清毒颗粒抑制血管新生防治乳腺癌的药理机制

【作者】 赵欣；

【导师】 邹忠梅； 蔡大勇；

【作者基本信息】 北京协和医学院 ， 生药学， 2014， 博士

【摘要】 血管新生参与乳腺癌发生发展、浸润、转移和复发。肿瘤血管新生的关键步骤包括血管内皮激活、增殖、迁移与成管。血管内皮生长因子(vascular endothelial growth factor, VEGF)主导肿瘤血管新生。肿瘤缺氧、炎症与凝血参与调节VEGF分泌,通过VEGF受体2(VEGFR2)信号通路诱导血管新生。其中,VEGFR2介导的钙调神经磷酸酶(calcineurin, CaN)/活化T细胞核因子(nuclear factor of activated T cells, NFAT)信号通路在乳腺癌血管新生中起主导作用。清毒颗粒由柴胡、郁金、陈皮、牡丹皮、黄芪、甘草、槐花、紫草、莪术、丹参十味中药组成,有疏肝解郁、滋阴凉血、活血散结的功效,中医临床用于治疗乳腺癌。本研究在前期工作的基础上,采用动物、细胞及分子实验,从VEGFR2及肿瘤相关炎症两条途径,探讨清毒颗粒抗乳腺癌血管新生的药效及药理。第一部分清毒颗粒抗裸鼠荷人乳腺癌血管新生的药效1.裸鼠荷人乳腺癌模型的复制目的：复制裸鼠荷人乳腺癌模型。方法：将浓度为1×106～8×106个/200μL(k=0.5)的MCF-7单细胞悬液接种于裸鼠乳房脂肪垫,测量肿瘤体积,回归MCF-7接种的半数有效浓度(EC50)。结果：MCF-7的EC50及95%置信区间分别为4.75×106个/200μL和3.50x106-6.30×106个/200μL,相关方程为Y=2.93+0.41/(1+10(26.41-3.96x)),R=0.99。结论：成功复制了裸鼠荷人乳腺癌模型。2.槲皮素抑制裸鼠荷人乳腺癌的量效目的：槲皮素抑制裸鼠荷人乳腺癌肿瘤生长的量效关系。方法：荷人乳腺癌裸鼠随机分为8组。槲皮素组以剂量3162.28～3162277.66nmol/kg/d (k=0316)连续灌胃21d。每周2次测量体重与肿瘤体积,回归槲皮素抑制肿瘤生长的量效关系。结果：槲皮素抑制肿瘤生长的ED50为45380.0Onmol/kg/d,其95%置信区间为34316.00-60011.00nmol/kg/d,量效方程为Y=9.27+64.41/(1+10(8.88-1.91x)),R=0.99。结论：槲皮素抑制乳腺癌生长具有剂量依赖性。3.清毒颗粒抑制裸鼠荷人乳腺癌的量效目的：清毒颗粒抑制裸鼠荷人乳腺癌肿瘤生长的量效关系。方法：荷人乳腺癌裸鼠随机分为8组。槲皮素界定剂量(31.62～31622.78nmol/kg/d,k=0.316)的清毒颗粒连续灌胃21d。每周2次测量体重与肿瘤体积,回归清毒颗粒抑制肿瘤生长的量效关系。结果：清毒颗粒抑制肿瘤生长的ED50为1403nmol/kg/d,其95%置信区间为435.90～4518.00nmol/kg/d,量效方程为Y=72.57+70.41/(1+10(0.94x-2.95)),R=0.99。结论：清毒颗粒抑制乳腺癌生长具有剂量依赖性,ED50为槲皮素单独给药的约1/40。4.清毒颗粒抑制裸鼠荷人乳腺癌血管新生的药效机制目的：采用荷人乳腺癌裸鼠,分析清毒颗粒抑制CaN活性抗乳腺癌血管新生的作用。方法：荷人乳腺癌裸鼠随机分为7组,模型组、阳性药三苯氧胺(TAM)组和他克莫司(FK506)组、槲皮素(Qu)组、清毒颗粒低(QDL)、中(QDL)、高(QDL)剂量组,给药21d。检测肿瘤体积、肿瘤坏死体积比、癌细胞增殖、血清VEGF含量、肿瘤血管密度和CaN活性,采用免疫组化、实时定量RT-PCR和Western blot法检测肿瘤组织内VEGF、VEGFR2和NFATc3的表达水平。结果：清毒颗粒抑制肿瘤生长,抑瘤率分别为20.70%、22.43%、22.27%,促进肿瘤坏死、抑制癌细胞增殖、减少血清VEGF含量、降低血管密度、下调CaN活性。清毒颗粒不同程度地降低VEGF、VEGFR2和NFATc3的原位蛋白表达、基因水平和蛋白含量。结论：清毒颗粒下调CaN活性,抑制裸鼠荷人乳腺癌血管新生。第二部分清毒颗粒抑制促癌炎症抗乳腺癌血管新生的药理1.大鼠眼前房荷人乳腺癌模型的建立目的：建立大鼠眼前房荷人乳腺癌模型。方法：将梯度密度MCF-7单细胞悬液(2.5×104～8×105个/mL,k=0.5)减压接种于大鼠眼前房,每日观察记录眼球红色强度,d28处死动物。HE染色观察眼前房肿瘤病理特征、形态计量肿瘤体积比,回归细胞接种密度与肿瘤体积比的量效关系；免疫组化染色检测肿瘤组织内vWF与CD4原位表达。结果：do-d1o眼球逐渐变红,伴有云雾状血管形成；d1o以后红色逐渐消退。病理观察显示随着细胞接种密度升高,肿瘤体积增加；癌细胞起始生长在睫状体,逐渐占据眼前房,随后破坏玻璃体和晶状体,肿瘤组织内有大量含铁血黄素沉积。MCF-7接种的EC50及95%置信区间分别为5×105个/mL和2.31×105-1.05×106个/mL。随着细胞接种密度升高,内皮vWF阳性表达增加,T淋巴细胞CD4阳性颗粒增多。结论：前房红色强度半定量时效与肿瘤体积比定量量效,可建立大鼠眼前房乳腺癌血管新生模型,该模型可用于分析血管新生与肿瘤免疫。2.清毒颗粒抑制乳腺癌炎性血管新生的药理机制目的：采用眼前房荷人乳腺癌大鼠,探索清毒颗粒抑制炎性血管新生的药理机制。方法：眼前房荷人乳腺癌模型大鼠随机分为8组,对照组(接种PBS)、模型组、阳性药三苯氧胺组和FK506组、槲皮素组、清毒颗粒低、中、高剂量组,给药28d。HE染色观察眼前房肿瘤组织,形态计量肿瘤体积比；免疫组化染色显示前房肿瘤组织中vWF,浸润炎性细胞中COX-2、IL-1β、IL-6、STAT3、STAT1、IRF1的细胞定位,形态计量原位阳性表达水平。结果：清毒颗粒不同程度抑制眼前房肿瘤生长、降低血管密度、抑制COX-2、IL-1p、IL-6和STAT3表达,上调STAT1和IRF1表达。结论：清毒颗粒通过下调促癌炎症信号抗乳腺癌血管新生。第三部分清毒颗粒抑制乳腺癌血管新生的药理机制1.