蒲公英根水提物诱导MDA-MB-231细胞凋亡及其作用机制研究
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摘要
目的探究蒲公英根水提物对人三阴性乳腺癌细胞MDA-MB-231增殖和凋亡的影响及其作用机制。方法采用Alamar Blue法检测蒲公英根水提物的细胞毒性,以及对MDA-MB-231细胞增殖的抑制作用;通过形态学观察、DAPI染色、流式细胞术,检测蒲公英根水提物对MDA-MB-231细胞凋亡的影响;Western blot分析蒲公英根水提物诱导MDA-MB-231细胞凋亡的作用机制。结果蒲公英各部位提取物无细胞毒性;且7.5、15 g·L~(-1)的蒲公英根水提物明显抑制MDA-MB-231细胞的增殖,细胞出现明显的凋亡特征,流式细胞术进一步证实了蒲公英根水提物能够诱导MDA-MB-231细胞凋亡;蒲公英根水提物呈浓度依赖性降低Bcl-2的蛋白表达,增加Bax、p53、活性c-PARP、caspase-8、caspase-9和caspase-3表达量; caspase-8抑制剂(Z-IETD-FMK)和caspas-9抑制剂(Z-LEHD-FMK)能够逆转蒲公英水提物对MDA-MB-231细胞的增殖抑制活性。结论蒲公英根水提物对人三阴性乳腺癌细胞MDA-MB-231具有明显的抑制作用,对治疗三阴性乳腺癌具有潜在的临床应用价值。
Aim To discuss the effects of water extract of dandelion root on the proliferation and apoptosis of human triple negative breast cancer cell line MDA-MB-231 and its mechanism. Methods In this study, the cytotoxicity of dandelion root extract and the inhibition of MDA-MB-231 cell proliferation were detected by Alamar Blue method. Effects of water extracts of dandelion root on apoptosis of MDA-MB-231 cells were assessed by morphological observation, DAPI staining and flow cytometry; The mechanism of dandelion root water extract induced apoptosis of MDA-MB-231 cells was analysed by Western blot. Results Extracts from various parts of dandelion were not cytotoxic; furthermore, 7.5 and 15 g·L~(-1) water extract of dandelion significantly inhibited the proliferation of MDA-MB-231 cells, and the cells showed obvious apoptotic characteristics. Flow cytometry further confirmed the aqueous extract of dandelion root could induce the apoptosis of MDA-MB-231 cells. In terms of molecular mechanism, the water extract of dandelion root decreased the expression of Bcl-2 protein in a concentration-dependent manner, while the expressions of Bax, p53, active c-PARP, caspase-8, caspase-9 and caspase-3 increased. Caspase-8 inhibitor(Z-IETD-FMK) and caspase-9 inhibitor(Z-LEHD-FMK) were able to reverse the proliferation inhibitory activity of dandelion water extract on MDA-MB-231 cells. Conclusions The water extract of dandelion has a significant inhibitory effect on human triple negative breast cancer MDA-MB-231 cells, pointing to its potential clinical application value for the treatment of triple negative breast cancer.
Aim To discuss the effects of water extract of dandelion root on the proliferation and apoptosis of human triple negative breast cancer cell line MDA-MB-231 and its mechanism. Methods In this study, the cytotoxicity of dandelion root extract and the inhibition of MDA-MB-231 cell proliferation were detected by Alamar Blue method. Effects of water extracts of dandelion root on apoptosis of MDA-MB-231 cells were assessed by morphological observation, DAPI staining and flow cytometry; The mechanism of dandelion root water extract induced apoptosis of MDA-MB-231 cells was analysed by Western blot. Results Extracts from various parts of dandelion were not cytotoxic; furthermore, 7.5 and 15 g·L~(-1) water extract of dandelion significantly inhibited the proliferation of MDA-MB-231 cells, and the cells showed obvious apoptotic characteristics. Flow cytometry further confirmed the aqueous extract of dandelion root could induce the apoptosis of MDA-MB-231 cells. In terms of molecular mechanism, the water extract of dandelion root decreased the expression of Bcl-2 protein in a concentration-dependent manner, while the expressions of Bax, p53, active c-PARP, caspase-8, caspase-9 and caspase-3 increased. Caspase-8 inhibitor(Z-IETD-FMK) and caspase-9 inhibitor(Z-LEHD-FMK) were able to reverse the proliferation inhibitory activity of dandelion water extract on MDA-MB-231 cells. Conclusions The water extract of dandelion has a significant inhibitory effect on human triple negative breast cancer MDA-MB-231 cells, pointing to its potential clinical application value for the treatment of triple negative breast cancer.
