咖啡酸3，4-二羟基—苯乙基酯诱导乳腺癌细胞凋亡的作用研究

发布时间：2018-02-25 00:27

本文关键词： 咖啡酸3 4-二羟基-苯乙基酯乳腺癌细胞凋亡线粒体凋亡途径　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：乳腺癌是常见的恶性肿瘤，严重威胁女性健康。在乳腺癌中，生长因子及其受体的异常激活起着关键的作用，如何抑制该途径发挥作用一直是研究的热点。植物药副作用小、对正常细胞几乎无毒性，所以人们一直试图从植物中提取出对抗生长因子作用的有效成分。咖啡酸3,4-二羟基-苯乙基酯（CADPE）是从植物长毛香科（Teucriumpilosum，又名铁马鞭）和草珊瑚中分离出来的抗肿瘤药物。由于CADPE毒副作用较少、安全性好，且具有广谱的抗肿瘤作用，近年受到广泛关注。但CADPE是否具有抗乳腺癌作用及其相关的作用机制，仍缺乏相关报道。因此，本研究采用人乳腺癌细胞系，探讨了CADPE的抗乳腺癌作用及其相关机制，发现CADPE可以抑制乳腺癌细胞增殖、诱导凋亡，并有望成为有效的生长因子拮抗剂。一、方法（一）乳腺癌细胞增殖人乳腺癌细胞系MCF-7、MCF-7ADR、MDA-MB-231和MDA-MB-435细胞体外培养，同时添加CADPE处理细胞，采用单细胞增殖检测试剂盒检测乳腺癌细胞增殖情况。（二）乳腺癌细胞凋亡检测 Hoechst33342染色法、流式细胞术Annexin V-PE/7-AAD双染色检测CADPE作用于人乳腺癌细胞系MDA-MB-231和MDA-MB-435细胞的形态学变化及凋亡率。（三）乳腺癌细胞线粒体凋亡途径相关测定采用不同浓度CADPE作用于人乳腺癌细胞系MDA-MB-231和MDA-MB-435细胞，利用DCFH-DA（细胞活性氧检测探针）测定细胞内的活性氧变化，JC-1法测定线粒体膜电位改变，Western blot检测caspase-3，Bcl-2和Bax蛋白表达。（四）生长因子受体及相关酪氨酸激酶磷酸化检测 Western blot方法检测CADPE对VEGF、EGF、PDGF、HGF刺激的人乳腺癌细胞系MDA-MB-231和MDA-MB-435细胞VEGFR、EGFR、Src及C-Met磷酸化水平的影响。（五）Her-2、c-myc及maz蛋白表达分析 Western blot方法检测CADPE处理乳腺癌细胞系MDA-MB-231和MDA-MB-435细胞后Her-2、c-myc及maz蛋白表达。（六）裸鼠体内荷瘤实验将人乳腺癌细胞系MDA-MB-231细胞接种裸鼠皮下，待成瘤后，每周两次腹腔注射2.5mg/kg的CAPDE，观察CADPE对肿瘤生长及裸鼠体重影响。二、结果（一）CADPE抑制乳腺癌细胞增殖采用CADPE处理乳腺癌细胞系MDA-MB-231、MDA-MB-435、MCF-7和MCF-7ADR细胞，结果显示CADPE10～80μmol/L的CADPE作用24h、48h和72h，均能明显抑制乳腺癌细胞增殖。（二）CADPE诱导乳腺癌细胞凋亡采用Hoechst33342染色乳腺癌MDA-MB-231和MDA-MB-435细胞，荧光显微镜下观察CADPE处理后细胞形态变化，结果显示存在细胞核浓缩等细胞凋亡特征。Annexin V-PE/7-AAD双染色结果显示20μmol/LCADPE作用48小时后细胞的凋亡率较对照组显著增高（p0.01）。（三）CADPE促进乳腺癌细胞活性氧产生 CADPE（0～80μmol/L）处理人乳腺癌细胞系MDA-MB-231及MDA-MB-435细胞48h，结果显示CADPE能明显增加细胞内活性氧（ROS）产生。（四）CADPE降低乳腺癌细胞线粒体膜电位 JC-1法测定线粒体膜电位，结果显示CADPE处理后乳腺癌细胞的线粒体膜电位明显降低。（五）CADPE对乳腺癌细胞Caspase-3、Bax和Bcl-2表达的影响 Western blot检测结果显示CADPE处理后乳腺癌细胞Caspase-3和Bax表达上调，，而Bcl-2表达下调。（六）CADPE抑制配体刺激的乳腺癌细胞VEGFR、EGFR及C-Met磷酸化 MDA-MB-231和MDA-MB-435细胞分别经相应配体刺激后，VEGFR、EGFR、Src、C-Met磷酸化增加，而预先使用CADPE处理组，VEGFR、EGFR和C-Met磷酸化水平相对未处理组增幅较小，但CADPE对Src磷酸化水平无明显影响。（七）CADPE影响乳腺癌细胞EGFR磷酸化的时间及剂量依赖性 MDA-MB-435细胞经EGF刺激后，EGFR磷酸化水平增高，这种作用随CADPE孵育时间延长或者CADPE浓度增加而降低。（八）CADPE抑制乳腺癌细胞Her-2、c-myc及maz蛋白表达 CADPE处理乳腺癌MDA-MB-231和MDA-MB-435细胞后，可以降低Her-2、c-myc及maz蛋白表达。（九）CADPE对裸鼠体内乳腺癌细胞瘤生长的影响裸鼠接种乳腺癌细胞系MDA-MB-231细胞成瘤后，给予CADPE处理，第38d、42d肿瘤体积明显小于对照组（P＝0.046,0.025），两组瘤重差别具有统计学意义（P＝0.017），但两组小鼠体重的变化无明显差别。三、结论 1. CADPE能抑制人乳腺癌细胞系MCF-7、MCF-7ADR、MDA-MB-231和MDA-MB-435细胞增殖，并具有时间及剂量依赖性。裸鼠体内实验进一步表明CADPE具有抗乳腺癌的作用。 2. CADPE可以增加Bax和caspase-3表达、下调Bcl-2表达、降低线粒体膜电位和提高细胞内活性氧水平，触发线粒体凋亡通路引起乳腺癌细胞凋亡。 3. CADPE可以降低生长因子诱导的VEGFR、EGFR和C-Met磷酸化，提示其可能通过抑制生长因子作用进而抑制乳腺癌细胞增殖。 4. CADPE抑制Her-2和c-myc及其上游基因maz蛋白产物的表达，表明其对这些乳腺癌中具有标志性作用的致癌基因具有负性调控作用。综上所述，CADPE可能通过抑制生长因子受体磷酸化，进而抑制下游相关癌基因Her-2、c-myc和maz的表达，从而抑制乳腺癌细胞增殖及诱导乳腺癌细胞凋亡。体内、外实验均显示CADPE具有抗乳腺癌作用，有望进一步开发成乳腺癌治疗药物。
