The Molecular Mechanism Study Of EGCG On The Inhibition Of Nasopharyngeal Carcinoma Angiogenesis

PART ONE THE MOLECULAR MECHANISM UNDERLYING ANGIOGENESIS IN NASOPHARYNGEAL CARCINOMA: THE IMBALANCE ACTIVATION OF SIGNALING PATHWAYSObject: Angiogenesis plays a key role in the process of tumor growth by providing abundant oxygen and nutrients to neoplasm and making a channel for the tumor invasion and metastasis. Pro-angiogenic factors released from tumor cells can promote the formation of new blood vessels by functioning on host endothelial cells. Vascular endothelial growth factor (VEGF) and its kinase insert domain containing receptor KDR take part in the process of angiogenesis. The mature and stabilization of neovascular are dependent on another subfamily of tyrosine kinase receptor Tie2 and its ligand angiopoietins. Nasopharyngeal carcinoma (NPC) characterized by rapid growth, early metastasis and high mortality is an intensive vascular-dependent marlignant tumor. Angiogenesis is an initial step for these malignant features, and lots of pro- or anti-angiogenic factors and receptors take part in this process. It is suggested that targeting at angiogenic signaling pathways may be a potent treatment for the prohibition of tumor invasion and metastasis. The molecular mechanism of these pathways during angiogenesis is still unclear. In this study the method of RT-PCR and Western blot were employed to evaluate the expression patterns of VEGF/KDR and Angiopoietins/Tie2, which are considered as two important signal conductive pathways, and the molecular mechanism underlying these factors and receptors in NPC growth and metastasis.Method1. Patients: Three were 23 patients with primary NPC (mean age 50.86±12.78 years). The samples were divided into 4 groups: group A (malignant tissue of NPC), group B (pharyngeal portion tissue within 1.0cm around tumor), group C (pharyngeal portion tissues 5cm beyond the tumor) and group D(pharyngeal portion tissues of health adult) respectively. The samples collected under nasal endoscope were put in liquid nitrogen and kept at-80℃in a deep refrigerator. They were diagnosed pathologically by H-E staining and graded by Edmonson's method. The samples were gradedΙ-Ⅱin 18 patients andⅢ-Ⅳin 5 patients.vessel invasion in 7, and lymph metabasis of neck in 7.2. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR):RNA was isolated from the specimens by using Trizol reagent (Gibco,USA). Briefly, the samples were weighed, snap frozen, and ground with a mortar and pestle in Trizol reagent, then allowed warming to room temperature. RNA was then treated with RNase-free 1μl DNase to remove any contaminating DNA. One microgram of total RNA was reverse transcribed using Revert-AidTM M-MuLV reverse transcriptase and Random primer (Sangong, Shanghai,China). PCR was performed using 2μl of the cDNA and primers specific for Ang-1, Ang-2, VEGF, KDR, Tie2 and the housekeeping gene G3PDH. 13μl PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. Images were captured using Kodak DNA Analysis (Gibco BRL, USA) and density values assessed using Kodak digital science 1S 2.0 software. The identity of PCR products was confirmed by sequencing.3. Western Blot:Samples were homogenized in lysis buffer. Proteins were dissolved in SDS-Laemmli buffer by boiling for 5 min. In each lane 50μg of total proteins were loaded and separated by SDS-PAGE (6% gel for Tie2 and KDR, 8% gel for Ang1, Ang2 and VEGF). The electrophoresed proteins were then transferred to a PVDF membrane by a semi-dry electrophoretic transfer procedure for 1.5 h at 1 mA/cm2. Ponceau staining was done to check if all proteins were transferred to a comparable extent in all lanes. The membranes were blocked with 5% defatted milk prepared in TBS buffer, pH 7.5. Then, primary antibodies were added to bind specially with the membrane overnight. After washing 3×15min with TBST, the corresponding second antibodies were added and incubated for 1 hour. Wash the membrane as described above. The immuno-complex was visualised by enhanced chemiluminescence detection by autoradiography on a X-ray film.4. Immunohistochemistry:Sections (4-6mm) were cut from paraffin blocks fixed with formalin, deparaffinized in xylene and then hydrated in ethanol step by step. Sections were boiled in citric acid for 3mins to repair antigen and incubated in 3% hydrogen peroxide for 5 min to quench of endogenous peroxidase activity. After incubating in 10% sheep serum for 5min, anti-CD34 mouse monoclonal antibody diluted at 1: 25 was used and incubated in 37°C for 1hour. The sections were developed using DAB method following the manufacture`s instructions. The positive cells were counted on at 200-fold in five fields.5. Statistical analysis:Data were expressed as mean±standard deviations. The absorbency for RT-PCR and vascular density was performed using one-way ANOVA with SPSS 11.0 software. P values≤0.05 were considered significant.Results1. The relationship between expression of the two signaling pathways and mircovessel density:In this study, Ang-1 mRNA was detected in all samples and no significant difference was found in all groups. The level of VEGF mRNA expression was the highest in group A and showed remarkable difference compared to other groups (p<0.05). Ang2 expression in groups A and B were significantly higher