马钱子碱对人乳腺癌细胞MDA-MB-231作用的实验研究
摘要
目的:马钱子碱(Brucine)为吲哚型生物碱,是马钱子的主要有效成份之一。本研究旨在观察Brucine对人乳腺癌细胞MDA-MB-231的增殖抑制和诱导凋亡作用,并分析Brucine在诱导细胞凋亡过程中对Bcl-2、Bax和Caspase-3蛋白表达的调控作用,为Brucine用于乳腺癌的临床治疗提供实验依据。
方法:人乳腺癌细胞株MDA-MB-231(雌激素受体阴性)以L-15培养基孵育,至对数生长期时,分别以Brucine刺激24h、48h和72h,Brucine浓度为50mg/L、100mg/L、200 mg/L、400 mg/L。用MTT法测定Brucine对细胞的增殖抑制作用;倒置显微镜观察细胞形态;Brucine处理结束后,收集细胞制成单细胞悬液,加入碘化丙啶(PI)后以流式细胞仪分析细胞周期并计算凋亡率的变化:加入AnnexinV-FITC和PI后以流式细胞术分析细胞凋亡。为检测Bcl-2、Bax和Caspase3分子在Brucine诱导乳腺癌细胞凋亡过程中的作用,以中和性抗体进行阻断实验。为检测Brucine作用后Bcl-2、Bax和Caspase3蛋白水平的变化,以50mg/L、100mg/L、200 mg/L、400 mg/L的Brucine处理乳腺癌细胞48h后,裂解乳腺癌细胞后收集蛋白并进行定量分析,经聚丙烯酞胺凝胶电泳分离蛋白后以半干式电转移将蛋白转移到硝酸纤维素膜上,以Western蛋白印迹法检测Bcl-2、Bax和Caspase3蛋白水平。
结果:(1)细胞形态学观察结果:空白对照组细胞贴壁均匀,状态良好,各干预组贴壁细胞明显减少,培养液内细胞碎片增多,且随着药物剂量的增加和干预时间的延长更明显。(2)MTT比色试验结果:各剂量组OD值较空白对照组显著降低(P＜0.01),而且与干预剂量、干预时间成反比,有显著统计学差异(p＜0.01)。(3)PI流式细胞仪检测结果:随着Brucine浓度的增加和作用时间的延长细胞凋亡率逐渐增高并呈明显的浓度、时间依赖,与对照组相比差异有显著性(P＜0.05);同时S期细胞逐渐减少,G0/G1期细胞逐渐增多(P＜0.05),细胞被阻滞在G1/S期。(4)Annexin V—FITC/P1分析结果:与空白对照组相比,各干预组早期调亡细胞所占比例显著增加(p＜0.01),各剂量组间也有显著差异(p＜0.01)。(5)Western blotting检测结果:作用48h后,各剂量组与空白对照组相比,抗凋亡基因Bcl-2表达水平明显降低,而促凋亡基因Bax明显增高,凋亡执行因子中Caspase-3表达增加。
结论:Brucine能够有效的抑制乳腺癌细胞株MDA-MB-231细胞的增殖,呈明显浓度、时间依赖;Brucine能够诱导乳腺癌MDA-MB-231细胞凋亡,其分子机制可能与激活Bax及抑制Bcl-2等凋亡调控基因有关;Bcl-2的表达降低有可能诱发Caspase-3激活,促使细胞凋亡。
Objective: Brucine, a main component of nux vomica, is one of indole alkaloids. In the study, we observe the effect of Brucine on proliferation inhibition and apoptosis induction on breast cancer cell MDA-MB-231. And analyze the regulation of Brucine on the expression of Bcl-2, Bax and Caspase-3 protein, thus provide an experimental basis for the clinic.
Methods: The breast cancer cell MDA-MB-231 was cultured in L-15 medium. At the logarithmic growth phase, Brucine was added, the concentration of which was 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L, to stimulate the cells for 24h, 48h, 72h. MTT assay was used to measure the proliferation inhibitory effect of Brucine on the cells. The changes of cellular morphology were observed under inverted microscope. After treatment, cells were collected to prepare single cell suspension. Breast cancer cells were collected and resuspended in PBS. PI was added and the cell cycle was analyzed with flow cytometry while the apoptotic rate was calculated at the same time. PI and Annexin V-FITC were added to analyze the early apoptosis. To analyze the function of Bcl-2, Bax, Caspase-3 protein in the process of apoptosis induction, blocking assay was applied by neutralized antibody. Breast cancer cells were treated with Brucine for 48h at the concentrations of 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L. Then the cells were lysed and protein was quantified and separated with SDS-PAGE and transferred to a nitrocelular membrane with a semi-dry apparatus. The level of Bcl-2, Bax, Caspase-3 protein was detected by Western blotting assay.