清毒颗粒抑制乳腺癌诱导血管新生目的：分析清毒颗粒对人乳腺癌分泌VEGF的影响。方法：以正常、TAM、FK506、Qu、QDL、QDM、QDH含药血清作用于MCF-7细胞,MTT法检测增殖,Elisa法检测培养基上清中VEGF含量,实时定量RT-PCR和Western blot法检测VEGF表达。结果：清毒颗粒抑制MCF-7细胞增殖、减少VEGF分泌、降低VEGF基因与蛋白表达。结论：清毒颗粒阻抑MCF-7细胞VEGF的恶性循环。2.清毒颗粒抑制癌源性内皮血管形成的机制目的：探讨清毒颗粒影响癌源性内皮血管新生的药理机制。方法：(1)分离培养人脐静脉血管内皮细胞(HUVEC)；(2)回归VEGF刺激内皮增殖的量效关系；(3)以培养细胞接触性抑制的时效与量效,建立癌细胞-内皮共培养肿瘤血管新生离体模型。MCF-7以102.5～104.5个/mL(k=0.316)5个浓度接种,培养6h、12h、24h、36h、48h、72h、96h、120h、144h、168h、192h、240h、288h、336h后终止；HUVEC以103-105个/mL(k=0.316)5个浓度接种,培养12h、24h、36h、48h、72h、96h、120h、144h、168h后终止；HE染色后对不同浓度各时间点的细胞数目进行计数,回归细胞发生接触性抑制的浓度(y)与时间(x)的关系；(4)以正常、TAM、FK506、Qu、QDL、QDM、QDH含药血清作用于VEGF诱导或MCF-7共培养的内皮,MTT法检测增殖,transwell法分析迁移,Magial基质胶观察成管,检测CaN活性,免疫组化染色检测NFATc3入核,实时定量RT-PCR和Western blot检测NFATc3表达。结果：(1)HUVEC检出率>90%；(2)VEGF刺激HUVEC增殖的EC50为15.58ng/mL；(3)终点时程决定MCF-7细胞接种密度的回归方程为y=5.07x2-1949x+185642(R=0.99)；终点时程决定HUVEC细胞接种密度的回归方程为y=0.50x2-722.30x+249774(R=0.87)；(4)清毒颗粒抑制VEGF诱导或MCF-7共培养的内皮迁移、成管,下调CaN活性,减少NFATc3入核,降低NFATc3的基因与蛋白表达。结论：清毒颗粒通过抑制内皮NFAT通路抗乳腺癌血管新生。更多还原

【Abstract】 Angiogenesis plays a key role in the development, invasion, metastasis, and recurrence of breast cancer. The angiogenic process includes vascular endothelial activation, proliferation, migration and tube formation. Vascular endothelial growth factor (VEGF) plays leading role in tumor angiogenesis. Tumor hypoxia, inflammation and coagulation involve in the regulation of VEGF secretion, and induce angiogenesis via VEGF receptor2(VEGFR2) signaling pathway. The VEGFR2mediated calcineurin (CaN)/nuclear factor of activated T cells (NFAT) pathway plays key roles in angiogenesis of breast cancer.Qingdu granules is a Chinese herbal formula for treating breast cancer in clinic. The component herbs of this formula were as follows:Radix bupleuri, Radix curcumae, Dried tangerine Peel, Tree Peony Bark, Radix Astragali, Radix Glycyrrhiza, Flos Sophorae, Radix Lithospermi, Curcuma zedoary, and Radix Salviae Miltiorrhizae. In this study, we applied three methods:animal models, cell experimental models and molecular biological methods to explore the underlying mechanisms of Qingdu granules on angiogenesis of breast cancer.Part1Qingdu granules inhibited angiogenesis in nude mice-bearing breast cancer1. Replication of nude mice bearing-breast cancer modelAim:To replicate a nude mice bearing-breast cancer model.Methods:MCF-7cells of1×106～8×106/200μL (k=0.5) were injected to the mammary fat pad of female nude mice. The tumor volumes were measured and the median effect concentration (EC50) of MCF-7cell injection density was calculated.Results:EC50of