冬凌草甲素诱导人多发性骨髓瘤H929细胞增殖和凋亡实验研究
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摘要
目的:观察冬凌草甲素(oridonin,ORI)对人骨髓瘤H929细胞株增殖、凋亡的影响,探讨其可能的作用机制。方法:分别用ORI 0、4、8、12、16、20、24、28、32μmol/L处理H929细胞12、24和36 h,应用四甲基偶氮唑蓝(MTT)检测H929细胞增殖抑制率,TUNEL法和荧光显微镜检观察Here29细胞形态变化及凋亡,AnnexinV-FITC/PI双集流式细胞术检测细胞凋亡率,蛋白印迹技术(Western blot)检测BAX、BCL-2、caspase-3、PARP、p38、JNK、ERK以及PI3K、Akt的表达变化。结果:ORI 8-16μmol/L体外显著抑制H929细胞增殖,并与浓度及时间呈正相关(P<0.05,P<0.05;r=0.961,r=0.973)。TUNEL染色法检出细胞核固缩、核碎裂;流式细胞术显示,H929细胞凋亡率呈浓度依赖性递增(P<0.05,r=0.88);Western blot检测结果提示,pro-caspase-3、BCL-2、p-PI3K、p-Akt表达呈浓度依赖性递减(r=0.9861,r=0.9725,r=0.9413,r=0.9373),而BAX、Cleaved PARP及p-p38、p-JNK、p-ERK表达递增(r=0.9178, r=0.8877,r=0.882,r=0.9645,r=0.8623)。结论:一定浓度ORI在体外能有效抑制骨髓瘤细胞H929的增殖,并诱导细胞凋亡,其可能的分子机制与上调MAPK及下调PI3K/Akt信号通路相关蛋白有关。
Objective: To investigate the effects of oridonin(ORI) on the proliferation and apoptosis of human multiple myeloma cell line H929 and its possible mechanism. Methods: H929 cells were exposed to ORI 0、4、8、12、16、20、24、28、32 μmol/L for 12, 24 and 36 hours respectively. The prolifcration inhibitory effect of ORI on H929 cells was determined by MTT assay and then the working concentrations of ORI were determined. The morphological changes and apoptosis of H929 cells were observed by TUNEL(TdT-mediated dUTP Nick-End Labeling)and fluorescence microscopy. The apoptosis rate of H929 cells was detected by flow cytometry with Annexin V-FITC/PI staining. The protein expressions of pro-caspase-3, BCL-2,p-PI3 K, p-Akt, BAX, Cleaved PARP and p-JNK, p-ERK and p-p38 in H929 cells were detected by Western blot. Results: Compared with the control group, the proliferation of H929 cells treated with the ORI of 8-16 μmol/L was significantly inhibited and the apoptosis of H929 cells was obviously increased in dose-and time-dependent manners. As for morphological changes, the characteristics of apoptotic cells were presented in H929 cells treated with ORI for 24 hours. The protein levels of pro-caspase-3, BCL-2,p-PI3 K, p-Akt were down-regulated with increasing of ORI concentration(r=0.9861, r=0.9725, r=0.9413, r=0.9373), while the BAX,Cleaved PARP and p-JNK, p-ERK and p-p38 were up-regulated(r=0.9178, r=0.8877, r=0.882, r=0.9645, r=0.8623).Conclusion: The ORI possesses anti-myeloma effects, can inhibit the proliferation and induce the apoptosis of H929 cell line in vitro. Its potential mechanism may be related with up-regulating the MAPK and down-regulating the PI3K/Akt signal pathways.
Objective: To investigate the effects of oridonin(ORI) on the proliferation and apoptosis of human multiple myeloma cell line H929 and its possible mechanism. Methods: H929 cells were exposed to ORI 0、4、8、12、16、20、24、28、32 μmol/L for 12, 24 and 36 hours respectively. The prolifcration inhibitory effect of ORI on H929 cells was determined by MTT assay and then the working concentrations of ORI were determined. The morphological changes and apoptosis of H929 cells were observed by TUNEL(TdT-mediated dUTP Nick-End Labeling)and fluorescence microscopy. The apoptosis rate of H929 cells was detected by flow cytometry with Annexin V-FITC/PI staining. The protein expressions of pro-caspase-3, BCL-2,p-PI3 K, p-Akt, BAX, Cleaved PARP and p-JNK, p-ERK and p-p38 in H929 cells were detected by Western blot. Results: Compared with the control group, the proliferation of H929 cells treated with the ORI of 8-16 μmol/L was significantly inhibited and the apoptosis of H929 cells was obviously increased in dose-and time-dependent manners. As for morphological changes, the characteristics of apoptotic cells were presented in H929 cells treated with ORI for 24 hours. The protein levels of pro-caspase-3, BCL-2,p-PI3 K, p-Akt were down-regulated with increasing of ORI concentration(r=0.9861, r=0.9725, r=0.9413, r=0.9373), while the BAX,Cleaved PARP and p-JNK, p-ERK and p-p38 were up-regulated(r=0.9178, r=0.8877, r=0.882, r=0.9645, r=0.8623).Conclusion: The ORI possesses anti-myeloma effects, can inhibit the proliferation and induce the apoptosis of H929 cell line in vitro. Its potential mechanism may be related with up-regulating the MAPK and down-regulating the PI3K/Akt signal pathways.