Results: (1) Cellular morphology: Cell adherence in control group were homogeneous and in good condition. Cell adherence in intervention group fall Off obviously , cell debris increased, and had doses and time dependent. (2)MTT: The optical density degraded significantly in every intervention group(p<0.01), and had doses and time dependent. (3)PI simple staining and flow cytometry analysis: Cell apoptotic rate increased with the increment of the concentration and treatment time, and has doses and time dependent. It had significant deviation to control group (p<0.05). S phase cells decreased while G0/G1 phase cells increased (p<0.05) and cells were blocked at G1/S phase. (4) Annexin V-FITC/PI analysis: In contrast to the control group, the early apoptosis cells increased significantly in all intervention groups (p<0.01) and had significantly differences between each intervention groups. (5) Western blotting: After treatment for 48 hours, Bcl-2 level was decreased, Bax level and Caspase-3 level were increased.
Conclusion: Brucine can inhibit the proliferation of MDA-MB-231 cells effectively with doses and time dependent. Brucine can induce the apoptosis of MDA-MB-231 cells, the molecular mechanism of which is related with the activation of Bax and inhibition of Bcl-2. The decreased expression of Bcl-2 stimulates the activation of Caspase-3. The decreased expression of Bcl-2 may stimulate the release of cytochrome C, and activate Caspase-3 and Caspase-3 cascade through a key point in its downstream, then induce apoptosis.
方法:人乳腺癌细胞株MDA-MB-231(雌激素受体阴性)以L-15培养基孵育,至对数生长期时,分别以Brucine刺激24h、48h和72h,Brucine浓度为50mg/L、100mg/L、200 mg/L、400 mg/L。用MTT法测定Brucine对细胞的增殖抑制作用;倒置显微镜观察细胞形态;Brucine处理结束后,收集细胞制成单细胞悬液,加入碘化丙啶(PI)后以流式细胞仪分析细胞周期并计算凋亡率的变化:加入AnnexinV-FITC和PI后以流式细胞术分析细胞凋亡。为检测Bcl-2、Bax和Caspase3分子在Brucine诱导乳腺癌细胞凋亡过程中的作用,以中和性抗体进行阻断实验。为检测Brucine作用后Bcl-2、Bax和Caspase3蛋白水平的变化,以50mg/L、100mg/L、200 mg/L、400 mg/L的Brucine处理乳腺癌细胞48h后,裂解乳腺癌细胞后收集蛋白并进行定量分析,经聚丙烯酞胺凝胶电泳分离蛋白后以半干式电转移将蛋白转移到硝酸纤维素膜上,以Western蛋白印迹法检测Bcl-2、Bax和Caspase3蛋白水平。
结果:(1)细胞形态学观察结果:空白对照组细胞贴壁均匀,状态良好,各干预组贴壁细胞明显减少,培养液内细胞碎片增多,且随着药物剂量的增加和干预时间的延长更明显。(2)MTT比色试验结果:各剂量组OD值较空白对照组显著降低(P＜0.01),而且与干预剂量、干预时间成反比,有显著统计学差异(p＜0.01)。(3)PI流式细胞仪检测结果:随着Brucine浓度的增加和作用时间的延长细胞凋亡率逐渐增高并呈明显的浓度、时间依赖,与对照组相比差异有显著性(P＜0.05);同时S期细胞逐渐减少,G0/G1期细胞逐渐增多(P＜0.05),细胞被阻滞在G1/S期。(4)Annexin V—FITC/P1分析结果:与空白对照组相比,各干预组早期调亡细胞所占比例显著增加(p＜0.01),各剂量组间也有显著差异(p＜0.01)。(5)Western blotting检测结果:作用48h后,各剂量组与空白对照组相比,抗凋亡基因Bcl-2表达水平明显降低,而促凋亡基因Bax明显增高,凋亡执行因子中Caspase-3表达增加。
结论:Brucine能够有效的抑制乳腺癌细胞株MDA-MB-231细胞的增殖,呈明显浓度、时间依赖;Brucine能够诱导乳腺癌MDA-MB-231细胞凋亡,其分子机制可能与激活Bax及抑制Bcl-2等凋亡调控基因有关;Bcl-2的表达降低有可能诱发Caspase-3激活,促使细胞凋亡。
Objective: Brucine, a main component of nux vomica, is one of indole alkaloids. In the study, we observe the effect of Brucine on proliferation inhibition and apoptosis induction on breast cancer cell MDA-MB-231. And analyze the regulation of Brucine on the expression of Bcl-2, Bax and Caspase-3 protein, thus provide an experimental basis for the clinic.
