining showed that the chromatin color of apoptotic cells was white,showing condensed or fragmented chromatin,chromatin staining uneven,and nuclear morphology. Flow cytometry showed that the apoptosis rates of HOS cells treated with 3μmol/L Evodiamine at 0,24,and 48 h were(5.32±1.62)%,(10.85±1.49)%,and(12.47±0.59)%,respectively. Compared with the control group,the difference was statistically significant(P<0.01).Evodiamine can up-regulate the expression of Caspase 3 protein in HOS cells and down-regulate the expression of Bcl-2 protein. Compared with the control group,the difference was statistically significant(P<0.05). Conclusion Evodiamine can decrease the cell viability of human osteosarcoma HOS cells,inhibit its proliferation in vitro,and induce apoptosis. The mechanism may be related to up-regulation of Caspase 3 protein expression and down-regulation of Bcl-2 protein expression.
Objective To study the effects of Evodiamine on proliferation and apoptosis of osteosarcoma HOS cell line and its possible mechanisms,several cell experiments in vitro were performed. Methods HOS cells were treated with0,1,2,4,and 8 μmol/L concentrations of Evodiamine for 24 and 48 h,and HOS cell activity was measured by CCK-8 method. HOS cells were treated with 3μmol/L Evodiamine for 0,24,and 48 h,and the morphology of nuclear chromatin in HOS cells was observed by apoptosis-Hoechst staining kit. After treatment with 3 μmol/L Evodiamine for 0,24,and 48 h,the apoptosis rate of HOS cells was detected by flow cytometry. After treatment with 3 μmol/L Evodiamine for 0,24 and48 h,the expression of Caspase 3 and Bcl-2 protein in HOS cells was detected by Western-blot. Results 2~8 μmol/L of Evodiamine inhibited the cell viability of HOS cells and inhibited their proliferation in a dose-time dependent manner. The cell viability was only(0.453±0.071)after 48 h of treatment at 8 μmol/L. Compared with the control group,the difference was statistically significant(P<0.01). Hoechst-33258 staining showed that the chromatin color of apoptotic cells was white,showing condensed or fragmented chromatin,chromatin staining uneven,and nuclear morphology. Flow cytometry showed that the apoptosis rates of HOS cells treated with 3μmol/L Evodiamine at 0,24,and 48 h were(5.32±1.62)%,(10.85±1.49)%,and(12.47±0.59)%,respectively. Compared with the control group,the difference was statistically significant(P<0.01).Evodiamine can up-regulate the expression of Caspase 3 protein in HOS cells and down-regulate the expression of Bcl-2 protein. Compared with the control group,the difference was statistically significant(P<0.05). Conclusion Evodiamine can decrease the cell viability of human osteosarcoma HOS cells,inhibit its proliferation in vitro,and induce apoptosis. The mechanism may be related to up-regulation of Caspase 3 protein expression and down-regulation of Bcl-2 protein expression.
引文
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[10]Chiou WF,Chou CJ,Shum AYC,et al. The vasorelaxant effect of evodiamine in rat isolated mesenteric arteries:mode of action[J]. Eur J Pharmacol,1992,215(2-3):277-283.
[11]Hu Y,Fahmy H,Zjawiony JK,et al. Inhibitory effect and transcriptional impact of berberine and evodiamine on human white preadipocyte differentiation[J]. Fitoterapia,2010,81(4):259-268.
[12]裘杨溢,徐盛涛,徐进宜.吴茱萸碱类衍生物的生物活性研究进展[J].药学与临床研究,2017,5(3):221-226.
[13]宋英,盛蓉,李娟,等.吴茱萸碱治疗痛风的药效学研究[J].中药药理与临床,2011,27(6):17-19.
[14]Huang YC,Guh JH,Teng CM. Induction of mitotic arrest and apoptosis by evodiamine in human leukemicT-lymphocytes[J].LifeSci,2004,75(1):35-49.
[15]Liao CH,Pan SL,Guh JH,et al. Antitumor mechanism of evodiamine, a constituent from Chinese herb Evodiae fructus,in human multiple-drug resistant breast cancer NCI/ADR-RES cells in vitro and in vivo[J]. Carcinogenesis,2005,26(5):968-975.
[16]Wang C,Wang MW,Tashiro S,et al. Evodiamine induced human melanoma A375-S2 cell death partially through interleukin 1 mediated pathway[J].Chinese Pharmacological Bulletin,2005. 28(6):984-9.
[17]农丽,伍钢,戴晓芳,等.吴茱萸碱逆转人肺癌细胞株A549/DDP耐药机理的实验研究[J].临床肿瘤学杂志,2010,15(6):487-492.
[18]张辉,吕家驹,丁森泰,等.吴茱萸碱诱导前列腺癌PC-3细胞凋亡机制的实验研究[J].中国老年学,2007,27(13):1230-1231.
