黄芪多糖对人乳腺癌MDA-MB-231裸鼠移植瘤生长及肿瘤相关凋亡蛋白的影响
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摘要
目的:观察黄芪多糖(Astragalus polysaccharides,APS)对人乳腺癌MDA-MB-231裸鼠移植瘤的生长抑制作用及其对肿瘤细胞凋亡的影响,研究APS抑制三阴性乳腺癌MDA-MB-231生长、诱导其凋亡的作用及其分子机制。方法:通过将人乳腺癌细胞MDA-MB-231接种于BALB/c-nu雌性裸鼠右侧腋窝皮下建立人乳腺癌裸鼠移植瘤模型; 18只乳腺癌荷瘤裸鼠随机分为3组:模型组(每天予生理盐水),APS低、高剂量组(200,400 mg·kg~(-1)),每组6只;每天灌胃给药200μL,连续21 d。实验结束,观察比较APS低、高剂量组与模型组荷瘤鼠肿瘤质量、瘤体体积的变化,计算肿瘤抑制率;苏木精-伊红(HE)染色观察瘤体组织细胞形态学变化;原位末端标记法(TUNEL)检测乳腺癌肿瘤组织的凋亡;蛋白免疫印迹法检测肿瘤组织中B细胞淋巴瘤/白血病-2蛋白(Bcl-2),Bcl-2相关X蛋白(Bax)、半胱天冬氨酸蛋白酶(Caspase)等凋亡相关蛋白表达。结果:与模型组比较,APS低、高剂量组中的乳腺癌肿瘤体积、瘤重减小(P <0. 05,P <0. 01),其肿瘤抑制率分别为37. 9%,57. 57%; HE染色观察APS干预组肿瘤组织均呈现明显形态学改变,组织细胞出现凋亡状态; TUNEL染色显示APS组肿瘤组织细胞凋亡率增加;蛋白免疫印迹法发现,与模型组比较,APS低、高剂量组肿瘤组织中Bcl-2蛋白水平下降(P <0. 05,P <0. 01),Bax,Caspase-9,Caspase-7蛋白表达水平上调(P <0. 05,P <0. 01)。结论:APS能有效抑制MDA-MB-231乳腺癌裸鼠移植瘤生长,诱导人乳腺癌MDA-MB-231细胞发生凋亡,其作用机制与APS影响乳腺癌细胞中凋亡相关蛋白Bcl-2,Bax,Caspase-9,Caspase-7蛋白表达水平有关。
Objective: To observe the inhibitory effect of astragalus polysaccharides( APS) on growth of human breast cancer MDA-MB-231 xenograft tumor in nude mice and its effect on the apoptosis of tumor cells,in order to study the effect of APS on growth and induction of apoptosis of triple negative breast cancer MDA-MB-231 and its possible molecular mechanism. Method: Human breast cancer cell MDA-MB-231 was inoculated into the right axillary subcutaneous of BALB/c-nu female nude mice to establish the transplanted tumor model of breast cancer. Eighteen nude mice were randomly divided into 3 groups: model group( saline per day),low-dose APS group( 200 mg·kg~(-1) APS per day),and high-dose APS group( 400 mg·kg~(-1) APS per day),with 6 rats in each group. The drug was administered by gavage( 200 μL) daily for 21 days. In the experiment,the length and diameter of breast cancer transplanted tumor were measured every two days,and the tumor volume was recorded and calculated. At the end of the experiment,the changes of tumor mass and tumor volume of the low and highdose APS groups and the model group were observed and compared,and the tumor inhibition rate was calculated.The cell morphology in tumor tissue was observed by hematoxylin-eosin( HE) staining, and Terminaldeoxynucleoitidyl transferase mediated nick end labeling( TUNEL) was used to verify the apoptosis of breast cancer tissues. The expressions of apoptosis-related proteins,such as B-cell lymphoma/leukemia-2 protein( Bcl-2),Bcl-2 associated X protein( Bax),Caspase in tumor tissues was detected by Western blot. Result: The tumor volume of breast cancer decreased in the low and high-dose APS groups,and the tumor inhibition rates were 37. 9% and 57. 57%,respectively,with statistically significant differences from the model group( P < 0. 05,P < 0. 01). HE of tumor tissue cells showed that APS led to obvious morphological changes,with apoptosis in the tissue cells. TUNEL staining showed that the apoptosis rate of tumor cells in APS intervention groups was higher than that in control group. Western blot showed that expression of Bcl-2 protein decreased( P < 0. 05,P < 0. 01),and expressions of Bax,Caspase-9 and Caspase-7 were up-regulated( P < 0. 05,P < 0. 01) in tumor tissue of APS intervention groups. Conclusion: APS can effectively inhibit the growth of MDA-MB-231 breast cancer xenografts in nude mice and induce apoptosis in human breast cancer MDA-MB-231 cells. The mechanism may be related to the effect of APS on expressions of apoptosis-related proteins Bcl-2,Bax,Caspase-9 and Caspase-7 in breast cancer cells.