Methods: The breast cancer cell MDA-MB-231 was cultured in L-15 medium. At the logarithmic growth phase, Brucine was added, the concentration of which was 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L, to stimulate the cells for 24h, 48h, 72h. MTT assay was used to measure the proliferation inhibitory effect of Brucine on the cells. The changes of cellular morphology were observed under inverted microscope. After treatment, cells were collected to prepare single cell suspension. Breast cancer cells were collected and resuspended in PBS. PI was added and the cell cycle was analyzed with flow cytometry while the apoptotic rate was calculated at the same time. PI and Annexin V-FITC were added to analyze the early apoptosis. To analyze the function of Bcl-2, Bax, Caspase-3 protein in the process of apoptosis induction, blocking assay was applied by neutralized antibody. Breast cancer cells were treated with Brucine for 48h at the concentrations of 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L. Then the cells were lysed and protein was quantified and separated with SDS-PAGE and transferred to a nitrocelular membrane with a semi-dry apparatus. The level of Bcl-2, Bax, Caspase-3 protein was detected by Western blotting assay.
Results: (1) Cellular morphology: Cell adherence in control group were homogeneous and in good condition. Cell adherence in intervention group fall Off obviously , cell debris increased, and had doses and time dependent. (2)MTT: The optical density degraded significantly in every intervention group(p<0.01), and had doses and time dependent. (3)PI simple staining and flow cytometry analysis: Cell apoptotic rate increased with the increment of the concentration and treatment time, and has doses and time dependent. It had significant deviation to control group (p<0.05). S phase cells decreased while G0/G1 phase cells increased (p<0.05) and cells were blocked at G1/S phase. (4) Annexin V-FITC/PI analysis: In contrast to the control group, the early apoptosis cells increased significantly in all intervention groups (p<0.01) and had significantly differences between each intervention groups. (5) Western blotting: After treatment for 48 hours, Bcl-2 level was decreased, Bax level and Caspase-3 level were increased.
Conclusion: Brucine can inhibit the proliferation of MDA-MB-231 cells effectively with doses and time dependent. Brucine can induce the apoptosis of MDA-MB-231 cells, the molecular mechanism of which is related with the activation of Bax and inhibition of Bcl-2. The decreased expression of Bcl-2 stimulates the activation of Caspase-3. The decreased expression of Bcl-2 may stimulate the release of cytochrome C, and activate Caspase-3 and Caspase-3 cascade through a key point in its downstream, then induce apoptosis.
引文
1.Cai BC,Wu H,Yang X W,et al.Analysis of special data for 13NMR of sixteen strychnos alkaloids[J].Acta Pharm Sin,1994,29(1):44-48.
2.迟德彪.钩吻素子体外抑制人结肠腺癌细胞生长并诱导其凋亡及其机制的探讨[D].广州:第一军医大学硕士学位论文.2000:25-26.
3.邓旭坤,蔡宝昌.马钱子抗肿瘤药理研究及临床应用进展[C].21世纪中医药-2004江苏省博士研究生学术论坛文集.2004:168-173.
4.Cai BC,Wang TS,Kurokawa M,et al.Cytotoxicities of alkaloids from processed and unprocessed seeds of Strychnos nux vomica[J].Zhong guo Yao Li Xue Bao,1998,19(5):425-428.
5.Cai BC,Chen L,Namba T.et al.A comparison of nine alkaloids from processed seeds of Strychnos nux vomica on tumor cell lines[J].Nat Med,1995,49(1):39-42.
6.陆跃鸣,陈龙,蔡宝昌,等.Brucine与异Brucine氮氧化物抗肿瘤细胞生长及抗氧化损伤作用的比较[J].南京中医药大学学报,1998,14(6):349-351.
7.邓旭坤,蔡宝昌,殷武,等.马钱子碱对小鼠肿瘤的抑制作用[J].中国天然药物,2005,3(6):392-395.
8.Yin W,Wang TS,Yin FZ,et al.Analgesic and anti-inflammatory properties of brucine and brucine N-oxide extracted from seeds of Strychnos nux vomica[J].J Ethnopharmacol,2003,2-3(88):205-214.
9.宋爱英,张国烈,刘松江,等.马钱子抗肿瘤作用的实验研究[J].中国中医药科技,2004,11(6):363.
10.宋爱英,谭辉,毕俊芳,等.马钱子浓缩液加碘油肝动脉灌注栓塞治疗原发性肝癌的临床观察[J].中国药学报,2007,35(5):28-29.
11.邓旭坤,蔡宝昌,殷武,等.Brucine对Heps荷瘤小鼠的抗肿瘤作用和毒性的研究[J].中国药理学通报,2006,22(1):35-39.
12.张蕻,李燕玲,任连生,等.马钱子天南星对小鼠移植性肿瘤H22的抑瘤作用 [J].中国药物与临床,2005,5(4):272.
13.殷武.马钱子生物碱类成分镇痛作用研究.南京:南京中医药大学硕士学位论文,2000:7-60.
14.朱燕娜,鲍梦周,曹永舒,等.马钱子碱和士的宁镇痛作用研究.河南医科大学学报,1989,2(44):288-291.
15.张国华,朱燕娜,鲍梦周,等.小鼠对马钱子碱耐受性及身体依赖性的初步探讨河南医科大学学报,1993,2(81):10-11.
