Unraveling Potential Candidate Targets Associated with Expression of p16INK4a or p16 Truncated Fragment by Comparative Proteomics Analysis

P16 is a tumor suppressor protein that is significantly involved in cycle regulation through the reduction of cell progression from G1 phase to S phase via CDK-cyclin D/p16INK4a/pRb/E2F cascade. The minimum functional domain of p16 has been uncovered that may function comparable to wild type p16. To expand the knowledge on molecules and mechanisms by which p16 or p1666-156 fragment suppresses human fibrosarcoma cell line growth, differential proteome profiles of fibrosarcoma cells following p16 full length or the functional domain overexpression were analyzed. Following transfecting HT-1080 fibrosarcoma cells with p16 full length, p1666-156 truncated form, and pcDNA3.1 empty vector, protein extract of each sample was harvested and clarified by centrifugation, and then the protein content was determined via Bradford assay. All protein extract of each sample was analyzed by two-dimensional gel electrophoresis. Immunoblot analysis was performed as further validation of the expression status of identified proteins. Expression of p16 or p1666-156 fragment could induce mostly common alterations (up/down-regulation) of proteome profile of HT-1080 cells. Mass spectrometry identification of the differentially expressed protein spots revealed several proteins that were grouped in functional clusters, including cell cycle regulation and proliferation, cell migration and structure, oxidative stress, protein metabolism, epigenetic regulation, and signal transduction. The minimum functional domain of p16 could act in the same way as p16 full length. Also, these new findings can significantly enrich the understanding of p16 growth-suppressive function at the molecular level by the introduction of potential candidate targets for new treatment strategies. Furthermore, the present study provides strong evidence on the functional efficacy of the identified fragment of p16 for further attempts toward peptidomimetic drug design or gene transfer to block cancer cell proliferation.