引文
[1] Torre L A, Bray F, Siegel R L, et al. Global cancer statistic, 2012[J]. CA Cancer J Clin, 2015, 65(2):87-108.
[2] Kumar P, Aggarwal R. An overview of triple-negative breast cancer[J]. Arch Gynecol Obstet, 2016, 293(2):247-69.
[3] 胡人杰. 中药方剂抗肿瘤作用的药理学研究进展——动物模型建立与评价指标的选择[J]. 亚太传统医药, 2014,10(5):59-63.[3] Hu R J. Advances in pharmacological research on antitumor effects of traditional chinese medicine formulas——selection of animal models and evaluation indexes[J]. Asia-Pacific Tradit Med, 2014, 10(5): 59-63.
[4] Mingarro D M, Plaza A, Galán A, et al. The effect of five Taraxacum species on in vitro and in vivo antioxidant and antiproliferative activity [J]. Food Funct, 2015, 6(8):2787-2793.
[5] Ovadje P, Ammar S, Guerrero J A, et al. Dandelion root extract affects colorectal cancer proliferation and survival through the activation of multiple death signalling pathways[J]. Oncotarget, 2016, 7(45):73080-100.
[6] Ovadje P, Hamm C, Pandey S. Efficient induction of extrinsic cell death by Dandelion root extract in human chronic myelomonocytic leukemia (CMML) cells[J]. PLoS One, 2012, 7(2):e30604.
[7] Chungmu P, Hyunjoo Y, Chang H K, et al. TOP1 and 2, polysaccharides from Taraxacum officinale, attenuate CCl4-induced hepatic damage through the modulation of NF-κB and its regulatory mediators[J]. Food Chem Toxicol, 2010, 48(5): 1255-61.
[8] Jeon H J, Kang H J, Jung H J, et al. Anti-inflammatory activity of taraxacum officinale[J]. J Ethnopharmacol, 2008, 115(1): 82-8.
[9] Hudec J, Burdová M, Kobida L, et al. Antioxidant capacity changes and phenolic profile of Echinacea purpurea, nettle (Urtica dioica L.), and dandelion (Taraxacum officinale) after application of polyamine and phenolic biosynthesis regulators[J]. J Agric Food Chem, 2007, 55(14): 5689-96.
[10] Yang S, Liu J, Chen Y, et al. Reversal effect of Tween-20 on multidrug resistance in tumor cells in vitro[J]. Biomed Pharmacother, 2012, 66(3):187-94.
[11] Koo H N, Hong S H, Song B K, et al. Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in HepG2 cells[J]. Life Sci, 2004, 74(9):1149-57.
[12] 王婷婷,赵丽,李晓丹,等. 胡黄连苷Ⅱ对脑缺血损伤后神经细胞凋亡和超微结构的影响[J]. 中国药理学通报,2015,31(3):400-6.[12] Wang T T,Zhao L,Li X D,et al.Effect of picroside Ⅱ on neuronal apoptosis and ultrastructure in cerebral ischemic injury in rats[J]. Chin Pharmacol Bull,2015,31(3):400-6.
[13] 张红艳,翟丽,王婷婷,等.胡黄连苷Ⅱ通过抑制cyto C/caspase-9/caspase-3通路发挥神经保护作用[J]. 中国药理学通报, 2017,33(5):668-74.[13] Zhang H Y,Zhai L,Wang T T,et al. Picroside Ⅱ plays a neuroprotective effect by inhibiting cyto C/caspase-9/caspase-3 signal pathway following ischemia/reperfusion injury in rats[J]. Chin Pharmacol Bull,2017,33(5):668-74.
[14] 张贝贝,刘文洪,李俊峰,叶志青.铁皮石斛多糖对高糖诱导的血管内皮细胞Bax、Bcl-2表达的影响[J]. 中国药理学通报, 2015,31(1):64-70.[14] Zhang B B, Liu W H, Li J F,Ye Z Q.Effects of Dendrobium candidum polysaccharide on the expression of Bax and Bcl-2 in vascular endothelial cells induced by high glucose[J]. Chin Pharmacol Bull, 2015, 31(1):64-70.