Objective: To observe the inhibitory effect of astragalus polysaccharides( APS) on growth of human breast cancer MDA-MB-231 xenograft tumor in nude mice and its effect on the apoptosis of tumor cells,in order to study the effect of APS on growth and induction of apoptosis of triple negative breast cancer MDA-MB-231 and its possible molecular mechanism. Method: Human breast cancer cell MDA-MB-231 was inoculated into the right axillary subcutaneous of BALB/c-nu female nude mice to establish the transplanted tumor model of breast cancer. Eighteen nude mice were randomly divided into 3 groups: model group( saline per day),low-dose APS group( 200 mg·kg~(-1) APS per day),and high-dose APS group( 400 mg·kg~(-1) APS per day),with 6 rats in each group. The drug was administered by gavage( 200 μL) daily for 21 days. In the experiment,the length and diameter of breast cancer transplanted tumor were measured every two days,and the tumor volume was recorded and calculated. At the end of the experiment,the changes of tumor mass and tumor volume of the low and highdose APS groups and the model group were observed and compared,and the tumor inhibition rate was calculated.The cell morphology in tumor tissue was observed by hematoxylin-eosin( HE) staining, and Terminaldeoxynucleoitidyl transferase mediated nick end labeling( TUNEL) was used to verify the apoptosis of breast cancer tissues. The expressions of apoptosis-related proteins,such as B-cell lymphoma/leukemia-2 protein( Bcl-2),Bcl-2 associated X protein( Bax),Caspase in tumor tissues was detected by Western blot. Result: The tumor volume of breast cancer decreased in the low and high-dose APS groups,and the tumor inhibition rates were 37. 9% and 57. 57%,respectively,with statistically significant differences from the model group( P < 0. 05,P < 0. 01). HE of tumor tissue cells showed that APS led to obvious morphological changes,with apoptosis in the tissue cells. TUNEL staining showed that the apoptosis rate of tumor cells in APS intervention groups was higher than that in control group. Western blot showed that expression of Bcl-2 protein decreased( P < 0. 05,P < 0. 01),and expressions of Bax,Caspase-9 and Caspase-7 were up-regulated( P < 0. 05,P < 0. 01) in tumor tissue of APS intervention groups. Conclusion: APS can effectively inhibit the growth of MDA-MB-231 breast cancer xenografts in nude mice and induce apoptosis in human breast cancer MDA-MB-231 cells. The mechanism may be related to the effect of APS on expressions of apoptosis-related proteins Bcl-2,Bax,Caspase-9 and Caspase-7 in breast cancer cells.
引文
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[8] LUO J, CHENG Z, RONG L, et al. Ganoderma lucidum polysaccharide alleviating colorectal cancer by alteration of special gut bacteria and regulation of gene expression of colonic epithelial cells[J]. J Funct Foods,2018,47:127-135.
[9]朱元章,张贵彪,朱国福.中药复方抗肿瘤机制研究进展[J].中国实验方剂学杂志,2017,23(16):227-234.