MCF-7cell injection density was4.75×106/200μL and its95%confidence interval (CI95) was3.50×106～6.30×106/200μL. The equation was Y=2.93+0.41/(1+10(26.41-3.96X)),R=0.99.Conclusion:The nude mice bearing-breast cancer model was successfully replicated.2. Effect of Quercetin on tumor growth in nude mice-bearing breast cancerAim:To observe effect of quercetin on tumor growth of breast cancer-bearing nude mice.Methods:Female nude mice-bearing breast cancer were divided into8groups and intragastrically administration daily with quercetin of316.22～3162277.66nmol/kg/d (k=0.316) for21days. The dose effect relationship of quercetin inhibiting tumor growth was analyzed.Results:ED50of quercetin was45380.00nmol/kg/d and its CI95was34316.00-60011.00nmol/kg/d. The equation was Y=9.27+64.41/(1+10(8.88-191X)), R=0.99.Conclusion:Quercetin inhibited breast tumor growth with dose dependent tendency.3. Effect of Qingdu granules on tumor growth in nude mice-bearing breast cancerAim:To observe effect of Qingdu granules on tumor growth of breast cancer bearing-nude mice.Methods:Female nude mice-bearing breast cancer were divided into8groups and intragastrically administration daily with Qingdu granules containing quercetin of31.62～31622.78nmol/kg/d (k=0.316) for21days. Tumor volumes were measured twice a week. The dose effect relationship of Qingdu granules inhibiting tumor growth was analyzed.Results:ED50of Qingdu granules was1403nmol/kg/d and its CI95was435.90～4518.00nmol/kg/d. The equation was Y=72.57+70.41/(1+10(0.94x-2.95)), R=0.99.Conclusion:Qingdu granules inhibited breast tumor growth with dose dependent tendency, and its ED50is nearly1/40of quercetin used alone.4. Pharmacodynamic mechanisms of Qingdu granules on angiogenesis of nude mice-bearing breast cancerAim:To evaluate the effect of Qingdu granules on angiogenesis of breast cancer bearing-nude mice via inhibiting CaN activity.Methods:Female nude mice-bearing breast cancer were randomly divided into seven groups, i.e., model, tamoxifen (TAM), tacrolimus (FK506), quercetin(Qu), Qingdu granules with low(QDL), middle(QDM) and high(QDH) dose. Tumor inhibitory ratio, and necrotic or proliferative ratios in tumor tissue were evaluated. Serum VEGF levels, vessel density and calcineurin activity were measured. Expression of VEGF, VEGFR2, and NFATc3were determined with immunohistochemical staining, real time RT-PCR and Western blot.Results:Anti-tumor effects of Qingdu granules were shown as decreased tumor volume, the tumor inhibitory ratio in QDL, QDM and QDH was20.70%,22.43%and22.27%, respectively. Qingdu