Metastasis Inhibition And Receptors’ Screening Of Peptide GEBP11in Gastric Cancer

【Background】Due to its advantages of low resistance, wide inhibition spectrum, easy accessibility,low toxicity etc., tumor vascular targeted therapy has become a hotspot in the field oftumor research and treatment. At present, one of the prominent problems of targetedagents is lack of specific molecules targeted to tumor blood vessels. Because of lowimmunogenicity, high affinity of ligand, low toxicity, easy synthesis and modified and soon, short peptide has become the attention focus. Early, we had got GEBP11-a cyclicnonapeptide homing specifically to vascular endothelial cells of human gastric cancer byphage display peptide library technology, and confirmed its well targeting and inhibitionof tumor angiogenesis.Studies also found that peptide can regulate the expression ofmolecular group in tumor vascular endothelial cells, and affect apoptosis, proliferation,matrix degradation, migration and adhesion of tumor vascular endothelial cells.Therefore,we speculated that the peptide may intervene tumor vascular endotheliacells proliferation,matrix degradation, apoptosis, migration and adhesion ability,through regulating tumor vascular endothelial cells molecular group expression, then affect the formation of tumorblood vessels, eventually inhibit tumor growth and metastasis.But the potential molecularmechanism of peptide inhibiting tumor is still unclear. The molecules initially combinedwith peptide, in other words the receptors of peptide on the surface of tumor bloos vesselsare also unkown. So this study is proposed to investigate the influences of short peptide ongastric cancer cells migration, invasion, mobility and proliferation potential, and isolation,purification and identification the specific receptors of GEBP11,aims to lay solidfoundation for the molecular mechanisms of inhibiting tumors of peptide and its receptors.【Objectives】1. To explore the influences of GEBP11on gastric cancer cells migration, invasion,mobility, proliferation potential analyzing the mechanism of inhibiting tumor.2. To screen the receptor of GEBP11making foundation for the mechanism research ofinhibiting angiogenesis.【Methods】1. In vitro migration/invasion/scratch tests were done to exam the influences of differentpeptide concentrations on gastric cancer cells migration and invasion abilities.2. In vitro high content screening and MTT experiments were tested the influences ofdifferent peptide concentrations on gastric cancer cells mobility and proliferationcapacities.3. Western blot analyses were used to detect the expression of MMP1and MMP10inCo-HUVECs after teated with GEBP11.4. Mass spectroscopy, high pressure liquid chromatograph and immunofluorescentstaining were performed to confirm the identity of Bio-2PEG-(GEBP11)3.5. Western blot analyses were detected the receptors of trimeric GEBP11peptide inCo-HUCECs cells.6. Co-immunoprecipitates and mass spectroscopyassays were used to screen and identifythe receptors of trimeric GEBP11peptide.【Results】1. Exploring the effects of peptide GEBP11on the biological behavior of gastric cancer cells.(1) Concentration up to20μg/ml, GEBP11can inhibit the capacities of migration andinvasion in gastric cancer cells in vitro migration、invasion and scratch experiments,compared with the control group without peptide. These inhibiting effects were stillobvious, when the concentration up to50μg/ml and100μg/ml. But these inhibitingresults were no longer increasing, while the concentration of peptide continued togrow.(2) GEBP11can inhibit the potential of lung metastasis in gastric cancer cells in vivo.(3) When the concentration up to20μg/ml, GEBP11can inhibit the mobility andproliferation abilities of gastric cancer cells in vitro HCS and MTT tests, comparedwith the control group without peptide. These inhibiting effects were still obvious,when the concentration up to50μg/ml and100μg/ml. Similar to the results of invitro migration、invasion and scratch experiments, these inhibiting effect were nolonger increasing, while the concentration of peptide continued to grow.(4) Western blot analyses showed that GEBP11can reduce the expression of MMP1and MMP10in Co-HUVECs.2. Separation、purification and identification of the receptor of GEBP11.(1) Mass spectroscopy, high pressure liquid chromatograph and immunofluorescentstaining affirmed the identification of Bio-2PEG-(GEBP11)3and2PEG-(GEBP11)3.(2) Western blot analyses confirmed that about36KD could be consistently identified byGEBP11.(3) Co-immunoprecipitation and mass spectroscopy assays suggested that receptor foractivated C kinase1(RACK1) might be the candidate of receptors for GEBP11.【Conclusion】1. GEBP11can inhibitgastric cancer cells’ migration, invasion, mobility, proliferationabilitiesin vitro and metastases formation in vivo.2. We successfully got Bio-2PEG-(GEBP11)3and preliminarily identified RACK1was thereceptor of GEBP11peptide by co-immunoprecipitation and mass spectrometry.3. GEBP11inhibits the metastasis of gastric cancer, partly through the following mechanism: GEBP11directly or indirectly reduces the expression of MMP1、 MMP10and other moleculars after combined with RACK1of Co-HUVECs, and finally displaysthe role of inhibition.