番荔枝内酯类化合物对乳腺癌细胞抑制作用及机理的研究
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摘要
研究背景：
随着人们生活水平的不断提高,乳腺癌发病率呈上升趋势,世界银行及世界卫生组织全球疾病负担报告所示：2002年乳腺癌在全球所造成的损失约614万伤残调整寿命年(Disability adjust Life years, DALYs),居女性恶性肿瘤疾病负担的首位,控制乳腺癌被认为是降低全球癌症负担的最有效的措施之一。中医药作为综合治疗的重要组成部分,有一定的疗效和独特的优势,但是长期以来,中医药在乳腺癌综合治疗中的地位不高,探讨中医药防治乳腺癌在综合治疗中的切入点和时机,运用分子生物学方法研究中医药防治乳腺癌机理,一直是我国乳腺癌研究的重要课题。番荔枝被称为“明日抗癌之星”,近年来国内外在番荔枝抗肿瘤的研究方面,取得了一定的进展,番荔枝内酯化合物被认为是继紫杉醇后的又一个有前途的、天然来源的抗肿瘤化合物,从番荔枝中分离出的抗癌活性最强的内酯为Bullatacin,研究表明：Bullatacin在小鼠体内活性比紫杉醇强400-600倍,本课题从番荔枝种子的醇浸膏中,分离出Bullatacin,研究其对人乳腺癌细胞的作用及其作用机理,为抗乳腺癌的中药新药开发提供理论和实验依据,进一步为中西结合治疗乳腺癌奠定理论基础。
研究目的：
1、阐明癌毒是乳腺癌发生发展的关键因素,规范化和个体化,辨病论治和辨证论治矛盾统一,为中西结合治疗乳腺癌的方向和思路提供一定的理论依据；
2、从番荔枝种子的醇浸膏中,筛选出抗癌活性最强的番荔枝内酯单体Bullatacin,研究其对人乳腺癌细胞的作用及其作用机理,为抗乳腺癌的中药新药开发提供理论和实验依据。
研究方法：
采用文献研究和现代实验研究相结合的方法。
1、文献研究：
1)对乳腺癌的古代与现代中医药文献进行研究,找寻出乳腺癌发生发展的关键因素,并对其进行阐发；
2)用循证医学的观点和方法,对现代医学关于乳腺癌的理论与临床实践进行一定的梳理,寻找中西结合诊治乳腺癌的切入点和时机。
2、实验研究：
2.1、番荔枝内酯单体Bullatacin的提取：
番荔枝种子粉碎,95%乙醇冷浸,回流提取3次,依次用石油醚、水饱和氯仿、甲醇萃取,通过反复硅胶柱层析、重结晶等方法从氯仿部分,分得番荔枝内酯类化合物,通过反复硅胶柱层析和重结晶等方法,得到番荔枝总内酯部位和Bullatacin单体化合物。
2.2、MTT法观察Bullatacin体外对ER(+)人乳腺癌细胞MCF-7和ER(-)人乳腺癌细胞MDA-MB-231的作用：
采用MTT法,,测试受试样品Bullacatin体外对ER(+)的人乳腺癌细胞MCF-7和ER(-)人乳腺癌细胞MDA-MB-231有无抑制作用及作用强度,并筛选出各自的有效剂量和作用时间。
2.3、观察Bullacatin对人乳腺癌细胞MDA-MB-231细胞形态学的影响：
倒置显微镜与透射电镜观察20ug/ml Bullacatin体外对ER(-)人乳腺癌细胞MDA-MB-231的形态学的影响。
2.4、流式细胞仪检测Bullacatin对人乳腺癌细胞MDA-MB-231细胞周期的影响：流式细胞仪分析,20ug/ml Bullacatin作用人乳腺癌细胞MDA-MB-231 0h、12h、24h、48h对细胞周期的影响
2.5、Annexin V-FITC染色流式细胞术考察Bullacatin对人乳腺癌细胞MDA-MB-231凋亡的影响：
Annexin V-FITC染色流式细胞仪检测凋亡,考察Bullacatin20ug/ml作用人乳腺癌细胞MDA-MB-231 48h对细胞凋亡的影响,分别加入MAPK通路激动剂与MAPK各主要通路的抑制剂后,观察药物对细胞凋亡的影响
2.6、Western Blot检测Bullacatin对人乳腺癌细胞MDA-MB-231 MAPK通路的作用：
Western Blotting检测Bullacatin 20ug/m作用人乳腺癌细胞MDA-MB-231 48h和分别加入MAPK通路激动剂与MAPK各主要通路的抑制剂后,不同实验组磷酸化与总蛋白的表达,观测Bullacatin对MAPK信号通路的作用。
研究结果：
1、癌毒贯穿乳腺癌发生与发展的始终,循证医学要求规范化和个体化,辨病论治和辩证论治的矛盾统一；
2、从番荔枝种子氯仿部分,分得番荔枝内酯类化合物,通过反复硅胶柱层析和重结晶等方法,得到的化合物,根据理化常数和波谱学数据鉴定了结构,为Bullatacin,纯度大于95%；
3、受试样品Bullacatin不管对ER(+)的MCF-7还是ER(-)MDA-MB-231细胞均有具有抑制作用,且有剂量和时间依赖性。20ug/ml的Bullacatin作用48小时,对于MDA-MB-231细胞的抑制率与紫杉醇组相比,没有差异；
4、20ug/ml的Bullacatin作用于细胞后,G1期的细胞比率减少,S期的细胞比率增加,细胞周期明显阻滞在S期。且G1期细胞比率随着Bullacatin作用的时间延长而减少,S期细胞比率随着Bullacatin作用的时间延长增加,Bullacatin对细胞周期的影响具有时间依赖性。
5、Bullacatin20ug/ml,作用于MDA-MB-231 48h细胞：
