In Vitro Induction Of Colon Cancer-reactive CTLs With Prostatic Acid Phosphatase

Colon cancer is the most common malignant tumor of the gastrointestinal tract, which has been causing many people dead per year. In the recent 20 years in China, especially in the big cities, incidence has been obviously increased. Colon cancer almost has no early special symptoms. The developed symptoms are often changed bowel evacuation habit and excrement feature, abdominal pain and bowel obstruction symptoms. Due to the rapid progress, concealment, initial few symptoms, the colon cancer is still lack of effective early diagnostic methods. When diagnosed, 60%～70% of the colon cancer have been late process. Therefore the mortality rate is very high. While for the early colon cancer patients after treatment, the survival rate can reach 90%. Therefore the early diagnosis methods and effective combination therapy is the key to reduce colorectal cancer mortality. Improving the level of early diagnosis and performing the early treatment are important to improve the prognosis.Prostatic Acid Phosphatase (PAP) is an enzyme secreted by prostate cancer epithelial cells. It is the main source for the APC. It is an isozyme coming from the Cytolysosome of the prostate cancer epithelial cells. Human prostate is the main source for the enzyme. As the prostate cancer markers, concentration measurement for serum PAP is widely used in the prostate cancer diagnosis and the monitoring for the treatment. Compared with prostate-specific antigen (PSA), PAP is a more accurate indicator for tumor micrometastases. In addition, the PAP is the immune therapy target for the metastatic non-androgen dependence prostatic carcinoma. PAP was once considered as the prostate specific tumor markers. Several articles report that PAP also exists in non-prostate organ. It seems that PAP is not an prostate-specific tumor marker. In previors study, We have confirmed that the positive expression of PAP in the stomach, ovarian and breast cancer. PAP is the new immune therapy target for the stomach, ovarian and breast epithelial tissue cancer. In this study, the PAP expression in colon cancer cell lines and colon cancer tissue and the possibility of PAP as a immunotherapy target of colon cancer were investigated .(1)The expression of PAP in colon cancer cell lines1)The expression of PAP mRNA in Colon carcinoma cell linesThe expression of PAP mRNA in various colon carcinoma cell lines was investigated by the RT-PCR method. Total RNA was isolated from 1×10⁷ colon carcinoma cells, cDNA was synthesized by RT-PCR. PAP cDNA was detected by PCR amplification using a set of oligonucleotide primers specific to PAP. The mRNA expression of PAP was detected in 3(colo201.colo205.colo320)of 5 colon adenocarcinoma cell lines(colo201, colo 205, colo 320, HCT116 and LoVo ).2)The expression of PAP protein in colon carcinoma cell linesThe expression of the PAP protein in colon carcinoma cell lines was examined by Western blot analysis. The cytoplasmic protein of different colon carcinoma cell lines were extracted according to the manual. The protein was separated by SDS-PAGE. The proteins in the acrylamide gel were blotted to a Hybond-polyvinylidene difluoride membraneand probed with Anti-PAP mouse monoclonal antibody. The PAP protein was found to be expressed in colo201,colo205 and colo320.3)Expression of PAP in colon cancer tissuesThe PAP expression of colon adenocarcinoma tissues were examined by immunohistochemical staining. As a result, colon cancer tissues were positive for PAP.(2)Induction of Peptide-Specific CTLs and Assay of Cytotoxicity1)HLA-A2-restricted PAP epitope peptide18 PAP epitope peptides which have the binding motif to HLA-A2 molecules, were required from the amino acid sequence of PAP through software analysis. All peptides were dissolved with dimethyl sulfoxide at a concen-tration of 10 mg/mL.2)Detection of anti- PAP peptide antibodyAnti- PAP peptide antibody(IgG) in the plasma of colon cancer patients and healthy donors were examined by ELISA analysis. High level anti- PAP_135-143,PAP_112-120,PAP_33-41,PAP_201-210 or PAP_196-205 IgG could be found in the plasma of most colon cancer patients.3)Induction of PAP peptide-Specific IFN-γsecreting CTLsPBMCs of HLA-A2⁺ cancer patients or HLA-A2⁺ healthy donors were incubated with PAP_135-143,PAP_112-120,PAP_33-41,PAP_201-210 or PAP_196-205 peptide respectively. The IFN-γsecretion were examined by the ELISPOT test. It is showed that peptide-specific IFN-γsecretion could be induced by PAP_112-120 or PAP_201-210 peptide.4)⁵¹Cr release assay of CytotoxicityThe cytotoxicity of PAP_112-120 or PAP_201-210 peptide-specific CTLs was examined by ⁵¹Cr-release assay. As a result, the PAP_112-120-specific CTLs lysed the PAP+/HLA-A2+ colon cancer cell colo 205 specificly.In conclusion, PAP mRNA and PAP protein were revealed to be expressed in colon cancer cell lines. PAP protein were positive in colon cancer tissue. PAP112-120 peptide-specific CTLs induced from colon cancer patients lysed the PAP+/HLA-A2+ colon cancer cell colo 205 specificly. HLA-A2-restricted PAP112-120 peptide could be used in the immunotherapy of colon cancer.