Identification of biomarkers for the initiation of apoptosis in human preneoplastic colonocytes by proteome analysis

Development of resistance of cells toward proapoptotic signals is regarded as one of the key processes that allow tumor development. To identify proteins that are crucial for the initiation of apoptosis in NCOL-I human preneoplastic colonocytes, we analyzed the proteome of cells exposed to the flavonoids flavone and quercetin that differ in their ability to induce apoptosis although they possess similar structures. Both flavonoids inhibited proliferation and induced differentiation of NCOL-I cells but only quercetin committed the cells to apoptosis. The accessible proteome of NCOL-I cells was separated by 2D-polyacrylamide-gelelectrophoresis and proteins with changed expression level were identified by peptide mass fingerprints of tryptic digests of the protein spots. A pre-fractionation of soluble and lipophilic proteins was used to enhance the resolution of analysis. After exposure to the test compounds for 24 hr, 73 proteins displayed changed steady state levels in case of quercetin and 32 in case of flavone. Several heat-shock proteins, annexins and cytoskeletal caspase substrates were regulated by quercetin but not by flavone and these protein classes are known to play a role in apoptosis induction and execution. Whereas proteins like lamin A, C and desmoplakin, are indicators that apoptosis has already proceeded, others, such as annexin IV or protein kinase C-beta play a pivotal role in the early phases of apoptosis. In conclusion, proteome analysis allowed the identification of marker proteins that are involved in the initiation of apoptotic cell death in preneoplastic colonocytes and those may help to develop new strategies for cancer prevention.