1)普通倒置显微镜下见：细胞密度较空白组减低,增殖变慢,细胞逐渐变大、变圆,细胞间接触变松,胞浆中颗粒增多,空泡化,脱壁,细胞周围碎片增多；
2)电镜显示：细胞表面微绒毛部分或全部脱失,胞质内细胞器明显扩张,以线粒体扩张明显,并有许多空泡形成,内质网扩张,细胞器有外渗现象,未见典型的凋亡和凋亡小体；
3)Bullacatin20ug/ml作用于MDA-MB-231细胞48小时,流式细胞仪检测,Annexin V染色的细胞为27.1%与阴性对照组4.2%比较,有显著差异；MAPK通路的激动剂能够促进MDA-MB-231 Annexin V染色的细胞的出现,作用较Bullacatin弱,激动剂与Bullacatin有协同作用；分别加入MAPK的3条主要通路的抑制剂,三者虽然与药物组Annexin V染色的细胞27.1%比有所下降,与阴性对照组4.2%比较,仍有显著差异。
6、Bullacatin20ug/ml，作用于MDA-MB-231细胞48小时Western Blotting检测发现：
1)激活ERK通路,导致P-ERK的增强,增强作用大于激动剂对ERK的作用；
2)激活JNK通路,导致P-ERK的增强,增强作用小于激动剂对ERK的作用；
3)激活P38-MAPK通路,导致P-P38的表达增强,增强作用大于激动剂对P38通路的作用。
4)JNK与ERK通路有协同作用；
5)ERK抑制剂PD98059和JNK抑制剂SP600125亦能轻度抑制P38的磷酸化,P38抑制剂SB203580对Bullatacin激活P38通路有抑制作用,但不能完全抑制其表达
研究结论：
1、乳腺癌现代医学要求的规范化和个体化治疗,中医理论所倡导的同病异治、异病同治,一定程度上可以通过癌毒理论和靶向治疗理论实现；
2、Bullatacin体外对人乳腺癌细胞MDA-MB-231具有较强的抑制作用,使G1期的细胞比率减少,S期的细胞比率增加,细胞周期明显阻滞在S期,这种抑制作用,同时有量效与时效关系；
3、Bullatacin诱导了细胞程序性死亡中的副凋亡形式,机理可能与MAPK通路的上游激活有关。
Background:
With the continuous development of living standards, the incidence of breast cancer keeps rising. The paper shows that advanced breast cancer 5-year survival rate is only between 10% and 15%.There is a close relationship between MAPK signal transduction system and breast cancer, so the research with related signal transduction pathway are expected to find a new target for curing breast cancer. As an important component of comprehensive treatment, Chinese medicine has a certain effect and unique advantage. Based on molecular biology methods and Chinese medicine, preventing and treating breast cancer have always being an extraordinary topic on breast cancer in China. Recently, there are some progresses on Annona anti-cancer, which is known as the "Star of Tomorrow Anti-cancer". Annona lactone compound, followed by the taxol, is considered as another promising and natural source of anti-tumor compounds. Bullatacin is the strongest anti-cancer activity lactone isolated from Annona lactone compound and its activity in mouse vivo is 400 to 600 times stronger than taxol. This project was to explain inhibition and mechanism of inhibition in human breast cancer cells of Bullatacin and to provide theoretical and experimental basis for the anti-cancer drug development of Chinese medicine. Furthermore, it established a theoretical basis for Chinese and Western combined treatment of breast cancer.