16.周建英,卞慧敏,马骋,等.马钱子碱和马钱子碱氮氧化物抗血小板聚集及抗血栓形成作用的研究.江苏中医,1998,19(4):41-42.
17.Fesus L,Thomazy V,Falus A.Induction and activation of tissue transglutaminase during programmed cell death.FEBS Lett,1987,24(1):104-108.
18.徐英含,恭西马俞主编.病理学·细胞凋亡.北京:人民卫生出版社,1996:336-343.
19.Kamesaki S,Kamesaki H,Jorgensen TJ,et al.Bcl-2 protein inhibits etoposide induces apoptosis through its effects on events subsequent to topoisomerase Ⅱinduces DNA strand breaks and their repair.Cancer Res,1993,53(18):4251-4256
20.Fulda S,Meyer E,Friesen C,et al.Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis.Oncogene,2001,20(9):1063-1075.
21.Green DR,Reed JC.Mitochondria and apoptosis.Science,1998,281:1308-1312
22.Swanton E,Savory P,Cosulich S,et al.Bcl-2 regulates caspase-3/caspase-2apoptotic cascade in cytosolic exytracts.Oncogene,1999,18(10):1781-1787
23.Werner AB,de Vries E,Tait SW,et al.Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax[J].J Biol Chem.2002,277(25):22781-22788.
24.Warnberg F,Nordgren H,Bergkvist L,et al.Tumour markers in breast carcinoma correlate with grade rather than with invasiveness[J].Br J Cancer,2001,85(6):869-874.
25.Hou Q,Cymbalyuk E,Hsu SC,et al.Apoptosis modulatory activities of transiently expressed Bcl-2:roles in cytochrome C release and Bax regulation[J].Apoptosis,2003,8(6):617-629.
26.周晓燕,许良中.Bcl-2基因家族与细胞凋亡[J].实用肿瘤杂志,2001,15(1):61-63.
27.杨红鹰,孙耘田,刘复生,等.Bcl-2基因表达与乳腺癌组织学类型、分级及其预后的关系[J].中华肿瘤杂志,2000,22(6):490-492.
28.Joensuu H,Pylkkanen L,Toikkanen S.Bcl-2 protein expression and longterm survival in breast cancer[J].Am J Pathol,1994,145(5):1191-1198.
29.Mustonen M,Raunio H,Paakko P,et al.The extent of apoptosis is inversely associated with bcl-2 expression in premalignant and malignant breast lesions[J].Histopathology,1997,31(4):347-354.
30.黄平,杨光伦,姚榛祥.Bcl-2和Bax表达在乳腺浸润性导管癌细胞凋亡中的作用[J].肿瘤防治杂志,2003,10(2):131-132.
1.张保宁.乳腺癌临床研究的回顾与展望[J].中华医学杂志,2005,81(1):1-5.
2.Gross A,Me Donnell JM,Konsmeyer SJ.Bcl-2 family members and the mitochondria in apoptosis.Genes Dev,1999,13(15):1899-1911.
3.Fam YD,Li KZ,Zhao YT.The morphological transform of apoplosis in human cholangio arcinoma cells induced by Bcl-2 mRNA cheaving ribozyme[J].Di Si Jun yi Daxue Xuebao,2000,21(3):324-327.
4.Yang LJ,Wang WL.Establishment of the hepatoma cells which express Bax protein stably and highly and observation of its apoptotic phenomena[J].Di Si Jun yi Daxue Xuebao,2002,23(5):455-458.
5.Hou Q,Cymbalyuk E,Hsu SC,et al.Apotosis modulatory activities of transiently expressed Bcl-2:roles in cytochrome C release and Bax regulation[J].Apoptosis,2003,8(6):617.
6.Soto T,Hanada M,Bodrug S,et al.Interactions among members of the bcl-2protein family analyzed with a yeast two-hybrid system.Proc Natl Acad Sci USA,1994,91:9238.
7.Cajewsk TF,Thompson CB.Apoptosis meets signol.Transduction:elimination of a BAD influence.Cell,1996,87:589
8.Vollerms HP,Dammtich J,Hensel F,et al.Differential expression of apoptosis receptor or diffuse and intestinal type stomach carcinoma[J].1997,79(3):433.
9.Sturm I,Papadopoulos S,Hillebrand T,et al.Impaired BAX protein expression in breast cancer:mutational and Lysis of the BAX and the P53 gene.Int J Cancer,2000,87:517
10.Kulig E,Jin L,Qian X,et al.Apoptosisin nontumorous and neoplastic human Pituitaries:expression of t he Bcl-2 family of Proteins.Am J pathol,1999,154:767
11.Oi-Long L u,Richard poulsom,Leslie Wong,et al.Bcl-2 expression in abult and embryonic non-haematopoietic tissues,J pat hol,1993,169:431
12.Gajewsk TF,Thompson CB.Apoptosis meets signol transduction:elimination of a BAD influence.Cell,1996,87:589
13.Yamaguchi H,Paranawit hana SR,Lee MW,et al.EpothiloneB nalogue (MS2247550) mdiated cytotoxicit y t hrough induc2tion of Bax conformational change in human breast cancer cell s[J].Cancer Res,2002,62(2):466
14.Sarkar FH,Rahman KM,Li Y.Bax t ranslocation to mitochondria is an important event in inducing apoptotic cell death by indole-3-carbinol(I3C) treatment of breast cancer cells[J].J Nutr,2003,133(7 Suppl):2434S-2439S.