than in groups C, D, E (p<0.01), but there were no difference between groups A and B. The level of KDR and Tie2 mRNA expression was not significantly different in all groups, but high absorbency was observed in groups A and B. The proteins synthesis of all factors examined in this study were up regulated consistently with their gene transcriptive level. In group A the level of VEGF and Ang1 protein were higher than those in other groups, while receptors KDR and Tie2 expression were increased simultaneously. The level of Ang2 and KDR were dominantly higher in group B than in other groups.Microvessel densitywasdetected with the method of immunohistochemistry. MVD were remarkably increased in groups A and B compared with other groups (p<0.05). The numbers of CD34 positive endothelial cells were maximal in group A on the immunohistochemistry photos under a microscope.2. The relationship between metastasis of NPC and angiogenic signaling pathways:The interaction between mRNA expression of the two angiogenic pathways and the invasiveness and metastasis of NPC was also analyzed. The expression VEGF, Ang2 and Tie2 with vessel cancerous embolism was significantly higher than that of those without vessel cancerous embolism in samples (p<0.05). At the same time, marked expression of VEGF and Ang2 was seen in NPC patients with lymph metabasis of neck compared to non- metabasis (p<0.01).Conclusion1. The distribution of vascular in the samples operated is well-regulated. The abundant vascular is seen in the NPC without necrosis but with chaos and the same as in margin of tumor.2. The signaling conductive pathways, VEGF/KDR and Angiopoietins/Tie2, which is characteristic of endothelial cell, are actived in the NPC samples. It is suggested that both pathways take part in the angiogenesis indispensably.3. The activation of the both pathways is inconsistent and lead to the abnormal vessels in morphology and function in the process of tumor angiogenesis. This is beneficial to the tumor growth and matestasis.4. The up-regulation of VEGF,Ang2 and Tie2 may be correlative with tumor invasion and matestasis. PART TWO THE EXPERIENCE STUDY OF EGCG ON THE INHIBITION OF NASOPHARYNGEAL CARCINOMA ANGIOGENESISObject:Tumor cells secrete many angiogenic factors. But until now, the cytokines specific to endothelia cell only include three families, VEGF, Angiopoietins and Ephrin. They play an angiogenic role through interaction with their receptors that are located on the membrane of endothelial cell. Among these cytokiones VEGF is a crucial factor that is able to increase vascular permeability, stimulate endothelial proliferation and migration and antiapopotosis. These biological effects are realized mainly through VEGFR2 (KDR) signal conducive pathway. Another member of receptor tyrosine kinase family, Tie2 takes part in maturating and stabilizing vessel structure during angiogenesis. EGCG, which is a major polyphenol constituent of green tea, is demonstrated to mediate many biological effects, such as anticancer, anti-oxidation, anti-inflammation and immuno-regulation. Accumulated experiments have elucidated the anticancer mechanism of EGCG by blocking the signal conductive pathway in tumor cells and inhibiting the activity of enzyme. In last years, research on angiogenic inhibition of by EGCG is gaining increasing attention. In this study, we aim at blocking the receptor tyrosing kinase phosphrylation of endothelial cell membrane with EGCG, observe the depression of angiogenesis in vitro and in vivo and explore the anti-angiogenesis mechanism of EGCG as well.Methed: 1. MTT: The endothelial cell line ECV304 and human NPC cell line HNE-1 were cultured respectively in DMEM containing 10%FBS at 37°C under a humidified atmosphere containing 5% CO2. After digested with 0.25% trypsin, cells were seeded into 96-well plate at 10,000 cells per well and EGCG was added simultaneously with different concentration. The plate was incubated at 37°C in 5% CO2 for 48hs, then co-cultured with 20ul dimethylthiazole 2, 5 diphenyl tetrazolium bromide (MTT 20 mg/ml) for another 4h. At last dimethylsulfoxide (DMSO) 100ul was added and set at room temperature for 15 minutes, and then the absorbance was read at 570nm.2. In vitro angiogenic assay: Collagen gels were prepared with Rat-tail typeⅠcollagen (2.5mg/ml) 4 volume and mixture (10×DMEM: 0.34N Na2CO3 2:1 and adjusted to pH 7.3 with 1N NaOH) 1volume. 100ul above solution was added to 96-well plate to gel at 37℃. ECV304 was seeded into the plate at 1×104 cell per well and incubated at 37℃in 5%CO2 for 24h. After washed twice with PBS, the cells were treated with various concentrations EGCG for another 24h, and then VEGF (final concentration 50ng/m) or HNE-1 CM 500ul was added to each well respectively. The area covered by the tube network was observed everyday and taken photos every other day.3. RT-PCR: ECV304 was seeded in 6-well plated (1×105/well) and cultured to be confluent. After the cells were washed twice with PBS, 1ml DMEM deprived from FBS with various concentrations of EGCG was added into each well and cultured for 24h. Then the cells were rinsed twice with PBS, VEGF (final concentration 50ng/ml) or HNE-1 CM 500ul was added into each well and cultured for 5 minutes. The cells were collected for RT-PCR, which was performed as one-step RT-PCR kit protocol. 