本文關(guān)鍵詞： 咖啡酸3 4-二羥基-苯乙基酯 乳腺癌 細(xì)胞凋亡 線粒體凋亡途徑　出處：《吉林大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：乳腺癌是常見的惡性腫瘤，嚴(yán)重威脅女性健康。在乳腺癌中，生長因子及其受體的異常激活起著關(guān)鍵的作用，如何抑制該途徑發(fā)揮作用一直是研究的熱點。植物藥副作用小、對正常細(xì)胞幾乎無毒性，所以人們一直試圖從植物中提取出對抗生長因子作用的有效成分。咖啡酸3,4-二羥基-苯乙基酯（CADPE）是從植物長毛香科（Teucriumpilosum，又名鐵馬鞭）和草珊瑚中分離出來的抗腫瘤藥物。由于CADPE毒副作用較少、安全性好，且具有廣譜的抗腫瘤作用，近年受到廣泛關(guān)注。但CADPE是否具有抗乳腺癌作用及其相關(guān)的作用機(jī)制，仍缺乏相關(guān)報道。因此，本研究采用人乳腺癌細(xì)胞系，探討了CADPE的抗乳腺癌作用及其相關(guān)機(jī)制，發(fā)現(xiàn)CADPE可以抑制乳腺癌細(xì)胞增殖、誘導(dǎo)凋亡，并有望成為有效的生長因子拮抗劑。 一、方法 （一）乳腺癌細(xì)胞增殖 人乳腺癌細(xì)胞系MCF-7、MCF-7ADR、MDA-MB-231和MDA-MB-435細(xì)胞體外培養(yǎng)，同時添加CADPE處理細(xì)胞，采用單細(xì)胞增殖檢測試劑盒檢測乳腺癌細(xì)胞增殖情況。 （二）乳腺癌細(xì)胞凋亡檢測 Hoechst33342染色法、流式細(xì)胞術(shù)Annexin V-PE/7-AAD雙染色檢測CADPE作用于人乳腺癌細(xì)胞系MDA-MB-231和MDA-MB-435細(xì)胞的形態(tài)學(xué)變化及凋亡率。 （三）乳腺癌細(xì)胞線粒體凋亡途徑相關(guān)測定 采用不同濃度CADPE作用于人乳腺癌細(xì)胞系MDA-MB-231和MDA-MB-435細(xì)胞，利用DCFH-DA（細(xì)胞活性氧檢測探針）測定細(xì)胞內(nèi)的活性氧變化，JC-1法測定線粒體膜電位改變，Western blot檢測caspase-3，Bcl-2和Bax蛋白表達(dá)。 （四）生長因子受體及相關(guān)酪氨酸激酶磷酸化檢測 Western blot方法檢測CADPE對VEGF、EGF、PDGF、HGF刺激的人乳腺癌細(xì)胞系MDA-MB-231和MDA-MB-435細(xì)胞VEGFR、EGFR、Src及C-Met磷酸化水平的影響。 （五）Her-2、c-myc及maz蛋白表達(dá)分析 Western blot方法檢測CADPE處理乳腺癌細(xì)胞系MDA-MB-231和MDA-MB-435細(xì)胞后Her-2、c-myc及maz蛋白表達(dá)。 （六）裸鼠體內(nèi)荷瘤實驗 將人乳腺癌細(xì)胞系MDA-MB-231細(xì)胞接種裸鼠皮下，待成瘤后，每周兩次腹腔注射2.5mg/kg的CAPDE，觀察CADPE對腫瘤生長及裸鼠體重影響。 二、結(jié)果 （一）CADPE抑制乳腺癌細(xì)胞增殖 采用CADPE處理乳腺癌細(xì)胞系MDA-MB-231、MDA-MB-435、MCF-7和MCF-7ADR細(xì)胞，結(jié)果顯示CADPE10～80μmol/L的CADPE作用24h、48h和72h，均能明顯抑制乳腺癌細(xì)胞增殖。 （二）CADPE誘導(dǎo)乳腺癌細(xì)胞凋亡 采用Hoechst33342染色乳腺癌MDA-MB-231和MDA-MB-435細(xì)胞，熒光顯微鏡下觀察CADPE處理后細(xì)胞形態(tài)變化，結(jié)果顯示存在細(xì)胞核濃縮等細(xì)胞凋亡特征。Annexin V-PE/7-AAD雙染色結(jié)果顯示20μmol/LCADPE作用48小時后細(xì)胞的凋亡率較對照組顯著增高（p0.01）。 （三）CADPE促進(jìn)乳腺癌細(xì)胞活性氧產(chǎn)生 CADPE（0～80μmol/L）處理人乳腺癌細(xì)胞系MDA-MB-231及MDA-MB-435細(xì)胞48h，結(jié)果顯示CADPE能明顯增加細(xì)胞內(nèi)活性氧（ROS）產(chǎn)生。 （四）CADPE降低乳腺癌細(xì)胞線粒體膜電位 JC-1法測定線粒體膜電位，結(jié)果顯示CADPE處理后乳腺癌細(xì)胞的線粒體膜電位明顯降低。 （五）CADPE對乳腺癌細(xì)胞Caspase-3、Bax和Bcl-2表達(dá)的影響 Western blot檢測結(jié)果顯示CADPE處理后乳腺癌細(xì)胞Caspase-3和Bax表達(dá)上調(diào)，，而Bcl-2表達(dá)下調(diào)。 （六）CADPE抑制配體刺激的乳腺癌細(xì)胞VEGFR、EGFR及C-Met磷酸化 MDA-MB-231和MDA-MB-435細(xì)胞分別經(jīng)相應(yīng)配體刺激后，VEGFR、EGFR、Src、C-Met磷酸化增加，而預(yù)先使用CADPE處理組，VEGFR、EGFR和C-Met磷酸化水平相對未處理組增幅較小，但CADPE對Src磷酸化水平無明顯影響。 （七）CADPE影響乳腺癌細(xì)胞EGFR磷酸化的時間及劑量依賴性 MDA-MB-435細(xì)胞經(jīng)EGF刺激后，EGFR磷酸化水平增高，這種作用隨CADPE孵育時間延長或者CADPE濃度增加而降低。 （八）CADPE抑制乳腺癌細(xì)胞Her-2、c-myc及maz蛋白表達(dá) CADPE處理乳腺癌MDA-MB-231和MDA-MB-435細(xì)胞后，可以降低Her-2、c-myc及maz蛋白表達(dá)。 （九）CADPE對裸鼠體內(nèi)乳腺癌細(xì)胞瘤生長的影響 裸鼠接種乳腺癌細(xì)胞系MDA-MB-231細(xì)胞成瘤后，給予CADPE處理，第38d、42d腫瘤體積明顯小于對照組（P＝0.046,0.025），兩組瘤重差別具有統(tǒng)計學(xué)意義（P＝0.017），但兩組小鼠體重的變化無明顯差別。 三、結(jié)論 1. CADPE能抑制人乳腺癌細(xì)胞系MCF-7、MCF-7ADR、MDA-MB-231和MDA-MB-435細(xì)胞增殖，并具有時間及劑量依賴性。裸鼠體內(nèi)實驗進(jìn)一步表明CADPE具有抗乳腺癌的作用。 2. CADPE可以增加Bax和caspase-3表達(dá)、下調(diào)Bcl-2表達(dá)、降低線粒體膜電位和提高細(xì)胞內(nèi)活性氧水平，觸發(fā)線粒體凋亡通路引起乳腺癌細(xì)胞凋亡。 3. CADPE可以降低生長因子誘導(dǎo)的VEGFR、EGFR和C-Met磷酸化，提示其可能通過抑制生長因子作用進(jìn)而抑制乳腺癌細(xì)胞增殖。 4. CADPE抑制Her-2和c-myc及其上游基因maz蛋白產(chǎn)物的表達(dá)，表明其對這些乳腺癌中具有標(biāo)志性作用的致癌基因具有負(fù)性調(diào)控作用。 綜上所述，CADPE可能通過抑制生長因子受體磷酸化，進(jìn)而抑制下游相關(guān)癌基因Her-2、c-myc和maz的表達(dá)，從而抑制乳腺癌細(xì)胞增殖及誘導(dǎo)乳腺癌細(xì)胞凋亡。體內(nèi)、外實驗均顯示CADPE具有抗乳腺癌作用，有望進(jìn)一步開發(fā)成乳腺癌治療藥物。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 付菊琴;曲瑋;梁敬鈺;;草珊瑚屬植物的研究進(jìn)展[J];海峽藥學(xué);2011年01期

2 ;Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro[J];World Journal of Gastroenterology;2008年15期

本文編號：1532344