Purpose:
1. Clarifying the toxicity of cancer is the key factor, standardized and individualized, unity on the contradiction between treatment of diseases and treatment of dialectical in the process of development of breast cancer and provide a theoretical basis for orientation and thinking of Chinese and Western combined treatment of breast cancer.
2. Selecting a stronger anti-cancer activity Annona lactone monomer, called Bullatacin, from Alcohol extract of Annona seed. The main purpose was to study on inhibition and mechanism of inhibition in human breast cancer cells of Bullatacin and to provide theoretical and experimental basis for the anti-cancer drug development of Chinese medicine
Methods:
The method was combination of literature and modern experimental research:
1. literature research:
1.1 Accessed to ancient and modern Chinese medical literature on breast cancer in order to find its occurrence and development factors, and then explained.
1.2 According to evidence-based Medicine, analysing theory and clinical practice on breast cancer, and exploring a point about Chinese and Western combined treatment of breast cancer.
2. Experimental study:
2.1 Extraction Annona lactone monomer Bullatacin: Crushed seeds of Annona, extracted 3 times with 95% ethanol, successively extracted with petroleum ether, chloroform saturated with water, methanol, Annona lactone compound was isolated from the chloroform part according to Silica gel column chromatography and recrystallizationh, Annona total lactones parts and Bullatacin monomer compound was obtained by the same way.
2.2 Observed the inhibition effect of Bullatacin on proliferation of ER (+)MCF-7 and ER (-) MDA-MB-231 cells with MTT method in vitro. Examined if Bullacatin had inhibiting or not and effect potency with ER(+)MCF-7 and ER(-)MDA-MB-231 cells, in addition selected their own effective dose and time.
2.3 Observed the effects of Bullatacin on cell morphology of MDA-MB-231: Observed the effect of 20ug/ml Bullatacin to MDA-MB-231 cells on morphology by inverted microscope and transmission electron microscopy.
2.4 The study on cell cycle of MDA-MB-231 treated with Bullacatin by flow cytometry: According to analysis of FCM, the effects on cell cycle of MDA-MB-23 at time 0h、12h、24h、48h after 20ug/ml Bullatacin treatment.
2.5 Observed the effects of Bullatacin on apoptosis of MDA-MB-231 with Annexin V-FITC staining: The apoptosis was measured by FCM, tested the apoptosis of MDA-MB-231 treated with 20ug/ml Bullatacin. After adding to MAPK pathway agonists and the inhibitor of main pathway respectively, testing the effects of apoptosis
2.6 Western Blotting detected on Bullacatin regulated MAPK pathway with MDA-MB-231 With treating 20ug/mlBullatacin,MAPKpathway agonists and the inhibitor of main pathway on MDA-MB-231 respectively, tested the expression of different experimental groups phosphorylation and the total protein as well as regulation of MAPK pathway according to western blotting.