引文
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9 Bai Q,Zhang X.Curcumin enhances cytotoxic effects of bortezomib in human multiple myeloma H929 cells:potential roles of NF-κB/JNK.Int J Mol Sci,2012;13(4):4831-4838.
10 Xu J,Pfarr N,Endris V,et al.Molecular signaling in multiple myeloma:association of RAS/RAF mutations and MEK/ERKpathway activation.Oncogenesis,2017;6(5):e337.
11 Xie B,Xu Z,Hu L,et al.Pterostilbene inhibits human multiple myeloma cells via ERK1/2 and JNK pathway in vitro and In vivo.Int J Mol Sci,2016;17(11):e1927.
12 Liu F,Yang X,Geng M,et al.Targeting ERK,an achilles'heel of the MAPK pathway,in cancer therapy.Acta Pharm Sin B,2018;8(4):552-562.
13 Ikeda H,Hideshima T,Fulciniti M,et al.PI3K/p110 is a novel therapeutic target in multiple myeloma.Blood,2010;116(9):1460-1468.
14 Zhu J,Wang M,Cao B,et al.Targeting the phosphatidylinositol3-kinase/AKT pathway for the treatment of multiple myeloma.Curr Med Chem,2014;(21):3173-3187.
15 Song M,Liu X,Liu K,et al.Targeting AKT with oridonin Inhibits growth of esophageal squamous cell carcinoma in vitro and patientderived xenografts in vivo.Mol Cancer Ther,2018;17(7):1540-1553.
2 Zollinger A,Stuhmer T,Chatterjee M,et al.Combined functional and molecular analysis of tumor cell signaling defines 2 distinct myeloma subgroups:Akt-dependent and Akt-independent multiple myeloma.Blood,2008;112(8):3403-3411.
3 Ramakrishnan V,Kumar S.PI3K/AKT/m TOR pathway in multiple myeloma:from basic biology to clinical promise.Leuk Lymphoma,2018;59(3):1-11.
4 Zhou G,Kang H,Wang L,et al.Oridonin,a diterpenoid extracted from medicinal herbs,targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t(8;21)leukemia in vitro and in vivo.Blood,2007;109(8):3441-3450.
5 Tsubaki M,Takeda T,Ogawa N,et al.Overexpression of survivin via activation of ERK1/2,Akt,and NF-κB plays a central role in vincristine resistance in multiple myeloma cells.Leuk Res,2015;39(4):445-452.
6 Munoz-Pinedo C,Guio-Carrion A,Goldstein JC,et al.Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration.Proc Natl Acad Sci U S A,2006;103(31):11573-11578.
7 Hu J,Hu WX.Targeting signaling pathways in multiple myelomapathogenesis and implication for treatments.Cancer Lett,2018;414(2):214-221.
8 Park WH,Seol JG,Kim ES,et al.Induction of apoptosis by vitamin D3 analogue EB1089 in NCI-H929 myeloma cells via activation of caspase 3 and p38 MAP kinase.Br J Haematol,2000;109(3):576-583.
9 Bai Q,Zhang X.Curcumin enhances cytotoxic effects of bortezomib in human multiple myeloma H929 cells:potential roles of NF-κB/JNK.Int J Mol Sci,2012;13(4):4831-4838.
10 Xu J,Pfarr N,Endris V,et al.Molecular signaling in multiple myeloma:association of RAS/RAF mutations and MEK/ERKpathway activation.Oncogenesis,2017;6(5):e337.
11 Xie B,Xu Z,Hu L,et al.Pterostilbene inhibits human multiple myeloma cells via ERK1/2 and JNK pathway in vitro and In vivo.Int J Mol Sci,2016;17(11):e1927.
12 Liu F,Yang X,Geng M,et al.Targeting ERK,an achilles'heel of the MAPK pathway,in cancer therapy.Acta Pharm Sin B,2018;8(4):552-562.
13 Ikeda H,Hideshima T,Fulciniti M,et al.PI3K/p110 is a novel therapeutic target in multiple myeloma.Blood,2010;116(9):1460-1468.
14 Zhu J,Wang M,Cao B,et al.Targeting the phosphatidylinositol3-kinase/AKT pathway for the treatment of multiple myeloma.Curr Med Chem,2014;(21):3173-3187.
15 Song M,Liu X,Liu K,et al.Targeting AKT with oridonin Inhibits growth of esophageal squamous cell carcinoma in vitro and patientderived xenografts in vivo.Mol Cancer Ther,2018;17(7):1540-1553.