[Abstract]:Breast cancer is a common malignant tumor, a serious threat to women's health. In breast cancer, growth factor and its receptor activation plays a key role, how to inhibit the pathway has been a research hotspot. Side effects of herbal medicine, almost non-toxic to normal cells, so people have been trying to extracted from plants to counter the effective components of growth factors. Caffeic acid 3,4- two hydroxy benzene ethyl ester (CADPE) from plant hair incense (Teucriumpilosum, also known as the iron whip) isolated and Caoshanhu anticancer drugs. Due to the side effects of CADPE less, good safety, and has a broad-spectrum anti-tumor effect in recent years, attracted widespread attention. But whether CADPE has the anti breast cancer effect and mechanism of the lack of relevant reports. Therefore, this study used human breast cancer cell line, investigate the CADPE effect and anti breast cancer It is found that CADPE can inhibit the proliferation of breast cancer cells and induce apoptosis, and it is expected to be an effective growth factor antagonist.
First, method
(I) proliferation of breast cancer cells
Human breast cancer cell line MCF-7, MCF-7ADR, MDA-MB-231 and MDA-MB-435 cells were cultured in vitro, and CADPE treated cells were added. The proliferation of breast cancer cells was detected by single cell proliferation detection kit.
(two) detection of apoptosis in breast cancer cells
Hoechst33342 staining, flow cytometry and Annexin V-PE/7-AAD double staining were used to detect the morphological and apoptotic rate of CADPE in human breast cancer cell line MDA-MB-231 and MDA-MB-435 cells.
(three) detection of mitochondrial apoptosis pathway in breast cancer cells
Human breast cancer cell line MDA-MB-231 and MDA-MB-435 cells were treated with different concentrations of CADPE. The reactive oxygen species in cells were detected by DCFH-DA (cell reactive oxygen probe), JC-1 membrane method was applied to detect mitochondrial membrane potential changes, Western blot was used to detect Caspase-3, Bcl-2 and Bax protein expression.
(four) detection of growth factor receptor and related tyrosine kinase phosphorylation
Western blot method was used to detect the effect of CADPE on VEGF, EGF, PDGF, HGF stimulated MDA-MB-231, MDA-MB-435 cells, VEGFR, EGFR, MDA-MB-435 and phosphorylation level of human breast cancer cell line.
(five) expression and analysis of Her-2, c-myc and maz protein
Western blot method was used to detect the expression of Her-2, c-myc and maz protein after CADPE treatment of breast cancer cell lines MDA-MB-231 and MDA-MB-435 cells.
