Characterization of colon cancer cell culture based screening assay to study effects of phenolic acids

In Canada, colorectal cancer is the second leading cause of death from cancer in men and the third leading cause of death from cancer in women. Several factors contribute to the development of cancer. Genetic predisposition, diet, and lifestyle habits are some of the major factors for colorectal cancer development. In the diet related factors, epidemiological studies suggest that consumption of whole grains rich in dietary fiber are associated with low incidence of human colon cancer. Recent studies have also shown that, in addition to dietary fiber, the type of dietary fiber and other compounds such as phenolic acids present in cereal grain bran may also have a role to play in colon cancer prevention. In a recent study, eleven major phenolic acids which differed in anti-oxidant activity were identified in wheat bran from wheat varieties belonging to six different market classes. The main objective of this study was to develop an in vitro cell culture based assay system to study the effect of phenolic acids on colon cancer development. Another objective was to study the effect of phenolic acids on selected molecular markers associated with cell proliferation, apoptosis and inflammation. Two well established colon carcinoma cell lines HT-29 and HCT 116 were treated with varying concentrations of fourteen phenolic acids to study their effect on cell survival and proliferation. In addition, immunohistochemical assays were performed on treated cells for two cell proliferation markers (Cyclin D1 and Ki67), an apoptosis marker (Bax) and three inflammatory markers (Beta-catenin, COX-2 and iNOS). Treatment of phenolic acids inhibited the growth of both the cell lines, however the effects varied with phenolic acid and cell line used in the assay. As determined by IC50, the growth of HCT 116 cells was inhibited the most by caffeic, ellagic, and gallic acids with IC50 of 0.22 mM, 0.17 mM, and 0.15 mM, respectively. On the other hand, caffeic, chlorogenic, and gallic acids are most effective in preventing the growth of HT-29 cells, with IC50 at 0.06 mM, 0.28 mM, and 0.30 mM, respectively. Immunohistochemical and Western Blotting studies revealed that phenolic acids differentially affected markers for cell proliferation, apoptosis and cell inflammation. In most of the cases, phenolic acid treatments up-regulated the pro-apoptosis marker Bax, while it down-regulated cell proliferation marker Cyclin D1. The results clearly show that a cell culture based assay can be used to study the effect of phenolic acids or other chemical constituents isolated from plants to study their effect on colon cancer cell lines. Statistical analysis revealed that only in very limited cases, results of molecular markers correlated to cell growth and proliferation. Therefore, to draw firm conclusions, more detailed and extensive studied need to be completed using different phenolic acids, the two cell lines and more replications. However, this study has developed the necessary protocols and provided some indicative results such as most of the phenolic acids induced pro-apoptosis pathway in both the colon cancer lines. Future studies with extracted phenolic acids from wheat bran using the cell culture system optimized in this study can be used to define the role of different wheat varieties in colon cancer prevention