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[12]李云祥,梁引库,高飞雄,等.中药治疗乳腺癌疾病研究进展[J].中国实验方剂学杂志,2019,25(3):211-219.
[13]张俊林.黄芪桃红汤联合CEF方案化疗对乳腺癌术后免疫功能的影响[J].国医论坛,2017,32(5):47-49.
[14] PU X,MA X,LIU L,et al. Structural characterization and antioxidant activity in vitro of polysaccharides from angelica and astragalus[J]. Carbohydr Polym,2016,137:154-164.
[15] LAI X,XIA W,WEI J,et al. Therapeutic effect of Astragalus polysaccharides on hepatocellular carcinoma H22-bearing mice[J]. Dose Response,2017,15(1):1-6.
[16]庄梦婕,刘丹,陈彦文,等.黄芪多糖联合顺铂对小鼠Lewis肺癌移植瘤Caspase-3、Smac/Diablo表达的影响[J].解剖学报,2018,49(1):63-69.
[17] ZHOU Z,MENG M,NI H. Chemosensitizing effect of astragalus polysaccharides on nasopharyngeal carcinoma cells by inducing apoptosis and modulating expression of Bax/Bcl-2 ratio and Caspases[J]. Med Sci Monit,2017,23:462-469.
[18]吴小进,米燕燕,杨辉,等.黄芪多糖抑制人乳腺癌MCF-7细胞增殖和诱导凋亡[J].中华生物医学工程杂志,2014,20(2):106-109.
[19] YE M N, CHEN H, ZHOU R, et al. Effects of astragalus polysaccharide on proliferation and Akt phosphorylation of the basal-like breast cancer cell line[J]. J Chin Integr Med,2011,9(12):1339-1346.
[20] LI W,SONG K,WANG S,et al. Anti-tumor potential of astragalus polysaccharides on breast cancer cell line mediated by macrophage activation[J]. Mater Sci Eng C Mater Biol Appl,2019,98:685-695.
[21]杜军辉.黄芪注射液辅助免疫化疗对子宫内膜癌患者免疫功能及血清肿瘤分子的影响[J].中国生化药物杂志,2017,37(11):101-103.
[22]吕心明,徐金芬,闫呈新,等.黄芪多糖联合放疗对小鼠H22肝癌移植瘤的放射增敏及免疫功能的影响[J].云南中医学院学报,2017,40(6):9-13.
[23]郭英.黄芪甲苷通过抑制Akt和NF-κB通路诱导胃癌MGC-803细胞凋亡[J].国际检验医学杂志,2018,39(19):2341-2344.
[24] JING Z,LIU L,JING W,et al. Formononetin,an isoflavone from Astragalus membranaceus inhibits proliferation and metastasis of ovarian cancer cells[J]. J Ethnopharmacol,2018,221:91-99.
[25]张虹,季有波,孙晓琪,等.黄芪多糖对乳腺癌细胞抑制作用实验研究[J].中国社区医师,2017,33(36):9-10,13.
[26]张虹,季有波,杨琪,等.黄芪多糖诱导乳腺癌细胞凋亡的实验研究[J].中国社区医师,2017,33(34):5,7.
[27]谷俊朝,余微波,王宇,等.黄芪多糖对TA2小鼠乳腺癌MA-891移植瘤生长及HSP70表达的影响[J].中华肿瘤防治杂志,2006,13(20):1534-1537.
[28]周丽菁,汪志学,龙婷婷,等.黄芪多糖在体外巨噬细胞-乳腺癌细胞共培养体系中的作用[J].免疫学杂志,2017,33(6):469-476.
[29] Hickman J A. Apoptosis induced by anticancer drugs[J]. Cancer Metastasis Rev,1992,11(2):121-139.
[30]张丙信,赵霞,陈雪,等. Caspase-9、Bcl-2和Bax在基底细胞样乳腺癌组织中的表达及意义[J].山东医药,2014,54(46):8-10.