(six) a tumor bearing experiment in nude mice
The human breast cancer cell line MDA-MB-231 cells were inoculated subcutaneously in nude mice. After tumor formation, 2.5mg/kg CAPDE was injected intraperitoneally two times a week to observe the effect of CADPE on tumor growth and body weight in nude mice.
Two, the result
(1) CADPE inhibits the proliferation of breast cancer cells
CADPE was used to treat breast cancer cell lines MDA-MB-231, MDA-MB-435, MCF-7 and MCF-7ADR cells. The results showed that CADPE action of CADPE10 to 80 mol/L could significantly inhibit the proliferation of breast cancer cells, including 24h, 48h and 72h.
(two) CADPE induced apoptosis in breast cancer cells
Using Hoechst33342 staining in breast cancer MDA-MB-231 and MDA-MB-435 cells. The morphological change of CADPE cells were observed under fluorescence microscope after treatment, the results showed the presence of nucleus concentration features of apoptosis.Annexin V-PE/7-AAD double staining showed that 48 hours after 20 mol/LCADPE cell apoptosis rate was significantly higher than the control group (P0.01).
(three) CADPE promotes the production of reactive oxygen species in breast cancer cells
CADPE (0~80 mu mol/L) treated human breast cancer cell line MDA-MB-231 and MDA-MB-435 cells 48h. The results showed that CADPE could significantly increase the production of intracellular reactive oxygen species (ROS).
(four) CADPE reduces the mitochondrial membrane potential of breast cancer cells
The mitochondrial membrane potential was measured by JC-1 method. The results showed that the mitochondrial membrane potential of breast cancer cells decreased significantly after CADPE treatment.
(five) the effect of CADPE on the expression of Caspase-3, Bax and Bcl-2 in breast cancer cells
The results of Western blot detection showed that the expression of Caspase-3 and Bax in breast cancer cells was up-regulated after CADPE treatment, and the expression of Bcl-2 was down.
(six) the phosphorylation of VEGFR, EGFR and C-Met in breast cancer cells with CADPE inhibitory ligand
After MDA-MB-231 and MDA-MB-435 cells were stimulated by corresponding ligands, the phosphorylation of VEGFR, EGFR, Src and C-Met increased, while the level of VEGFR, EGFR and C-Met phosphorylation in the pretreatment group was smaller than that in the untreated group, but CADPE had no significant effect on the phosphorylation level of the phosphorylation.
(seven) the effect of CADPE on the time and dose dependence of EGFR phosphorylation in breast cancer cells
When MDA-MB-435 cells were stimulated by EGF, the level of phosphorylation of EGFR was increased. This effect decreased with the prolongation of the incubation time of CADPE or the increase in the concentration of CADPE.
(eight) CADPE inhibits the expression of Her-2, c-myc and maz protein in breast cancer cells
After CADPE treatment of breast cancer MDA-MB-231 and MDA-MB-435 cells, the expression of Her-2, c-myc and maz protein can be reduced.
(nine) the effect of CADPE on the growth of breast cancer cell tumor in nude mice
After inoculation of breast cancer cell line MDA-MB-231 into nude mice, CADPE was treated. The tumor volume of 38d and 42d was significantly smaller than that of the control group (P = 0.046,0.025). The difference of tumor weight between the two groups was statistically significant (P = 0.017), but there was no significant difference in body weight between the two groups.
Three. Conclusion
1. CADPE inhibited the proliferation of human breast cancer cell lines MCF-7, MCF-7ADR, MDA-MB-231 and MDA-MB-435 in a time and dose-dependent manner. Further experiments in nude mice showed that CADPE has the effect of anti breast cancer.
2. CADPE can increase the expression of Bax and Caspase-3, down regulate Bcl-2 expression, decrease mitochondrial membrane potential and increase intracellular reactive oxygen species, triggering mitochondrial apoptosis pathway to induce apoptosis in breast cancer cells.
3. CADPE can reduce the phosphorylation of VEGFR, EGFR and C-Met induced by growth factors, suggesting that it may inhibit the proliferation of breast cancer cells by inhibiting the effect of growth factors.
4. CADPE inhibited the expression of maz and protein products of Her-2 and c-myc and their upstream genes, indicating that they play a negative regulatory role in these breast cancer markers.
In conclusion, CADPE may inhibit growth factor receptor phosphorylation, thereby inhibiting the downstream gene Her-2, expression of c-myc and maz, inhibit the proliferation of breast cancer cells and induce apoptosis of breast cancer cells. In vivo, experiments showed that CADPE has the effect of anti breast cancer, is expected to further development of drugs for the treatment of breast cancer.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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