15.Yu W,Sanders BG,Kline K.RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells involves Baxt ranslocation to mitochondria[J].Cancer Res,2003,63(10):2483-2491.
16.刘雪梅,黄剑飞.bcl-2、bad在乳腺癌中表达的临床意义[J].河南肿瘤学杂志,2005,18(1):14
17.Kapranos N,Karaiosifidi H,Valavanis C,et al.Prognostic significance of apoptosis related proteins Bcl-2 and Bax in node2negative breast cancer patients[J].Anticancer Res,1997,17(4A):2499
18.周晓燕,许良中.Bcl-2基因家族与细胞凋亡[J].实用肿瘤杂志,2001,15(1):61-63.
19.杨红鹰,孙耘田,刘复生,等.Bcl-2基因表达与乳腺癌组织学类型、分级及其预后的关系[J].中华肿瘤杂志,2000,22(6):490-492.
20.Joensu UH,Pylkkanen L,Toikkanen S.Bcl-2 protein expression and longterm survival in breast cancer[J].Am J Pathol,1994,145(5):1191-1198.
21.Mustonen M,Raunio H,Paakko P,et al.The extent of apoptosis is inversely associated with bcl-2 expression in premalignant and malignant breast lesions[J].Histopathology,1997,31(4):347-354.
22.黄平,杨光伦,姚榛祥.Bcl-2和Bax表达在乳腺浸润性导管癌细胞凋亡中的作用[J].月中瘤防治杂志,2003,10(2):131-132
23.Hellemans P,van Dam PA,Weyler J,et al.Prognostic value of bcl-2 expression in invasive breast cancer[J].Br J Cancer,1995,72(2):354-360.
24.Thompson CB.Apoptosis in t he pat hogenesis and treat ment of disease.Science 1995,26:1456
25.Cleary ML,Smit h SD,Sklar J.Cloning and structural analysis of CDNAS for Bcl-2and a hybrid Bcl-2 immunoglobulin transcript resulting from the t(14,18)translocation Cell,1986,47:19
26.Bakhshi A,Jensen J P,Goldman P,et al.Cloning t he chromosomal breakpoint of t(14/18)human lymphomas:clustering around J H onchromosomal 14 and nerar a transcri-Ptional unit on 18.Cell,1985,41:899
27.张燕,赵铭锋,刘同慎.乳腺癌bcl-2、bax基因的表达及其临床意义[J].山东医药,2006,46(31):42-43.
28.Hurlimann J,Larrinaga B,Vala DL.bcl-2 protein in invasive ductal breast carcinomas [J].Virchows Arch,1995,426:163-168.
29.van Slooten HJ,Clahsen PC,van Dierendonck J H,et al Expression of Bcl-2 in node negative breast cancer is associated with various prognostic factors,but does not predict response to one course of perioperative chemotherapy[J].Br J Cancer,1996,74:78-85.
30.Hellemans P,van Dam PA,Weyler J,et al.Prognostic value of Bcl-2expression in invasive breast cancer[J].Br J Cancer,1995,72:354-360.
31.Chai SJ,Tang WY,Eleatan J,et al.Prostate tumor from an Asia population:examination of Bax,Bcl-2,p53 and ras and identifyication of bax as a Prognostic marker[J].Br J Cancer,2000,83(6):761-768.
32.黄建华,吕新生,刘少华,等.Bcl-2,Bax基因蛋白在乳腺癌中的表达及临床病理学意义[J].湖南医学,2000,17(4):251-253.