Results:
1. Caner poison breakthrough the occurrence and development of breast carcinoma, Evidence-Based Medicine request standardized and individualized on the Treatment of Diseases and on the treatment of Syndrome should go to contradictory unity;
2. We get Annona lactones compound from chloroform part of Annona Seeds,by silicagel column chromatography and recrystallization,we get the compound, According to the physical constants and spectral data to identify the structure, learn to Bullatacin> 95% purity.
3.Samples of bullacatin have the inhibition on both MCF7 cells of ER(+)and M-DA-MB-231 ce 11-s of ER(-) and a dose and time dependence.Compared with taxol group, there is the no diffe-rence on the 20 ug/ml of Bullacatin roled in the MDA-MB-231 cell inhibition rate for 48h
4.20ug/ml of Bullacatin role in cells, cell ratio of G1 period reducecell ratio of S-period ad-d a nd the cell cycle stay on the S- period obviously. Alongwith the ti-me of the Bullacatin roles, t he cell ratio of G1 period reduces and the cell ratio of S-period a-dds. The bullacation influe nces the cell cycle with the time dependence.
5. Bullacatin 20ug/ml act on MDA-MB-231 cell for 48hours:
5.1 be detected by optical microscope:cell density and proliferation rate is lower in than that in control group,the cells in model group become larger and more round gradually.the space of cells is looser,cytoplasma granula is increasing,blebbing, dropped, so chips around cells is increasing.
5.2 be detected by electric mirror membrane microvilli is almost lost, cytoplasm of organelle show marked dilation.mitochondria is the worst one.they have apparent vacuole to form,endoplasmatic enlargement and organelle has exosmose,but typical apoptosis and apoptotic body are noshown.
Conclusions:
1. Breast cancer required the standardization of modern medicine and individualized therapy. Traditional Chinese medicine theory advocated by the same disease with different treatment, different diseases. To a certain extent, cancer by targeting therapy and the theory of caner poison.
2. Bullatacin on human breast cancer cells MDA-MB-231 has strong inhibitory effect.It can make enabling cell cycle arrest in S- phase. The inhibition has a dose and time dependent;
3. Bullatacin induced programmed cell death in the form of paraptosis, the mechanism may be upstream of MAPK pathway activation.
随着人们生活水平的不断提高,乳腺癌发病率呈上升趋势,世界银行及世界卫生组织全球疾病负担报告所示：2002年乳腺癌在全球所造成的损失约614万伤残调整寿命年(Disability adjust Life years, DALYs),居女性恶性肿瘤疾病负担的首位,控制乳腺癌被认为是降低全球癌症负担的最有效的措施之一。中医药作为综合治疗的重要组成部分,有一定的疗效和独特的优势,但是长期以来,中医药在乳腺癌综合治疗中的地位不高,探讨中医药防治乳腺癌在综合治疗中的切入点和时机,运用分子生物学方法研究中医药防治乳腺癌机理,一直是我国乳腺癌研究的重要课题。番荔枝被称为“明日抗癌之星”,近年来国内外在番荔枝抗肿瘤的研究方面,取得了一定的进展,番荔枝内酯化合物被认为是继紫杉醇后的又一个有前途的、天然来源的抗肿瘤化合物,从番荔枝中分离出的抗癌活性最强的内酯为Bullatacin,研究表明：Bullatacin在小鼠体内活性比紫杉醇强400-600倍,本课题从番荔枝种子的醇浸膏中,分离出Bullatacin,研究其对人乳腺癌细胞的作用及其作用机理,为抗乳腺癌的中药新药开发提供理论和实验依据,进一步为中西结合治疗乳腺癌奠定理论基础。
