Expression And Detection Of Chaperone Proteins In Human Gastric Cancer

ObjectiveChaperone proteins are also called stress proteins or heat shock proteins ( HSPs ) . Recently, it was found that chaperone proteins participated in the specific tumor immune response when studying the immunostimulatory principles of tumors. The expression of chaperone proteins closely related with tumor immu-nogenicity, it suggested that chaperone proteins have important roles in the im-munotherapy of tumor.Developing more effective anticancer vaccine has been one of the major goals of cancer immunotherapy. Purified tumor - derived chaperone proteins such as HSP70, HSP90, GRP94/gp96 and calreticulin (CRT) have shown promise as vaccine, capable of generating tumor - specific T - cell responses and protective antitumor immunity. With the clinical use of purified tumor - derived chaperone proteins as anti - cancer vaccines already in clinical trial stage, we have focused our attention on the utility of chaperone - rich cell lysates (CRCL) in cancer immunotherapy. CRCL', as prepared from tumor lysates via a free solution - isoelectric focusing (FS -IEF) technique, is a high -yield vaccine enriched for numerous chaperone proteins. With the aim of starting vaccine trials in human tumors using autologous tumor - derived CRCL, we investigated the expression of GRP94/gp96 and CRT in human gastric cancer and the relationship between the levels of expression and pathological factors. Furthermore, we used isoelectric focusing technique to establish the method of separating CRCL from the tissue of human gastric cancer and established foundation for the study and development of chaperone vaccine.Materials and Methods1. SpecimensAll specimens were obtained from 51 patients (27 males and 24 females) with gastric cancer who underwent operation in China Medical University between December 2002 and November 2003. The non - carcinomatous tissues beside the tumor were obtained as the normal control.2. ReagentsSP Immunohistochemistry Kit ( Fuzhou Maixin - Bio Company ) Mouse Anti - HSP70, Mouse Anti - HSP90, Rat Anti - GRP94/gp96 and Mouse Anti - CRT Monoclonal Antibody ( Stressgen, USA) Western blot Kit ( Wuhan Boster Company ) Ampholytes ( pH 4 -6, 3 -10; BIO -RAD, USA )3. Experimental Method1) HE Staining and Immunohistochemistry; HE Staining was used to determine histological type and differentiation degree of the samples. Immunohistochemistry was used to detect GRP94/gp96 and CRT expression and the relationship between the levels of expression and pathological factors.2) Separation, Extraction and Detection of CRCL; Isoelectric focusing technique was used to separate CRCL from the tumor tissue homogenate, then twenty fractions were harvested in 20 pipes by vaccum pump. Protein concentration of the dialysate was determined by Bradford method using bovine serum albumin as a standard. The pH of each fraction was determined with a standard pH meter, and the protein content was analyzed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and stained with cosmic S - 250 to detect the effect of separation. Then immunoblotting was performed with mouse anti -HSP70, mouse anti - HSP90, rat anti - GRP94/gp96 and mouse anti - CRT monoclonal antibody as primary antibody and human anti - mouse or rat IgG per-oxidase labeled antibody as second body separately to identify the molecular weight and the quality of the proteins.