Preliminary Screening Of The Receptor Of MDR Reversal Peptide GMBP42 And Its Functional Research In Gastric Cancer

【Background】In recent years, despite great advances in the treatment of cancer, the continue development of new anticancer drugs and emergence of new treatment schemes, the outcome of clinical treatment is still far from expectation. Multidrug resistance (MDR) is believed to be the major obstacle to successful chemotherapy in cancer patients. The multidrug resistance of gastric cancer hasn't have ideal judgement standard of drug-resistant degree and effective reversals or modulators. MDR is needed to tackle immediately. There are four generations of drugs which act directly on the p-gp to block their activity can reverse MDR, which was confirmed by experiments. Because of the toxic side effects and adverse pharmacokinetic interactions, the modulators are failed in clinical application. So, exploring new methods and strategies and developing new drugs which can reverse multidrug resistance of gastric cancers have very important significance in treatment of gastric cancer. In research and development of drugs, small molecule short peptide has many advantages in clinical application. The research on small molecule short peptide is a hot area. In previous study works, our group obtain GMBP42 specifically binding to drug resistance cells by screening with bio-panning method on MDR gastric cancer cells and drug sensitive gastric cancer cells. Through researching on pharmacodynamics in vivo, we preliminary witness that GMBP42 can inhibit the growth of the drug-resistant tumor in the nude mice, partly reverse the drug resistance of tumor, increase the drug sensibility of tumor. To further identify the receptor of GMBP42 and research intensively on function and mechanism in MDR have important significances on overall recognizing the mechanism of drug-resistant in cancer. The relevant study can provide experiment foundation and theory support for researching and developing new modulators to reverse MDR in gastric cancer. Thus, this subject will focus on them.【Objectives】1. To identify the binding of GMBP42 in drug-resistant gastric cancer cells and obtain the informations of the expression and location of receptor.2. To screen the binding receptor of GMBP42 in drug-resistant gastric cancer cells, which can make a solid basis for investigating the mechanism of GMBP42.3. To make clear the function of GMBP42 that reverse MDR and preliminary analysis the mechanism of GMBP42. 【Methods】1. The binding effect of GMBP42 to MDR gastric cancer cells and the location of receptor of GMBP42 were detected by immunocytochemical staining. The expression quantity of the receptor in MDR cells was verified by flow cytometry.2. Purification and enrichment of the binding receptor protein of GMBP42 by immunoprecipitation, then preliminary screen the receptor with mass spectroscopy analysis.3. Observation the effect of GMBP42 on the growth of MDR cells by MTT.4. The change of the IC50 values of MDR cells with GMBP42 for anticancer drugs was detected by in vitro drug sensitivity assay.5. The change of accumulation and retention of drugs of MDR cells with GMBP42 was detected by adriamycin accumulation and retention assay.6. The effect of GMBP42 on the apoptosis of MDR cells was measured by flow cytometry.7. Detection of the expression of some MDR relevant molecule in MDR cells with GMBP42 by RT-PCR.8. Detection of the expression of MDR1 and the apoptosis related molecule BCL-2, BAX in MDR cells with GMBP42 by Western-blot.【Results】1. The condition of GMBP42 binding to cells and the information of receptor of GMBP421) Immunocytochemical staining showed that GMBP42 has positive response with SGC7901/VCR and SGC7901/ADR cells and negative response with SGC7901 cells. There are positive particle at surfaces and in the cytoplasm of drug-resistant cells.2) FCM analysis revealed that the expression of the receptor of GMBP42 in drug-resistant cancer cells was higher than in drug-sensitive cancer cells.3) Purification and enrichment of the binding receptor protein of GMBP42 by immunoprecipitation, then preliminary screen the receptor with mass spectroscopy analysis. EEF1A, Elongation factor 1-alpha was detected in the two protein bands, so it will be the most possibility candidate of receptor.2. The function and molecule mechanism of GMBP42 in reversing MDR1) MTT results showed that GMBP42 has no effect on the growth of drug-resistant cells.2) In vitro drug sensitivity assay revealed that the IC50 values of SGC7901/VCR and SGC7901/ADR with GMBP42 for ADR ,5-FU and MMC were significantly decreased and for CDDP were not changed compared these cells without GMBP42.3) Adriamycin accumulation and retention assay by FCM showed that the fluorescence intensity was higher in drug-resistant cancer cells with GMBP42 than without GMBP42. GMBP42 can increase the accumulation and retention of adriamycin.4) The apoptosis of cells analysised by FCM showed that the apoptosis index of drug-resistant cells with GMBP42 was higher than without GMBP42. The results revealed that GMBP42 can promote drugs induced the apoptosis of cells.5) The RT-PCR showed that the expression of MDR1 in RNA was decreased in drug-resistant cancer cells with GMBP42. 6) The western-blot showed that the expression of MDR1, BCL-2 was decreased and the expression of BAX was increased in drug-resistant cancer cells with GMBP42.【Conclusion】1) GMBP42 can bind to drug-resistant cells with high affinity. The expression of receptor of GMBP42 in drug-resistant cells was higher than in drug–sensitive cells. Screen preliminary of the receptor of GMBP42, we obtain 19 candidate protein molecule. EEF1A ,Elongation factor 1-alpha was detected in the two bands, so it will be the most possibility candidate of receptor .2) GMBP42 has no influence on the growth of drug-resistant cells. It can increase the accumulation and retention of drugs in intracellular, decrease the IC₅₀ value of anticancer drugs, promote the apoptosis of cells. The function of reversing MDR make effect through change the expression of apoptosis related genes(BCL-2/BAX) and decrease the expression of MDR related molecule MDR1. The relevant molecule mechanism will need further study .