研究目的：
1、阐明癌毒是乳腺癌发生发展的关键因素,规范化和个体化,辨病论治和辨证论治矛盾统一,为中西结合治疗乳腺癌的方向和思路提供一定的理论依据；
2、从番荔枝种子的醇浸膏中,筛选出抗癌活性最强的番荔枝内酯单体Bullatacin,研究其对人乳腺癌细胞的作用及其作用机理,为抗乳腺癌的中药新药开发提供理论和实验依据。
研究方法：
采用文献研究和现代实验研究相结合的方法。
1、文献研究：
1)对乳腺癌的古代与现代中医药文献进行研究,找寻出乳腺癌发生发展的关键因素,并对其进行阐发；
2)用循证医学的观点和方法,对现代医学关于乳腺癌的理论与临床实践进行一定的梳理,寻找中西结合诊治乳腺癌的切入点和时机。
2、实验研究：
2.1、番荔枝内酯单体Bullatacin的提取：
番荔枝种子粉碎,95%乙醇冷浸,回流提取3次,依次用石油醚、水饱和氯仿、甲醇萃取,通过反复硅胶柱层析、重结晶等方法从氯仿部分,分得番荔枝内酯类化合物,通过反复硅胶柱层析和重结晶等方法,得到番荔枝总内酯部位和Bullatacin单体化合物。
2.2、MTT法观察Bullatacin体外对ER(+)人乳腺癌细胞MCF-7和ER(-)人乳腺癌细胞MDA-MB-231的作用：
采用MTT法,,测试受试样品Bullacatin体外对ER(+)的人乳腺癌细胞MCF-7和ER(-)人乳腺癌细胞MDA-MB-231有无抑制作用及作用强度,并筛选出各自的有效剂量和作用时间。
2.3、观察Bullacatin对人乳腺癌细胞MDA-MB-231细胞形态学的影响：
倒置显微镜与透射电镜观察20ug/ml Bullacatin体外对ER(-)人乳腺癌细胞MDA-MB-231的形态学的影响。
2.4、流式细胞仪检测Bullacatin对人乳腺癌细胞MDA-MB-231细胞周期的影响：流式细胞仪分析,20ug/ml Bullacatin作用人乳腺癌细胞MDA-MB-231 0h、12h、24h、48h对细胞周期的影响
2.5、Annexin V-FITC染色流式细胞术考察Bullacatin对人乳腺癌细胞MDA-MB-231凋亡的影响：
Annexin V-FITC染色流式细胞仪检测凋亡,考察Bullacatin20ug/ml作用人乳腺癌细胞MDA-MB-231 48h对细胞凋亡的影响,分别加入MAPK通路激动剂与MAPK各主要通路的抑制剂后,观察药物对细胞凋亡的影响
2.6、Western Blot检测Bullacatin对人乳腺癌细胞MDA-MB-231 MAPK通路的作用：
Western Blotting检测Bullacatin 20ug/m作用人乳腺癌细胞MDA-MB-231 48h和分别加入MAPK通路激动剂与MAPK各主要通路的抑制剂后,不同实验组磷酸化与总蛋白的表达,观测Bullacatin对MAPK信号通路的作用。
研究结果：
1、癌毒贯穿乳腺癌发生与发展的始终,循证医学要求规范化和个体化,辨病论治和辩证论治的矛盾统一；
2、从番荔枝种子氯仿部分,分得番荔枝内酯类化合物,通过反复硅胶柱层析和重结晶等方法,得到的化合物,根据理化常数和波谱学数据鉴定了结构,为Bullatacin,纯度大于95%；
3、受试样品Bullacatin不管对ER(+)的MCF-7还是ER(-)MDA-MB-231细胞均有具有抑制作用,且有剂量和时间依赖性。20ug/ml的Bullacatin作用48小时,对于MDA-MB-231细胞的抑制率与紫杉醇组相比,没有差异；
4、20ug/ml的Bullacatin作用于细胞后,G1期的细胞比率减少,S期的细胞比率增加,细胞周期明显阻滞在S期。且G1期细胞比率随着Bullacatin作用的时间延长而减少,S期细胞比率随着Bullacatin作用的时间延长增加,Bullacatin对细胞周期的影响具有时间依赖性。
5、Bullacatin20ug/ml,作用于MDA-MB-231 48h细胞：
1)普通倒置显微镜下见：细胞密度较空白组减低,增殖变慢,细胞逐渐变大、变圆,细胞间接触变松,胞浆中颗粒增多,空泡化,脱壁,细胞周围碎片增多；
2)电镜显示：细胞表面微绒毛部分或全部脱失,胞质内细胞器明显扩张,以线粒体扩张明显,并有许多空泡形成,内质网扩张,细胞器有外渗现象,未见典型的凋亡和凋亡小体；
3)Bullacatin20ug/ml作用于MDA-MB-231细胞48小时,流式细胞仪检测,Annexin V染色的细胞为27.1%与阴性对照组4.2%比较,有显著差异；MAPK通路的激动剂能够促进MDA-MB-231 Annexin V染色的细胞的出现,作用较Bullacatin弱,激动剂与Bullacatin有协同作用；分别加入MAPK的3条主要通路的抑制剂,三者虽然与药物组Annexin V染色的细胞27.1%比有所下降,与阴性对照组4.2%比较,仍有显著差异。
6、Bullacatin20ug/ml，作用于MDA-MB-231细胞48小时Western Blotting检测发现：
1)激活ERK通路,导致P-ERK的增强,增强作用大于激动剂对ERK的作用；
2)激活JNK通路,导致P-ERK的增强,增强作用小于激动剂对ERK的作用；
3)激活P38-MAPK通路,导致P-P38的表达增强,增强作用大于激动剂对P38通路的作用。
4)JNK与ERK通路有协同作用；
5)ERK抑制剂PD98059和JNK抑制剂SP600125亦能轻度抑制P38的磷酸化,P38抑制剂SB203580对Bullatacin激活P38通路有抑制作用,但不能完全抑制其表达
研究结论：
1、乳腺癌现代医学要求的规范化和个体化治疗,中医理论所倡导的同病异治、异病同治,一定程度上可以通过癌毒理论和靶向治疗理论实现；
