| 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. |
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