granules promoted tumor necrosis, limited oncocyte proliferation, decreased serum VEGF levels, reduced vessel density and CaN activities. Qingdu granules reduced existed levels, gene levels, and protein amounts of VEGF, VEGFR2and NFATc3.Conclusion:Qingdu granules downregulated CaN activities and inhibited angiogenesis of breast cancer.Part2Qingdu granules inhibited inflammation induced angiogenesis in rat anterior chamber-bearing breast cancer1. Establish of rat anterior chamber-bearing breast cancer modelAim:To establish the anterior chamber-bearing breast cancer model in rats.Methods:MCF-7cells of2.5×l04～8×105/mL (k=0.5) were injected into the anterior chamber of rats. The red change of eye was daily recorded. All rats were carried euthanasia at d28in each group. The pathological changes of anterior chamber were investigated with HE staining. The tumor volume ratio was morphometried. vWF and CD4expression were measured with immunohistochemical staining.Results:d0～d10, the red of eyes become thicker. After d10, the red gradually become thinner. As the elevated of MCF-7injected density, the tumor volume ratios increased, gradually occupied anterior chamber, then destroyed vitreous and lens, and numerous of hemosiderin deposited in tumor tissues. The EC50of MCF-7injected density is5×105/mL with its CI95ranged from2.31×105-1.05×106/mL. As the MCF-7injected density increased, the vWF and CD4positive granules both gradually increased in tumor tissues.Conclusion:The established rat anterior chamber-bearing breast cancer model had the features of both angiogenesis and tumor immunity.2. Mechanisms of Qingdu granules inhibiting inflammatory angiogenesis of breast cancerAim:To explore the underlying mechanisms of Qingdu granules on inflammatory angiogenesis of anterior chamber-bearing breast cancer in rats.Methods:Anterior chamber-bearing breast cancer rats were divided into8groups, i.e., control, model, TAM, FK506, Qu, QDL, QDM and QDH. The animals were intragastrically administration daily for28days. The pathological changes of tumor tissues were investigated with HE staining. The tumor volume ratio was morphometried. Immuhistochemical staining was used for investigated vWF, COX-2, IL-lp, IL-6, STAT3, STAT1, and IRF1cellular location. The existed positive protein levels were analyzed.Results:Qingdu granules inhibited tumor growth, decreased vessel density, reduced COX-2, IL-1β, IL-6and STAT3existed levels. Qingdu granules upregulated STAT1and IRF1expression.Conclusion:Qingdu granules inhibited angiogenesis