Functional Research And Receptors’ Screening Of Peptide GMBP1in The Gastric Cancer Multidrug Resistance

[Background]Gastric cancer (GC) ranks fourth among the most commonly diagnosed tumors and isthe second leading cause of cancer-related deaths worldwide. Multidrug resistance (MDR)is a major clinical obstacle in the treatment of gastric cancer (GC). Since the1980s,many types of molecules have been investigated to be correlated with MDR. According toresearch, a variety of molecules have been found associated with MDR, such as ABCtransporter P-Glycoprotein, MRP、BCRP，GSH/GST，P53，TopoII. However, due to sideeffects and adverse pharmacokinetic interactions, most of these drugs failed in clinicalapplication. As a result, the mechanisms of MDR are still not fully understood. Therefore,exploring novel agents that can reverse MDR in GC is necessary for the improvement ofchemotherapy in GC patients. In the previous study, using a phage display bio-panningapproach combined with MTT assays, we screened a specific peptide GMBP1(gastriccancer MDR cell-specific binding peptide), ETAPLSTMLSPY, which could bind to thesurface of gastric cancer MDR cells specifically and internalized into MDR cellscompared with control cells SGC7901and GES. Immunofluoresence staining showed thatphage GMBP1bound preferably to MDR cells rather than SGC7901or GES, whichindicated that peptide GMBP1may be closely associated with GC MDR. Thus, it is of great importance to explore the functional of GMBP1in GC, identify the properties ofGMBP1receptors and further research the potential signal mechanisms of GMBP1in thereversal of GC MDR, which may provides new insight into research on the emergence ofMDR in GC cells and new targets for molecular targeted therapy of GC.【Objectives】1. To identify the specific binding ability of peptide GMBP1to GC MDR cells andanalyze the function of GMBP1in GC MDR.2. To screen the receptor of GMBP1providing new insights into the mechanism researchof GMBP1.3. To identify the receptor of GMBP1and preliminary analyze the mechanism ofGMBP1and its own receptor in GC MDR.【Methods】1. Immunocytochemistry staining assay was performed to identify the specific bindingability of GMBP1to MDR cell and observe the subcellular localization ofGMBP1-receptor.2. MTT assay was used to detect the effect of GMBP1on MDR cell growth.3. In vitro drug sensitivity method was examined to detect the effect of GMBP1on MDRcells to chemotherapeutic drugs.4. The effect of GMBP1on MDR cells to chemotherapeutic drugs induced death wasanalyzed by flow cytometry and hoechst staining methods.5. Western blot analyses were used to identify the receptors of GMBP1in MDR cells.6. To obtain a high amount of the GMBP1-receptor, lysates from MDR cells weresubjected to co-immunoprecipitation assays. The co-immunoprecipitates were thenanalyzed by silver staining, in-gel digestion and MALDI-TOF/TOF methods toidentify the receptor of GMBP1.7. Confocal laser, western blot, and siRNA experiments were performed to furtheridentify the receptor of GMBP1.8. The effect of GMBP1on the expression levels of MDR and apoptosis associatedmolecules (P-gp, MRP, Bcl-2and Bax) was detected by western blot. 9. The GMBP1-receptor levels in control and overexpressed-GRP78MDR cells whichpre-incubated with GMBP1or not were examined by Western blot. Similarly, FACSanalyzed the effect of GRP78expression levels on the apoptosis of correspondingcells.10. To study the effect of down-regulation of GRP78by siRNA on MDR associatedmolecules, we examined P-gp, Bcl-2, and Bax expression levels in siGRP78MDRcells and FACS analyzed the effect of GRP78on the cells to chemotherapeutic drugsinduced apoptosis.【Results】1. The effect of peptide GMBP1on the biological behavior of target cells.1) Immunocytochemistry and immunofluorescence staining showed that peptideGMBP1bound to SGC7901/ADR and SGC7901/VCR was positively stained darkbrown and located at the cytoplasm and the surface of MDR cells, in contrast toSGC7901and GES.2) MTT assays revealed that GMBP1had no significant influence on cell growthcompared with controls; in vitro drug sensitivity assays indicated all IC50values ofMDR cells pre-incubated with GMBP1and then treated with chemotherapeuticdrugs were significantly decreased compared with cells incubated with URP, whichsuggests that GMBP1could modulate the sensitivity of MDR cells tochemotherapeutic drugs.3) The results of Hoechst and FACS analysis suggested that GMBP1could increasethe apoptosis of GC MDR cells by re-sensitizing GC MDR cells tochemotherapeutic drugs.2. Screening and identification of the receptor for GMBP1.1) Western blot analyses showed that two bands, at approximately80KD and130KD, could be consistently identified by GMBP1.2) Co-immunoprecipitation and MALDI-TOF/TOF methods preliminary suggestedthat78KD glucose regulated protein (GRP78) might be the candidate of receptorsfor GMBP1. 3. Identification of the receptor of GMBP1and mechanism research the function ofGMBP1in GC MDR.1) Confocal laser and western blot methods showed that GMBP1and GRP78antibodies had the same cellular localization and protein bands, and western blotrevealed that the expressions of GRP78in GC MDR cells were markedly higherthan SGC7901and GES.2) Western blot analysis reveals that GMBP1could down-regulate the expression ofP-gp and Bcl-2; conversely, no obvious difference in MRP expression was found.3) Western blot and FACS analysis suggested that GMBP1could down-regulateGRP78expression sensitizing GC MDR cells to a variety of chemotherapeuticagents.4) Western blot and siRNA methods initially revealed that GMBP1could suppressGRP78expression to re-sensitize GC MDR cells to a variety of chemotherapeuticagents, which may be mediated partly through P-gp expression.【Conclusion】1. GMBP1could bind specifically to the cytoplasm and surface of MDR cells.2. GMBP1itself had no significant influence on MDR cells proliferation, however,GMBP1could modulate the sensitivity of MDR cells to chemotherapeutic drugs.3. GRP78was the receptor for GMBP1.4. The reversal effect of GMBP1on MDR was implemented on the one hand may be bysuppression the expression of GRP78and further depleting of the expression of P-gp,on the other hand may be through downregulating the expression of apoptosisassociated molecules Bcl-2/Bax to re-sensitize GC MDR to chemotherapeutic drugs.