[15] Schuler M, Green D R. Mechanisms of p53-dependent apoptosis[J]. Biochem Soc Trans, 2001, 29(6):684-8.
[2] Kumar P, Aggarwal R. An overview of triple-negative breast cancer[J]. Arch Gynecol Obstet, 2016, 293(2):247-69.
[3] 胡人杰. 中药方剂抗肿瘤作用的药理学研究进展——动物模型建立与评价指标的选择[J]. 亚太传统医药, 2014,10(5):59-63.[3] Hu R J. Advances in pharmacological research on antitumor effects of traditional chinese medicine formulas——selection of animal models and evaluation indexes[J]. Asia-Pacific Tradit Med, 2014, 10(5): 59-63.
[4] Mingarro D M, Plaza A, Galán A, et al. The effect of five Taraxacum species on in vitro and in vivo antioxidant and antiproliferative activity [J]. Food Funct, 2015, 6(8):2787-2793.
[5] Ovadje P, Ammar S, Guerrero J A, et al. Dandelion root extract affects colorectal cancer proliferation and survival through the activation of multiple death signalling pathways[J]. Oncotarget, 2016, 7(45):73080-100.
[6] Ovadje P, Hamm C, Pandey S. Efficient induction of extrinsic cell death by Dandelion root extract in human chronic myelomonocytic leukemia (CMML) cells[J]. PLoS One, 2012, 7(2):e30604.
[7] Chungmu P, Hyunjoo Y, Chang H K, et al. TOP1 and 2, polysaccharides from Taraxacum officinale, attenuate CCl4-induced hepatic damage through the modulation of NF-κB and its regulatory mediators[J]. Food Chem Toxicol, 2010, 48(5): 1255-61.
[8] Jeon H J, Kang H J, Jung H J, et al. Anti-inflammatory activity of taraxacum officinale[J]. J Ethnopharmacol, 2008, 115(1): 82-8.
[9] Hudec J, Burdová M, Kobida L, et al. Antioxidant capacity changes and phenolic profile of Echinacea purpurea, nettle (Urtica dioica L.), and dandelion (Taraxacum officinale) after application of polyamine and phenolic biosynthesis regulators[J]. J Agric Food Chem, 2007, 55(14): 5689-96.
[10] Yang S, Liu J, Chen Y, et al. Reversal effect of Tween-20 on multidrug resistance in tumor cells in vitro[J]. Biomed Pharmacother, 2012, 66(3):187-94.
[11] Koo H N, Hong S H, Song B K, et al. Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in HepG2 cells[J]. Life Sci, 2004, 74(9):1149-57.
[12] 王婷婷,赵丽,李晓丹,等. 胡黄连苷Ⅱ对脑缺血损伤后神经细胞凋亡和超微结构的影响[J]. 中国药理学通报,2015,31(3):400-6.[12] Wang T T,Zhao L,Li X D,et al.Effect of picroside Ⅱ on neuronal apoptosis and ultrastructure in cerebral ischemic injury in rats[J]. Chin Pharmacol Bull,2015,31(3):400-6.
[13] 张红艳,翟丽,王婷婷,等.胡黄连苷Ⅱ通过抑制cyto C/caspase-9/caspase-3通路发挥神经保护作用[J]. 中国药理学通报, 2017,33(5):668-74.[13] Zhang H Y,Zhai L,Wang T T,et al. Picroside Ⅱ plays a neuroprotective effect by inhibiting cyto C/caspase-9/caspase-3 signal pathway following ischemia/reperfusion injury in rats[J]. Chin Pharmacol Bull,2017,33(5):668-74.
[14] 张贝贝,刘文洪,李俊峰,叶志青.铁皮石斛多糖对高糖诱导的血管内皮细胞Bax、Bcl-2表达的影响[J]. 中国药理学通报, 2015,31(1):64-70.[14] Zhang B B, Liu W H, Li J F,Ye Z Q.Effects of Dendrobium candidum polysaccharide on the expression of Bax and Bcl-2 in vascular endothelial cells induced by high glucose[J]. Chin Pharmacol Bull, 2015, 31(1):64-70.
[15] Schuler M, Green D R. Mechanisms of p53-dependent apoptosis[J]. Biochem Soc Trans, 2001, 29(6):684-8.