2、Bullatacin体外对人乳腺癌细胞MDA-MB-231具有较强的抑制作用,使G1期的细胞比率减少,S期的细胞比率增加,细胞周期明显阻滞在S期,这种抑制作用,同时有量效与时效关系；
3、Bullatacin诱导了细胞程序性死亡中的副凋亡形式,机理可能与MAPK通路的上游激活有关。
Background:
With the continuous development of living standards, the incidence of breast cancer keeps rising. The paper shows that advanced breast cancer 5-year survival rate is only between 10% and 15%.There is a close relationship between MAPK signal transduction system and breast cancer, so the research with related signal transduction pathway are expected to find a new target for curing breast cancer. As an important component of comprehensive treatment, Chinese medicine has a certain effect and unique advantage. Based on molecular biology methods and Chinese medicine, preventing and treating breast cancer have always being an extraordinary topic on breast cancer in China. Recently, there are some progresses on Annona anti-cancer, which is known as the "Star of Tomorrow Anti-cancer". Annona lactone compound, followed by the taxol, is considered as another promising and natural source of anti-tumor compounds. Bullatacin is the strongest anti-cancer activity lactone isolated from Annona lactone compound and its activity in mouse vivo is 400 to 600 times stronger than taxol. This project was to explain inhibition and mechanism of inhibition in human breast cancer cells of Bullatacin and to provide theoretical and experimental basis for the anti-cancer drug development of Chinese medicine. Furthermore, it established a theoretical basis for Chinese and Western combined treatment of breast cancer.
Purpose:
1. Clarifying the toxicity of cancer is the key factor, standardized and individualized, unity on the contradiction between treatment of diseases and treatment of dialectical in the process of development of breast cancer and provide a theoretical basis for orientation and thinking of Chinese and Western combined treatment of breast cancer.
2. Selecting a stronger anti-cancer activity Annona lactone monomer, called Bullatacin, from Alcohol extract of Annona seed. The main purpose was to study on inhibition and mechanism of inhibition in human breast cancer cells of Bullatacin and to provide theoretical and experimental basis for the anti-cancer drug development of Chinese medicine
Methods:
The method was combination of literature and modern experimental research:
1. literature research:
1.1 Accessed to ancient and modern Chinese medical literature on breast cancer in order to find its occurrence and development factors, and then explained.
1.2 According to evidence-based Medicine, analysing theory and clinical practice on breast cancer, and exploring a point about Chinese and Western combined treatment of breast cancer.