4. Immunoprecipitation: ECV304 cells were lysed with ice-cold lysis buffer containing 1mM Na3VO4. After this, 200ug protein were co-cultured with the special antibody (2ug/ml) at 4℃overnight and the antigen elution was performed as immunoprecipitate kit protocol. The antigen was unwatered to the final volume 40ul and added 5×SDS buffer (5ul/20ul sample), boiled 5 minutes. After the protein was electrophoresed by 8% SDS-PAGE, the gel containing proteins was stained by silver nitrate.5. Western blot: After the eluted protein was electrophoresed, it was transferred to nitrate cellulose membrane. The membrane was blocked by 5% defatted milk for 1hour and probed with the first antibody p-Tyr (PY99) at1: 1000 dilution at 4℃overnight. Then it was washed with TBST for 1hour and incubated with horseradish peroxidase-conjugated anti-mouse IgG for 1 h. Bound antibody was visualized with the ECL system according to the manufacturer's directions.6. Animal experiment: The nasopharyngeal carcinoma cell line HNE-1 was digested with trypsin and injected 1×106 cells subcutaneously into the flank of 2 athymic nude mice. Xenograft tumors were excised from mice when it grew up to 5×5mm2 and cut into 1mm3 pieces. These pieces were implanted subcutaneously into the flank of the experimental mice respectively. The experiment of EGCG treatment didn't begin until the xenograft tumors grown about 5×5mm2. 21 mice were divided equally into three groups randomly, control group, high-dose group (EGCG 50mg/kg) and low-dose group (EGCG 25mg/kg). The athymic nude mice were treated with intraperitoneal injections of EGCG every other day whereas control group was injected with equal saline instead of EGCG. The weight of mice was recorded and the tumor size was measured with calipers. The tumor volume was calculated according to the formula 1/2width2×length and MVD was evaluated according to the number of CD34 positive stained cell.7. Statistical analysis: Data were expressed as mean±standard deviations. Student's t-tests were used for univariate analysis. Statistical significance was defined by p< 0.05.Result:1. The proliferation of HNE-1 cell line was obviously inhibited by EGCG and was dose-dependent. But in the cultural endothelial cell line ECV304 the inhibition of EGCG was shown weakly.2. In our study, the expression of gene transcription of KDR and Tie2 were shown in the quiescent ECV304 and it was up-regulated evidently by the stimulation of VEGF and HNE-1 CM. But after treated with EGCG the expression of these two receptors was inhibited in dose-dependent manner.3. When stimulated with VEGF or HNE-1 CM, the protein synthesis and phosphorylation of KDR and Tie2 increased more rapidly than those without the stimulation. If the cell was pre-intervened by EGCG, the protein synthesis and phosphorylation of the two receptors were both restrained in spite of the stimulation of VEGF or HNE-1 CM. The degree of inhibition was closely related with the dosage of EGCG.4. Tube-like structure had been observed on the collagen gel culture in this study. In control group the vessel formation of endothelial cell was established successfully and maintained about 4days. VEGF and HNE-1 CM can stimulate ECV304 cells to form capillary-like structure and keep this framework up to 8 days. But with treatment of EGCG ahead, the vessel formation was incomplete compared with control group. If treated with high dose of EGCG the vessel length reduced significantly. Tube-like structure was shrunk with the prolonged EGCG function.5. Studies from our laboratory indicated that the growth and angiogenesis of xenograft tumor in athymic nude mice were inhibited in high-dose EGCG group and showed difference (p<0.05) compared with control and low-dose groups. In high-dose group the volume of xenograft tumor was inhibited up to 39.8% at twenty-two days. But in low-dose group the tumor volume was not affected by EGCG. No metastasis in intraperitoneal cavity and lungs were seen in all groups at 22 day, but two cases were found focal necrosis of tumor with nude mouse in high-dose group. Under microscopy there were 3 animals with focal necrosis in high-dose group, 1 in low-dose group and none in control. The angiogenesis was evaluated by CD34 positive-stained in this study. It was clearly that the CD34 positive-stained endothelial cells were much more in control group than in therapy groups. CD34 positive-stained cells were shown minimum in high-dose group with. During the experiment, there was no difference in the weight of nude mouse among three groups. Hence, EGCG was able to inhibit angiogenesis and result in depression of tumor growth.Conclution:1. EGCG is able to inhibit the proliferation of human nasopharyngeal carcinoma cell line HNE-1 but not of normal endothelial cell line ECV304.2. EGCG can inhibit the gene transcription of receptor tyrosine kinase KDR and Tie2, which results in the depression of their protein synthesis and phosphorylation.3. EGCG reduced the vessel formation of endothelial cell in vitro angiogenic experiment, and it can also inhibit the growth of xenograft tumor and angiogenesis in athymic nude mouse model with slight side-effect.4. EGCG serves as antiangiogenic agent targeting particularly at tumor cell and angiogenesis. It will be one of potent anticancer drug in the future.