33.Buchholz TA,Davis DW,McConkey DJ,et al.Chemotherapy induced apoptosis and Bcl-2 levels correlate wit h breast cancer response to chemotherapy[J].Cancer J,2003,9(1):33
34.Manion MK,Hockenbery DM.Targeting Bcl-2 related proteins in cancer therapy [J].Cancer Biol Ther,2003,2(4Suppl 1):S105
35.Andersen M H,Svane IM,Kvist borg P,et al.Immunogenicity of Bcl-2 in patients with cancer[J].Blood,2005,105(2):728
36.Del Bufalo D,Biroccio A,Trisciuoglio D,et al.Bcl-2 has differing effects on the sensitivity of breast cancer cell s depending on the antineoplastic drug used[J].Eur J Cancer,2002,38(18):2455
37.Tanabe K,Kim R,Inoue H,et al.Antisense Bcl-2 and H ER-2 oligonucleotide treat ment of breast cancer cell s enhances their sensitivity to anticancer drugs[J].Int J Oncol,2003,22(4):875
38.沈镇宙,邵志敏主编.现代乳腺肿瘤学进展[M].第1版.上海:上海科技文献出版社,2002:353-355
39.Gee J M,Robert son J F,Ellis IO,et al.Immunocytochemical localization of Bcl-2protein in human breast cancers and it s relationship to a series of prognostic markers and response to endocrine therapy[J].IntJ ancer,1994,59(5):619
40.Milella M,Trisciuoglio D,Bruno T,et al.Trast uzumab down regulates Bcl-2expression and potentiates apoptosis induction by Bcl-2/Bcl-XL bispecific antisense oligonucleotides in HER-2 gene amplified breast cancer cells[J].Clin Cancer Res,2004,10(22):7747
41.Linjawi A,Kontogiannea M,Halwani F,et al.Prognostic significance of p53,bcl-2,and Bax expression in early breast cancer[J].J Am Coil Surg,2004,198(1):83.
2.迟德彪.钩吻素子体外抑制人结肠腺癌细胞生长并诱导其凋亡及其机制的探讨[D].广州:第一军医大学硕士学位论文.2000:25-26.
3.邓旭坤,蔡宝昌.马钱子抗肿瘤药理研究及临床应用进展[C].21世纪中医药-2004江苏省博士研究生学术论坛文集.2004:168-173.
4.Cai BC,Wang TS,Kurokawa M,et al.Cytotoxicities of alkaloids from processed and unprocessed seeds of Strychnos nux vomica[J].Zhong guo Yao Li Xue Bao,1998,19(5):425-428.
5.Cai BC,Chen L,Namba T.et al.A comparison of nine alkaloids from processed seeds of Strychnos nux vomica on tumor cell lines[J].Nat Med,1995,49(1):39-42.
6.陆跃鸣,陈龙,蔡宝昌,等.Brucine与异Brucine氮氧化物抗肿瘤细胞生长及抗氧化损伤作用的比较[J].南京中医药大学学报,1998,14(6):349-351.
7.邓旭坤,蔡宝昌,殷武,等.马钱子碱对小鼠肿瘤的抑制作用[J].中国天然药物,2005,3(6):392-395.
8.Yin W,Wang TS,Yin FZ,et al.Analgesic and anti-inflammatory properties of brucine and brucine N-oxide extracted from seeds of Strychnos nux vomica[J].J Ethnopharmacol,2003,2-3(88):205-214.
9.宋爱英,张国烈,刘松江,等.马钱子抗肿瘤作用的实验研究[J].中国中医药科技,2004,11(6):363.
10.宋爱英,谭辉,毕俊芳,等.马钱子浓缩液加碘油肝动脉灌注栓塞治疗原发性肝癌的临床观察[J].中国药学报,2007,35(5):28-29.
11.邓旭坤,蔡宝昌,殷武,等.Brucine对Heps荷瘤小鼠的抗肿瘤作用和毒性的研究[J].中国药理学通报,2006,22(1):35-39.
12.张蕻,李燕玲,任连生,等.马钱子天南星对小鼠移植性肿瘤H22的抑瘤作用 [J].中国药物与临床,2005,5(4):272.
13.殷武.马钱子生物碱类成分镇痛作用研究.南京:南京中医药大学硕士学位论文,2000:7-60.
14.朱燕娜,鲍梦周,曹永舒,等.马钱子碱和士的宁镇痛作用研究.河南医科大学学报,1989,2(44):288-291.
15.张国华,朱燕娜,鲍梦周,等.小鼠对马钱子碱耐受性及身体依赖性的初步探讨河南医科大学学报,1993,2(81):10-11.
16.周建英,卞慧敏,马骋,等.马钱子碱和马钱子碱氮氧化物抗血小板聚集及抗血栓形成作用的研究.江苏中医,1998,19(4):41-42.
17.Fesus L,Thomazy V,Falus A.Induction and activation of tissue transglutaminase during programmed cell death.FEBS Lett,1987,24(1):104-108.
18.徐英含,恭西马俞主编.病理学·细胞凋亡.北京:人民卫生出版社,1996:336-343.
19.Kamesaki S,Kamesaki H,Jorgensen TJ,et al.Bcl-2 protein inhibits etoposide induces apoptosis through its effects on events subsequent to topoisomerase Ⅱinduces DNA strand breaks and their repair.Cancer Res,1993,53(18):4251-4256
20.Fulda S,Meyer E,Friesen C,et al.Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis.Oncogene,2001,20(9):1063-1075.
21.Green DR,Reed JC.Mitochondria and apoptosis.Science,1998,281:1308-1312
22.Swanton E,Savory P,Cosulich S,et al.Bcl-2 regulates caspase-3/caspase-2apoptotic cascade in cytosolic exytracts.Oncogene,1999,18(10):1781-1787
23.Werner AB,de Vries E,Tait SW,et al.Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax[J].J Biol Chem.2002,277(25):22781-22788.