[31]黄兆宇,武睿超,冉江华,等.线粒体调控细胞凋亡的相关研究综述[J].吉林医学,2018,39(7):1356-1357.
[32]阎力君,洪涛,雒江菡,等.黄芪多糖对结肠癌SW620细胞增殖及凋亡作用的影响[J].中国实验方剂学杂志,2017,23(22):97-101.
[33] Knight T,Luedtke D,Edwards H,et al. A delicate balance-The BCL-2 family and its role in apoptosis,oncogenesis,and cancer therapeutics[J]. Biochem Pharmacol,2019,162:250-261.
[34] Mondal A, Bennett L L. Resveratrol enhances the efficacy of sorafenib mediated apoptosis in human breast cancer MCF7 cells through ROS,cell cycle inhibition,Caspase-3 and PARP cleavage[J]. Biomed Pharmacother,2016,84:1906-1914.
[35]杨涛,费振海,钟兴明. Caspase家族与细胞凋亡的研究进展[J].浙江医学,2018,40(18):2083-2087,2091.
[2] JIANG X, TANG H, CHEN T. Epidemiology of gynecologic cancers in China[J]. J Gynecol Oncol,2018,29(1):e7.
[3]文习武.三阴性乳腺癌的临床病理特征及治疗进展[J].三峡大学学报:自然科学版,2017,39(S1):120-121.
[4] Foulkes W D,Smith I E,Reisfilho J S. Triple-negative breast cancer[J]. N Engl J Med,2010,363(20):1938-1948.
[5]王蓓,傅健飞,吕晓皑. 74例三阴性乳腺癌临床特征及预后分析[J].肿瘤学杂志,2009,15(11):1005-1008.
[6] YANG Y,QIU S,QIAN L,et al. OCF can repress tumor metastasis by inhibiting epithelial-mesenchymal transition involved in PTEN/PI3K/Akt pathway in lung cancer cells[J]. PLo S One,2017,12(3):1-17.
[7] BAI H,SUN S,ZHANG Y,et al. Inhibitory effect of Dendrobium officinale polysaccharide on growth of human breast cancer MCF-7 cells and the related mechanism[J]. Biomed Res,2017,28(4):1922-1926.
[8] LUO J, CHENG Z, RONG L, et al. Ganoderma lucidum polysaccharide alleviating colorectal cancer by alteration of special gut bacteria and regulation of gene expression of colonic epithelial cells[J]. J Funct Foods,2018,47:127-135.
[9]朱元章,张贵彪,朱国福.中药复方抗肿瘤机制研究进展[J].中国实验方剂学杂志,2017,23(16):227-234.
[10]周思颖,张思杰,徐寒子,等.中医药治疗乳腺癌进展[J].中国中医基础医学杂志,2017,23(10):1489-1492.
[11]张奕萌,谢小红,高秀飞.中医药治疗三阴性乳腺癌进展[J].浙江中西医结合杂志,2018,28(1):77-80.
[12]李云祥,梁引库,高飞雄,等.中药治疗乳腺癌疾病研究进展[J].中国实验方剂学杂志,2019,25(3):211-219.
[13]张俊林.黄芪桃红汤联合CEF方案化疗对乳腺癌术后免疫功能的影响[J].国医论坛,2017,32(5):47-49.
[14] PU X,MA X,LIU L,et al. Structural characterization and antioxidant activity in vitro of polysaccharides from angelica and astragalus[J]. Carbohydr Polym,2016,137:154-164.
[15] LAI X,XIA W,WEI J,et al. Therapeutic effect of Astragalus polysaccharides on hepatocellular carcinoma H22-bearing mice[J]. Dose Response,2017,15(1):1-6.
[16]庄梦婕,刘丹,陈彦文,等.黄芪多糖联合顺铂对小鼠Lewis肺癌移植瘤Caspase-3、Smac/Diablo表达的影响[J].解剖学报,2018,49(1):63-69.
