Non-invasive detection of protein kinase C Beta II and [], putative biomarkers for colon cancer using fecal messenger RNA

Abstract

There is strong evidence that select dietary factors play a key role in the development of colon cancer. It has recently been shown that dietary fat and fiber can alter colonic protein kinase C (PKC) activity and isozyme expression, which may influence the malignant transformation process. The effect of diet on the expression of PKC [ ], and [ ]in fecal material was examined in this study. In addition, the effect of a carcinogen on PKC [ ],, and [ ] mRNA levels in feces was determined in order to develop non-invasive methodology to detect potential markers for colon cancer. Weanling rats were fed diets containing corn oil or fish oil, cellulose or pectin and injected twice at one-week intervals with saline or azoxymethane (AOM; 15 mg/kg body weight) in a 2 x 2 x 2 factorial design. Fresh fecal samples (n=6 per treatment) were collected 36 weeks after the second injection, poly A+ RNA was isolated and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) performed using primers specific for the regulatory subunit of PKC [ ], and [ ]. Fecal PKC mRNA isozyme expression was altered by the injection of carcinogen (p < 0.03), with AOM injected animals having 2.5-fold lower PKC @ expression compared to saline injected animals. Fecal PKC 01, mRNA expression was 3-fold higher in tumor bearing animals as compared to non-tumor bearing animals (p < 0.03). In addition, AOM injection increased mucosal PKC [ ] MRNA expression compared with saline controls. No main effect of tumor incidence on mucosal PKC [ ], expression was observed. Since tumor incidence exerts a reciprocal effect on fecal PKC [ ], and [ ]mMRNA expression, data were also expressed as a ratio between PKC [ ]and [ ]. The ratio of PKC [ ]was significantly higher in animals with tumors, 2.18 ︢1.25, as compared to animals without tumors, 0.50 ︢0.16 (p=0.025). We propose that expression of PKC [ ] and the ratio of [ ]in feces mayserve as non-invasive markers for colon carcinogenesis. A sensitive technique for thedetection of colon cancer is of importance since early diagnosis can substantially reduce mortality.