24.Warnberg F,Nordgren H,Bergkvist L,et al.Tumour markers in breast carcinoma correlate with grade rather than with invasiveness[J].Br J Cancer,2001,85(6):869-874.
25.Hou Q,Cymbalyuk E,Hsu SC,et al.Apoptosis modulatory activities of transiently expressed Bcl-2:roles in cytochrome C release and Bax regulation[J].Apoptosis,2003,8(6):617-629.
26.周晓燕,许良中.Bcl-2基因家族与细胞凋亡[J].实用肿瘤杂志,2001,15(1):61-63.
27.杨红鹰,孙耘田,刘复生,等.Bcl-2基因表达与乳腺癌组织学类型、分级及其预后的关系[J].中华肿瘤杂志,2000,22(6):490-492.
28.Joensuu H,Pylkkanen L,Toikkanen S.Bcl-2 protein expression and longterm survival in breast cancer[J].Am J Pathol,1994,145(5):1191-1198.
29.Mustonen M,Raunio H,Paakko P,et al.The extent of apoptosis is inversely associated with bcl-2 expression in premalignant and malignant breast lesions[J].Histopathology,1997,31(4):347-354.
30.黄平,杨光伦,姚榛祥.Bcl-2和Bax表达在乳腺浸润性导管癌细胞凋亡中的作用[J].肿瘤防治杂志,2003,10(2):131-132.
1.张保宁.乳腺癌临床研究的回顾与展望[J].中华医学杂志,2005,81(1):1-5.
2.Gross A,Me Donnell JM,Konsmeyer SJ.Bcl-2 family members and the mitochondria in apoptosis.Genes Dev,1999,13(15):1899-1911.
3.Fam YD,Li KZ,Zhao YT.The morphological transform of apoplosis in human cholangio arcinoma cells induced by Bcl-2 mRNA cheaving ribozyme[J].Di Si Jun yi Daxue Xuebao,2000,21(3):324-327.
4.Yang LJ,Wang WL.Establishment of the hepatoma cells which express Bax protein stably and highly and observation of its apoptotic phenomena[J].Di Si Jun yi Daxue Xuebao,2002,23(5):455-458.
5.Hou Q,Cymbalyuk E,Hsu SC,et al.Apotosis modulatory activities of transiently expressed Bcl-2:roles in cytochrome C release and Bax regulation[J].Apoptosis,2003,8(6):617.
6.Soto T,Hanada M,Bodrug S,et al.Interactions among members of the bcl-2protein family analyzed with a yeast two-hybrid system.Proc Natl Acad Sci USA,1994,91:9238.
7.Cajewsk TF,Thompson CB.Apoptosis meets signol.Transduction:elimination of a BAD influence.Cell,1996,87:589
8.Vollerms HP,Dammtich J,Hensel F,et al.Differential expression of apoptosis receptor or diffuse and intestinal type stomach carcinoma[J].1997,79(3):433.
9.Sturm I,Papadopoulos S,Hillebrand T,et al.Impaired BAX protein expression in breast cancer:mutational and Lysis of the BAX and the P53 gene.Int J Cancer,2000,87:517
10.Kulig E,Jin L,Qian X,et al.Apoptosisin nontumorous and neoplastic human Pituitaries:expression of t he Bcl-2 family of Proteins.Am J pathol,1999,154:767
11.Oi-Long L u,Richard poulsom,Leslie Wong,et al.Bcl-2 expression in abult and embryonic non-haematopoietic tissues,J pat hol,1993,169:431
12.Gajewsk TF,Thompson CB.Apoptosis meets signol transduction:elimination of a BAD influence.Cell,1996,87:589
13.Yamaguchi H,Paranawit hana SR,Lee MW,et al.EpothiloneB nalogue (MS2247550) mdiated cytotoxicit y t hrough induc2tion of Bax conformational change in human breast cancer cell s[J].Cancer Res,2002,62(2):466
14.Sarkar FH,Rahman KM,Li Y.Bax t ranslocation to mitochondria is an important event in inducing apoptotic cell death by indole-3-carbinol(I3C) treatment of breast cancer cells[J].J Nutr,2003,133(7 Suppl):2434S-2439S.
15.Yu W,Sanders BG,Kline K.RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells involves Baxt ranslocation to mitochondria[J].Cancer Res,2003,63(10):2483-2491.
16.刘雪梅,黄剑飞.bcl-2、bad在乳腺癌中表达的临床意义[J].河南肿瘤学杂志,2005,18(1):14
17.Kapranos N,Karaiosifidi H,Valavanis C,et al.Prognostic significance of apoptosis related proteins Bcl-2 and Bax in node2negative breast cancer patients[J].Anticancer Res,1997,17(4A):2499
18.周晓燕,许良中.Bcl-2基因家族与细胞凋亡[J].实用肿瘤杂志,2001,15(1):61-63.
