Cancer Angiogenesis Mechanism Of EGCG To Inhibit Angiogenesis In Experimental Research

Object: 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. Hepatocellular carcinoma (HCC) 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 HCC growth and metastasis. Method1, Patients: Samples were obtained from 23 patients with primary HCC (mean age 50.86 ?12.78 years) who had undergone partial hepatectomy. The Samples were divided into 3 groups: group A (malignant tissue of HCC), group B (liver tissue within 1.0cm around tumor), and group C (liver tissues 5cm beyond the tumor) respectively. Meanwhile group D (8 cases cirrhosis) and group E (4 donors liver) served as positive and negative control groups. The samples collected during operation were put in liquid nitrogen and kept at-80C in a deep refrigerator. They were diagnosed pathologically by H-E staining and graded by Edmonson' s method. The samples were graded II in 18 patients and III in 5. Incomplete capsule or no capsule was seen in 9 patients, vessel invasion in 7, and satellite lesion in 7.2, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): RNA was isolated from the specimen 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 lul DNase to remove any contaminating DNA. One microgram of total RNA was reverse transcribed using Revert-Aid? M-MuLV reverse transcriptase and Random primer (Sangon, Shanghai,China). PCR was performed using 2ul of the cDNA and primers specific for Ang-1, Ang-2, VEGF, KDR, Tie2 and the housekeeping gene G3PDH. 13(0.1 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 50ug of total proteins were loaded and separated by SDS-PAGE (6% gel for Tie2 and KDR, 8% gel for Angl, 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 3xl5min with TBST, the corresponding second antibodies were added and incubated for 1 hour. Washing the membrane as described above. The immuno-complexwas visualised by enhanced chemiluminescence detection b...