Effect Of Camellia Sinensis Ribosome-inactivation Protein CsRIP2 A Chain On Apoptosis Of MCF-7 Cells

CsRIP2 was obtained by RT-PCR from cotyledon of Camellia sinensis,which encodes a type Ⅱ Ribosome-inactivating Proteins. In this study, its full-length cDNA was cloned by RACE method; the A strand of CsRIP2 A was expressed in E.coli purified. The anti-tumor activity of A strand of CsRIP2 was verified in vitro.The full-length cDNA was cloned by RACE method from developing cotyledon of Camellia sinensis. CsRIP2 is 2095 bp in length, encoding a predicted protein of 570 amino acid residues. DNA sequence for A strand of CsRIP2 was inserted into pCznl plasmid. The CsRIP2 A-His expression vecter was transformed into Arctic ExpressTM. The recombinant CsRIP2 A chain protein was expressed with IPTG inducing. The fusion protein was verified maily in supernatant by SDS-PAGE electrophoresis. The soluble fusion protein were purified by Ni resin affinity chromatography. The specific polyclond antibodies against CsRIP2 A chain protein were obtained by rabbit vaccination.The RIP activity of CsRIP2 A protein was investigated based on rabbit reticulocyte lysate translation in vetro system and luciferase assay system. The result showed that CsRIP2 A protein could inhibited the protein synthesize in vitro, in a dose-dependent maner. To investigate the anti-tumor activity of CsRIP2 A protein. The breast cancer cell MCF-7 was treated with CsRIP2 A proteins of various concentration, it was showed that when the concentration of protein is higher than 4.5 μg/mL, the proliferation of cells is inhibited significantly. The flowcytometry was applied to detect the effect of CsRIP2 A chain protein on the cell cycle of MCF-7. The results showed that the CsRIP2 A chain protein could arrest MCF-7 cells in Go/Gi phase with increasing the cells in G0/G1 phase and decreasing the cells in S phase. When MCF-7 cells were treated with CsRIP2 A chain protein of 18μg/mL for 1 day, and then stained with Hoechst 33342, they exhibited features of apoptosis changes, including increased chromation condensation. When MCF-7 cells were treated with CsRIP2 A chain protein of 36 μg/mL, and stained with Hoechst 33342-PI, ration of PI positive cells increased significantly, in a time-dependent manner. Apoptosis is often accompanied by the activation of caspase. ELISA results showed that compared with the control group, the avtivation levels of caspase-2,3,6 and 8 were increased. These experiments confirmed that the high concentration of CsRIP2 A protein in vitro has the pharmacological activity of inducing apoptosis of breast cancer cell line MCF-7.In this study, RACE was used to clone CsRIP2 gene from Camellia sinensis Fuzao2 seed and the CsRIP2 A protein fusion protein was expressed in prokaryotic expression system, and purified by His-reisin affinity chromatography, the purified protein could inhibited the synthesis of protein in rabbit reticulocyte lysate translation in vetro system. The purified protein could inhibit cell proliferation at low concentrations, and induce apoptosis of MCF-7 cells by activity caspases at high concentrations, demonstrating CsRIP2 A chain protein has pharmacological activity against cancer cells, which laid the foundation for the next step in mice vivo experiments.