19.杨红鹰,孙耘田,刘复生,等.Bcl-2基因表达与乳腺癌组织学类型、分级及其预后的关系[J].中华肿瘤杂志,2000,22(6):490-492.
20.Joensu UH,Pylkkanen L,Toikkanen S.Bcl-2 protein expression and longterm survival in breast cancer[J].Am J Pathol,1994,145(5):1191-1198.
21.Mustonen M,Raunio H,Paakko P,et al.The extent of apoptosis is inversely associated with bcl-2 expression in premalignant and malignant breast lesions[J].Histopathology,1997,31(4):347-354.
22.黄平,杨光伦,姚榛祥.Bcl-2和Bax表达在乳腺浸润性导管癌细胞凋亡中的作用[J].月中瘤防治杂志,2003,10(2):131-132
23.Hellemans P,van Dam PA,Weyler J,et al.Prognostic value of bcl-2 expression in invasive breast cancer[J].Br J Cancer,1995,72(2):354-360.
24.Thompson CB.Apoptosis in t he pat hogenesis and treat ment of disease.Science 1995,26:1456
25.Cleary ML,Smit h SD,Sklar J.Cloning and structural analysis of CDNAS for Bcl-2and a hybrid Bcl-2 immunoglobulin transcript resulting from the t(14,18)translocation Cell,1986,47:19
26.Bakhshi A,Jensen J P,Goldman P,et al.Cloning t he chromosomal breakpoint of t(14/18)human lymphomas:clustering around J H onchromosomal 14 and nerar a transcri-Ptional unit on 18.Cell,1985,41:899
27.张燕,赵铭锋,刘同慎.乳腺癌bcl-2、bax基因的表达及其临床意义[J].山东医药,2006,46(31):42-43.
28.Hurlimann J,Larrinaga B,Vala DL.bcl-2 protein in invasive ductal breast carcinomas [J].Virchows Arch,1995,426:163-168.
29.van Slooten HJ,Clahsen PC,van Dierendonck J H,et al Expression of Bcl-2 in node negative breast cancer is associated with various prognostic factors,but does not predict response to one course of perioperative chemotherapy[J].Br J Cancer,1996,74:78-85.
30.Hellemans P,van Dam PA,Weyler J,et al.Prognostic value of Bcl-2expression in invasive breast cancer[J].Br J Cancer,1995,72:354-360.
31.Chai SJ,Tang WY,Eleatan J,et al.Prostate tumor from an Asia population:examination of Bax,Bcl-2,p53 and ras and identifyication of bax as a Prognostic marker[J].Br J Cancer,2000,83(6):761-768.
32.黄建华,吕新生,刘少华,等.Bcl-2,Bax基因蛋白在乳腺癌中的表达及临床病理学意义[J].湖南医学,2000,17(4):251-253.
33.Buchholz TA,Davis DW,McConkey DJ,et al.Chemotherapy induced apoptosis and Bcl-2 levels correlate wit h breast cancer response to chemotherapy[J].Cancer J,2003,9(1):33
34.Manion MK,Hockenbery DM.Targeting Bcl-2 related proteins in cancer therapy [J].Cancer Biol Ther,2003,2(4Suppl 1):S105
35.Andersen M H,Svane IM,Kvist borg P,et al.Immunogenicity of Bcl-2 in patients with cancer[J].Blood,2005,105(2):728
36.Del Bufalo D,Biroccio A,Trisciuoglio D,et al.Bcl-2 has differing effects on the sensitivity of breast cancer cell s depending on the antineoplastic drug used[J].Eur J Cancer,2002,38(18):2455
37.Tanabe K,Kim R,Inoue H,et al.Antisense Bcl-2 and H ER-2 oligonucleotide treat ment of breast cancer cell s enhances their sensitivity to anticancer drugs[J].Int J Oncol,2003,22(4):875
38.沈镇宙,邵志敏主编.现代乳腺肿瘤学进展[M].第1版.上海:上海科技文献出版社,2002:353-355
39.Gee J M,Robert son J F,Ellis IO,et al.Immunocytochemical localization of Bcl-2protein in human breast cancers and it s relationship to a series of prognostic markers and response to endocrine therapy[J].IntJ ancer,1994,59(5):619
40.Milella M,Trisciuoglio D,Bruno T,et al.Trast uzumab down regulates Bcl-2expression and potentiates apoptosis induction by Bcl-2/Bcl-XL bispecific antisense oligonucleotides in HER-2 gene amplified breast cancer cells[J].Clin Cancer Res,2004,10(22):7747
41.Linjawi A,Kontogiannea M,Halwani F,et al.Prognostic significance of p53,bcl-2,and Bax expression in early breast cancer[J].J Am Coil Surg,2004,198(1):83.