via downregulating pro-tumor inflammatory signals.Part3Pharmacological mechanism of Qingdu granules inhibiting angiogenesis of breast cancer1. The effect of Qingdu granules on breast cancer induced angiogenesisAim:To study the effect of Qingdu granules on VEGF secretion of MCF-7cells.Methods:Prepared rat normal, TAM, FK506, Qu, QDL, QDM, QDH contained sera, respectively. MCF-7cells were dealt with drug sera. Cell proliferation was detected with MTT, VEGF secreted by cell was measured with Elisa, and VEGF expression was evaluated with real time RT-PCR and Western blot.Results:Qingdu granules inhibited cell proliferation, decreased VEGF secretion, and reduced VEGF expression.Conclusion:Qingdu granules inhibited VEGF vicious cycle of MCF-7cells.2. The mechanism of Qingdu granules on breast cancer induced endothelia angiogenesisAim:To study the mechanisms of Qingdu granules on angiogenesis of breast cancer.Methods:(1) Isolated and cultured human umbilical vein endothelial cells (HUVEC).(2) The dose effect relationship of VEGF stimulating HUVEC proliferation was analyzed.(3) To establish MCF-7and endothelia cocultured angiogenic model in vitro. MCF-7cells were cultured with concentrations of102.5～104.5/mL (k=0.316) for6h,12h,24h,36h,48h,72h,96h,120h,144h,168h,192h,240h,288h,336h, respectively. HUVEC cells were cultured with concentrations of103～105/mL (k=0.316) for12h,24h,36h,48h,72h,96h,120h,144h,168h, respectively. Using HE staining to counts cell numbers of each concentration and time. The concentration(y) and time (x) for cell contact inhibition was regressed.(4)The VEGF induced or MCF-7cocultured HUVEC cells were dealt with normal, TAM, FK506, Qu, QDL, QDM, QDH drug sera. HUVEC proliferation was detected with MTT, the migration was analyzed by transwell, and tube formation was evaluated with Magial matrix. CaN activity was measured. NFATc3entering into nucleus was investigated by immunohistochemical staining. NFATc3expression was determined with real time RT-PCR and Western blot.Results:(1) Identified HUVEC>90%.(2) The EC50of VEGF was15.58ng/mL.(3) The MCF-7equation was y=5.07x2-1949x+185642(R=0.99). The HUVEC equation was y=0.50x2-722.30x+249774(R=0.87).(4) Qingdu granules inhibited VEGF induced HUVEC proliferation, inhibited VEGF or MCF-7induced HUVEC migration, tube formation, downregulated CaN activities, decreased the ratio NFATc3nuclear translocation, and reduced NFATc3expression with dose dependent tendency.Conclusion:The anti-angiogenic mechanisms of Qingdu granules involved in inhibiting of NFAT signal pathway.更多还原