2. Experimental study:
2.1 Extraction Annona lactone monomer Bullatacin: Crushed seeds of Annona, extracted 3 times with 95% ethanol, successively extracted with petroleum ether, chloroform saturated with water, methanol, Annona lactone compound was isolated from the chloroform part according to Silica gel column chromatography and recrystallizationh, Annona total lactones parts and Bullatacin monomer compound was obtained by the same way.
2.2 Observed the inhibition effect of Bullatacin on proliferation of ER (+)MCF-7 and ER (-) MDA-MB-231 cells with MTT method in vitro. Examined if Bullacatin had inhibiting or not and effect potency with ER(+)MCF-7 and ER(-)MDA-MB-231 cells, in addition selected their own effective dose and time.
2.3 Observed the effects of Bullatacin on cell morphology of MDA-MB-231: Observed the effect of 20ug/ml Bullatacin to MDA-MB-231 cells on morphology by inverted microscope and transmission electron microscopy.
2.4 The study on cell cycle of MDA-MB-231 treated with Bullacatin by flow cytometry: According to analysis of FCM, the effects on cell cycle of MDA-MB-23 at time 0h、12h、24h、48h after 20ug/ml Bullatacin treatment.
2.5 Observed the effects of Bullatacin on apoptosis of MDA-MB-231 with Annexin V-FITC staining: The apoptosis was measured by FCM, tested the apoptosis of MDA-MB-231 treated with 20ug/ml Bullatacin. After adding to MAPK pathway agonists and the inhibitor of main pathway respectively, testing the effects of apoptosis
2.6 Western Blotting detected on Bullacatin regulated MAPK pathway with MDA-MB-231 With treating 20ug/mlBullatacin,MAPKpathway agonists and the inhibitor of main pathway on MDA-MB-231 respectively, tested the expression of different experimental groups phosphorylation and the total protein as well as regulation of MAPK pathway according to western blotting.
Results:
1. Caner poison breakthrough the occurrence and development of breast carcinoma, Evidence-Based Medicine request standardized and individualized on the Treatment of Diseases and on the treatment of Syndrome should go to contradictory unity;
2. We get Annona lactones compound from chloroform part of Annona Seeds,by silicagel column chromatography and recrystallization,we get the compound, According to the physical constants and spectral data to identify the structure, learn to Bullatacin> 95% purity.
3.Samples of bullacatin have the inhibition on both MCF7 cells of ER(+)and M-DA-MB-231 ce 11-s of ER(-) and a dose and time dependence.Compared with taxol group, there is the no diffe-rence on the 20 ug/ml of Bullacatin roled in the MDA-MB-231 cell inhibition rate for 48h
4.20ug/ml of Bullacatin role in cells, cell ratio of G1 period reducecell ratio of S-period ad-d a nd the cell cycle stay on the S- period obviously. Alongwith the ti-me of the Bullacatin roles, t he cell ratio of G1 period reduces and the cell ratio of S-period a-dds. The bullacation influe nces the cell cycle with the time dependence.
5. Bullacatin 20ug/ml act on MDA-MB-231 cell for 48hours:
5.1 be detected by optical microscope:cell density and proliferation rate is lower in than that in control group,the cells in model group become larger and more round gradually.the space of cells is looser,cytoplasma granula is increasing,blebbing, dropped, so chips around cells is increasing.
5.2 be detected by electric mirror membrane microvilli is almost lost, cytoplasm of organelle show marked dilation.mitochondria is the worst one.they have apparent vacuole to form,endoplasmatic enlargement and organelle has exosmose,but typical apoptosis and apoptotic body are noshown.
Conclusions:
1. Breast cancer required the standardization of modern medicine and individualized therapy. Traditional Chinese medicine theory advocated by the same disease with different treatment, different diseases. To a certain extent, cancer by targeting therapy and the theory of caner poison.
2. Bullatacin on human breast cancer cells MDA-MB-231 has strong inhibitory effect.It can make enabling cell cycle arrest in S- phase. The inhibition has a dose and time dependent;
3. Bullatacin induced programmed cell death in the form of paraptosis, the mechanism may be upstream of MAPK pathway activation.
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