[17] ZHOU Z,MENG M,NI H. Chemosensitizing effect of astragalus polysaccharides on nasopharyngeal carcinoma cells by inducing apoptosis and modulating expression of Bax/Bcl-2 ratio and Caspases[J]. Med Sci Monit,2017,23:462-469.
[18]吴小进,米燕燕,杨辉,等.黄芪多糖抑制人乳腺癌MCF-7细胞增殖和诱导凋亡[J].中华生物医学工程杂志,2014,20(2):106-109.
[19] YE M N, CHEN H, ZHOU R, et al. Effects of astragalus polysaccharide on proliferation and Akt phosphorylation of the basal-like breast cancer cell line[J]. J Chin Integr Med,2011,9(12):1339-1346.
[20] LI W,SONG K,WANG S,et al. Anti-tumor potential of astragalus polysaccharides on breast cancer cell line mediated by macrophage activation[J]. Mater Sci Eng C Mater Biol Appl,2019,98:685-695.
[21]杜军辉.黄芪注射液辅助免疫化疗对子宫内膜癌患者免疫功能及血清肿瘤分子的影响[J].中国生化药物杂志,2017,37(11):101-103.
[22]吕心明,徐金芬,闫呈新,等.黄芪多糖联合放疗对小鼠H22肝癌移植瘤的放射增敏及免疫功能的影响[J].云南中医学院学报,2017,40(6):9-13.
[23]郭英.黄芪甲苷通过抑制Akt和NF-κB通路诱导胃癌MGC-803细胞凋亡[J].国际检验医学杂志,2018,39(19):2341-2344.
[24] JING Z,LIU L,JING W,et al. Formononetin,an isoflavone from Astragalus membranaceus inhibits proliferation and metastasis of ovarian cancer cells[J]. J Ethnopharmacol,2018,221:91-99.
[25]张虹,季有波,孙晓琪,等.黄芪多糖对乳腺癌细胞抑制作用实验研究[J].中国社区医师,2017,33(36):9-10,13.
[26]张虹,季有波,杨琪,等.黄芪多糖诱导乳腺癌细胞凋亡的实验研究[J].中国社区医师,2017,33(34):5,7.
[27]谷俊朝,余微波,王宇,等.黄芪多糖对TA2小鼠乳腺癌MA-891移植瘤生长及HSP70表达的影响[J].中华肿瘤防治杂志,2006,13(20):1534-1537.
[28]周丽菁,汪志学,龙婷婷,等.黄芪多糖在体外巨噬细胞-乳腺癌细胞共培养体系中的作用[J].免疫学杂志,2017,33(6):469-476.
[29] Hickman J A. Apoptosis induced by anticancer drugs[J]. Cancer Metastasis Rev,1992,11(2):121-139.
[30]张丙信,赵霞,陈雪,等. Caspase-9、Bcl-2和Bax在基底细胞样乳腺癌组织中的表达及意义[J].山东医药,2014,54(46):8-10.
[31]黄兆宇,武睿超,冉江华,等.线粒体调控细胞凋亡的相关研究综述[J].吉林医学,2018,39(7):1356-1357.
[32]阎力君,洪涛,雒江菡,等.黄芪多糖对结肠癌SW620细胞增殖及凋亡作用的影响[J].中国实验方剂学杂志,2017,23(22):97-101.
[33] Knight T,Luedtke D,Edwards H,et al. A delicate balance-The BCL-2 family and its role in apoptosis,oncogenesis,and cancer therapeutics[J]. Biochem Pharmacol,2019,162:250-261.
[34] Mondal A, Bennett L L. Resveratrol enhances the efficacy of sorafenib mediated apoptosis in human breast cancer MCF7 cells through ROS,cell cycle inhibition,Caspase-3 and PARP cleavage[J]. Biomed Pharmacother,2016,84:1906-1914.
[35]杨涛,费振海,钟兴明. Caspase家族与细胞凋亡的研究进展[J].浙江医学,2018,40(18):2083-2087,2091.