Colorectal carcinoma (CRC) is a serious complication of inflammatory bowel disease (IBD) and accounts for approximately 15% of all IBD-associated deaths. The likelihood of IBD-related carcinoma is greater than that of sporadic CRC. Over one half are diagnosed at stage III or IV when cancer cells have already invaded surrounding tissues and most conventional therapeutics are limited in their success. Therefore the early detection of cancer, which is difficult in IBD, is crucial for its ultimate control and prevention. While mining the colonic mucosal and submucosal layers for biomarkers that differentiate ulcerative colitis (UC) from Crohn's colitis (CC) using Matrix-assisted laser desorption/ionization (MALDI) profiling, we found a signal at m/z 5045 to be more intense in UC. Liquid chromatography–mass spectrometry (LC-MS/MS) analysis allowed identification of this signal as triply charged hemoglobin alpha chain.
Macrophages are highly versatile phagocytes active in multiple roles in the immune system and key players in the inflammatory response. Their presence within the inflammatory microenvironment, in some cases, has been proven to increase transformation, angiogenesis, and immunosuppression. In hemorrhagic situations (as in UC), macrophages engulf erythrocytes that are outside the vascular bed and as a result free hemoglobin is released. Hemoglobin induces DNA damage in human colonic cells and is genotoxic. The potential carcinogenic effects of hemoglobin were documented when it was shown that hemoglobin increases the number of aberrant crypt foci in colon mucosa. In the colon, free hemoglobin is expected to increase the production of reactive oxygen (O2) species (ROS) from peroxides via the Fenton reaction, which may be the cause of cellular toxicity and eventually pro-mutagenic lesions. Intracellular reactions with active O2 can result in the initiation and progression of carcinogenesis by induction of gene mutations, chromosomal damage and cytotoxic effects.
We hypothesize that elevated expression of mucosal free hemoglobin would be associated with an increased risk of CRC. To validate this hypothesis will require investigating whether hemoglobin could be classified as a proliferative or transforming agent for colon cancer cells by causing reactive oxygen species release and subsequent DNA damage. For this purpose, we will assess the cellular viability of normal colonic cell-lines, NCM 356 and NCM 460. These cell-lines will be treated with hemoglobin at different concentrations to determine the changes in levels of ROS. ROS production will be measured using C-400 staining assay and further analysis will be carried out using FACS. Additionally, we will also examine the potential cytotoxicity of hemoglobin.
Supported: MMC-VICC Cancer Partnership Grant#: 3U54CA091408-09S 1; MeTRC grant#: 5U54RR026140-03, and Vanderbilt SPORE in GI Cancer Grant#: P50CA095103.
