Roles Of PrPc And RPL6 In MDR Of Gastric Cancer And The Underlying Mechanisms

(Background] Multidrug resistance of tumors is a major cause for failure of chemotherapy and death of tumor patients. By suppression subtractive hybridization, we found that the Prion protein (PrP) and Ribosomal protein L6 (RPL6) were overexpressed in drug resistant gastric cancer cell line SGC7901/ADR. PrP is a glycoprotein, synthesized by the ribosome, transferred into and folded in the endoplasmic reticulum, modified in the Golgi apparatus and formed CLD structures (caveolae-like membrane domains, CLD) on the membrane with Caveolin, .COX (cyclooxygenase) and P-gp (P-glycoprotein). PrP also forms protein complex with laminin, 37-kDa/67-kDa laminin receptor, and plays important roles in proliferation, differentiation, adhesion, survival, apoptosis and signal transduction in neuron cells. There are two forms of PrPs, the normal or cellular form of prion protein (PrPc) and the pathogenic or scapie form of prion protein (PrPsc). Those two forms have distinct characteristics. PrPc is a normal component of cells, while the PrPsc can replicate in the cells by coerce the mutation of PrPc, and is pathogenic, infectious and replicative like virus. Studies on PrP (including knock-out mice model) were confined to the nerve system. Its functions outside of nerve system are barely known. Our study casts- 7 -nerve system. Its functions outside of nerve system are barely known. Our study casts some 1 ight on i ts functions outside of nerve system, enriches our knowledge of PrP, and provides a new sight into the mechanism of gastric malignancy transformation and its reversal.RPL6 is also upregulated i n the SGC7901/ADR cells. Ribosomal proteins have functions other than synthesis of proteins. RPL6 is localized at the amino-tRNA binding site of the 5OS ribosomal subunit and in the core of peptide transferase. The full-length cDNA of RPL6 is 959bp with a molecular weight of 33417, encoding 288 amino acids. There were only few studies about RPL6, and no correlation between RPL6 and gastric cancer MDR was found in previous studies. Studying the roles of RPL6 in gastric cancer MDR will further our understanding of non-ribosomal functions of RPL6 and the mechanisms of MDR.[Objective! 1. To study the differential expression of PrP and RPL6 in different tumors tissues and cancer cell lines of digestive system; 2. To examine the differential expression of PrP and RPL6 in gastric cancer tissues and cell lines and its significance梒orrelations with gastric cancer MDR; 3. To explore the possible mechanisms of PrP and RPL6-mediated gastric cancer MDR; 4. To study the interactions between PrP and RPL6.[Methods] (1) The cDNAs of PrP and RPL6 were cloned from SGC7901 cells by RT-PCR; (2) Differential expression of PrP and RPL6 was examined by Northern blot, RT-PCR and Western blot; (3) Sense and antisense eukaryotic expression vectors of PrP and RPL6 were constructed by cloning the cDNAs of PrP and RPL6 into pcDNA3.1-B. SGC7901 cells was transfected with sense vector, and SGC7901/ADR cells with antisense vector. Stable transfectants were obtained by G418 screening. mRNA and its corvesponding protein in the transfected cells were examined; (4) Theform of PrP was identified by protease digestion; (5) MTT assays were performed to determine the in vitro drug sensitivity of the transfected cells, and the IC₅₀ values and resistance index (RI) were calculated. In vivo drug sensitivity assays were performed in the SRCA mice with normal immunity. (6) Accumulation and detention of adriamycin in the transfected cells were examined by flow cytometry (FCM); (7) Apoptosis of the transfeceted cell lines was examined by Western blot, DNA Ladder electrophoresis and PI staining; (8) Using FCM, cell cycle distribution of the transfected cells was studied. The proliferous indexes (PI) were calculated, and the growth curves were drawn; (9) Correlations between PrP, RPL16 and P-gp were studied by Western blot and Verapamil block assay; (10)Correlations between PrP, RPL16 and COX-2 and Caveolin were observed by Western blot an...