Mechanism Study Of Peptide GMBP1 And Its Receptor GRP78 In Modulating Gastric Cancer MDR By ITRAQ-based Proteomic Analysis

【Background】Gastric cancer(GC) is one of the most common malignant tumors in the world, and it is a serious threat to human health and life. Chemotherapy is one of the main treatments of gastric cancer; however, the multidrug resistance is a major obstacle to the treatment of gastric cancer. The mechanisms of MDR are still unknown. MDR is needed to tackle immediately. To study MDR in gastric cancer, we used two drug-resistant cell lines, SGC7901/VCR and SGC7901/ADR, which were derived from the human gastric cancer cell line SGC7901 by stepwise selection in vitro using adriamycin and vincristine, respectively, as cellular models. Previously using a phage display approach, we obtained a peptide-GMBP1, which bind to the surface of MDR gastric cancer cells specifically andsubsequently intesrnalize into gastric cancer cells. Furthermore, GMBP1 was found to have the potential to reverse MDR phenotype of gastric cancer cells and GRP78 was identified as the receptor for this peptide. This study is aimed to study the mechanism of peptide GMBP1 and its receptor GRP78 in modulating MDR to gastric cancer.【Objectives】1. To identify the subcellular localization and analyze the internalization mechanism of peptide GMBP1 and its receptor GRP78. 2. To screen the peptide GMBP1-induced differentially expressed proteins in GC MDR cellines and preliminary analyze the mechanism of GMBP1 and its receptor in GC MDR.【Methods】1. Fluorescence-activated cell sorting(FACS) and immunofluorescence staining were used to observe the subcelluar location and the mechanism of GMBP1 internalization. 2. To build the down-regulated expression of GRP78, and transfect the si RNA-GRP78 and si RNA-Control into GC MDR cell lines SGC7901/ADR and SGC7901/VCR respectively, then using western blot and RT-PCR to verify the transfection efficiency. 3. Through FACS assay to explore the role of GRP78 in the internalization of the GMBP1 peptide in multidrug-resistant gastric cells. 4. Double immunofluorescence staining assay was used to characterize the colocalization of GMBP1 and transferrin in GC MDR cells. Intervention experiment was used to identify the mechanism of GRP78-mediated GMBP1 internalization in GC MDR cells. 5. Using i TRAQ-based proteomic analysis to screen the peptide GMBP1-induced differentially expressed proteins in GC MDR cellines. 6. The GO and KEGG pathway analysis were used to analyze the differentially expressed proteins. 7. Western blot was used to verify the expression of two differentially expressed proteins: EIF4 E and CTBP2. 8. To study the effect of peptide GMBP1-treated GC MDR cells on MDR associatedmolecules, we examined GRP78, P-gp, Bcl-2, and Bax expression levels.【Results】1. The mechanism of peptide GMBP1 internalization into multidrug resistance cells. 1) Immunofluorescence staining showed that GMBP1 and its receptor GRP78 were localized in the cytoplasm and the surface of MDR cells. 2) Flow cytometric assay showed that peptide GMBP1 bound to SGC7901/ADR and SGC7901/VCR was positively stained higher fluorescent intensity. 3) Western blot and RT-PCR assays showed that the transfection of SGC7901/ADR and SGC7901/VCR cells with specific GRP78 si RNA resulted in a marked inhibition of GRP78 protein expression and decreased m RNA levels compared to cells transfected with control si RNA(p<0.05). 4) Immunofluorescence staining showed that the fluorescent intensity decreased in GRP78 down-regulated expression cells. 5) Double immunofluorescence staining assay showed that FITC-GMBP1 and Alexa Fluor 594-transferrin were observed in the cytoplasm and perinuclear regions of the cells. Blocked transferrin uptake, the the internalization of FITC-GMBP1 was strongly decreased. 2. i TRAQ-based proteomic analysis to screen the peptide GMBP1-induced differentially expressed proteins in GC MDR cellines. 1) We included an additional 1.5-fold change and a 0.8-fold change cutoff for all i TRAQ ratios to reduce false positives for the selection of differentially expressed proteins. 143 proteins were differentially expressed in GMBP1-treated SGC7901/ADR cells compared with SGC7901/ADR cells: 95 proteins were upregulated in GMBP1-treated SGC7901/ADR cells, and 48 were downregulated. 217 proteins were expressed differently in GMBP1-treated SGC7901/VCR cells than in control cells: 129 were upregulated in GMBP1-treated SGC7901/VCR cells, and 88 were downregulated. The proteins that were the most downregulated in the two cell lines were identified. 2) Differentially proteins were cataloged according to biological processes(BPs), molecular functions(MFs), and cellular components(CCs) according to the GOdatabase. The differentially expressed proteins were involved in 38 KEGG pathways in GMBP1-treated SGC7901/ADR cells and in 79 KEGG pathways in GMBP1-treated SGC7901/VCR cells. 3. Identification differentially expressed proteins EIF4 E and CTBP2 and mechanism research the function of GMBP1 in GC MDR. 1) Western blot showed that the expression levels of proteins in EIF4 E and CTBP2 were clearly downregulated in GMBP1-treated SGC7901/ADR and SGC7901/VCR cells. This trend is similar to that observed for protein expression levels in the i TRAQ analysis. 2) Western blot analysis reveals that GMBP1 could down-regulate the expression of GRP78, MDR1 Bcl-2; up-regulates the expression of Bax.【Conclusion】1. GMBP1 and its receptor, GRP78, were localized in the cytoplasm of gastric cancer cells. The uptake of GMBP1 was mediated through a clathrin-independent transferrin-related pathway by its receptor GRP78. 2. i TRAQ-based proteomics method is useful in discovering gastric cancer drug resistance related proteins. 95 proteins were upregulated in GMBP1-treated SGC7901/ADR cells, and 48 were downregulated. 129 were upregulated in GMBP1-treated SGC7901/VCR cells, and 88 were downregulated. EIF4 E and CTBP2 were found to be the key GC MDR related molecules. 3. The reversal effect of GMBP1 on MDR was that by targeting GRP78.The expression of GRP78 modulate the expression of EIF4 E and MDR1 through PI3K/AKT pathway, and through downregulating the expression of apoptosis associated molecules Bcl-2/Bax to re-sensitize GC MDR to chemotherapeutic drugs.