[19]Smeland SR,Bruland OS,Hjorth L,et al. Results of the Scandinavian Sarcoma Group XIV protocol for classical osteosarcoma[J]. Acta Orthopaedica,2011,82(2):211-216.
[20]Holmboe L,Andersen AM,Morkrik L,et al. High dose methotrexate chemotherapy:pharmacokinetics,folate and toxicity in osteosarcoma patients[J]. Bri J Clin Pharmacol,2011,73(1):106-114.
[21]郑颖,王刚阳,陈瑞玲,等.骨肉瘤多药耐药的研究进展[J].现代生物医学进展,2017,17(33):6570-6573.
[2] Ferrari S,Ruggieri P,Cefalo G,et al. Neoadjuvant chemotherapy with methotrexate,cisplatin,and doxorubicin with or without ifosfamide in nonmetastatic osteosarcoma of the extremity:An Italian Sarcoma Group Trial ISG/OS-1[J]. J Clin Oncol,2012,30(17):2112-2118.
[3] Dean DC,Shen S,Hornicek FJ,et al. From genomics to metabolomics:emerging metastatic biomarkers in osteosarcoma[J]. Cancer Metastasis Rev,2018,37(4):719-731.
[4] Isakoff MS,Bielack SS,Meltzer P,et al. Osteosarcoma:Current treatment and a collaborative pathway to success[J]. Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology,2015,33(27):3029-3035.
[5] Thompson PA,Chintagumpala M. Targeted therapy in bone and soft tissue sarcoma in children and adolescents[J]. Curr Oncol Rep,2012,14(2):197-205.
[6] van Dalen EC,van der Pal HJ,Kremer LC,et al.Different dosage schedules for reducing cardiotoxicity in people with cancer receiving anthracycline chemotherapy[J]. Cochrane Database of Systematic Reviews,2009,6(4):CD005008.
[7] Zhang W,Zhang Q,Zheng TT,et al. Delayed high-dose methotrexate excretion and influencing factors in osteosarcoma patients[J]. Chin Med J,2016,129(21):2530-2534.
[8] Zhang Y,Yang J,Zhao N,et al. Progress in the chemotherapeutic treatment of osteosarcoma[J]. Oncol Lett,2018,16(5):6228-6237.
[9] Morrow JJ,Khanna C. Osteosarcoma genetics and epigenetics:emerging biology and candidate therapies[J]. Critical Reviews in Oncogenesis,2015,20(3-4):173.
[10]Chiou WF,Chou CJ,Shum AYC,et al. The vasorelaxant effect of evodiamine in rat isolated mesenteric arteries:mode of action[J]. Eur J Pharmacol,1992,215(2-3):277-283.
[11]Hu Y,Fahmy H,Zjawiony JK,et al. Inhibitory effect and transcriptional impact of berberine and evodiamine on human white preadipocyte differentiation[J]. Fitoterapia,2010,81(4):259-268.
[12]裘杨溢,徐盛涛,徐进宜.吴茱萸碱类衍生物的生物活性研究进展[J].药学与临床研究,2017,5(3):221-226.
[13]宋英,盛蓉,李娟,等.吴茱萸碱治疗痛风的药效学研究[J].中药药理与临床,2011,27(6):17-19.
[14]Huang YC,Guh JH,Teng CM. Induction of mitotic arrest and apoptosis by evodiamine in human leukemicT-lymphocytes[J].LifeSci,2004,75(1):35-49.
[15]Liao CH,Pan SL,Guh JH,et al. Antitumor mechanism of evodiamine, a constituent from Chinese herb Evodiae fructus,in human multiple-drug resistant breast cancer NCI/ADR-RES cells in vitro and in vivo[J]. Carcinogenesis,2005,26(5):968-975.
[16]Wang C,Wang MW,Tashiro S,et al. Evodiamine induced human melanoma A375-S2 cell death partially through interleukin 1 mediated pathway[J].Chinese Pharmacological Bulletin,2005. 28(6):984-9.
[17]农丽,伍钢,戴晓芳,等.吴茱萸碱逆转人肺癌细胞株A549/DDP耐药机理的实验研究[J].临床肿瘤学杂志,2010,15(6):487-492.
[18]张辉,吕家驹,丁森泰,等.吴茱萸碱诱导前列腺癌PC-3细胞凋亡机制的实验研究[J].中国老年学,2007,27(13):1230-1231.
[19]Smeland SR,Bruland OS,Hjorth L,et al. Results of the Scandinavian Sarcoma Group XIV protocol for classical osteosarcoma[J]. Acta Orthopaedica,2011,82(2):211-216.
[20]Holmboe L,Andersen AM,Morkrik L,et al. High dose methotrexate chemotherapy:pharmacokinetics,folate and toxicity in osteosarcoma patients[J]. Bri J Clin Pharmacol,2011,73(1):106-114.
[21]郑颖,王刚阳,陈瑞玲,等.骨肉瘤多药耐药的研究进展[J].现代生物医学进展,2017,17(33):6570-6573.