Protein carbonylation is an irreversible post-translational modification induced by severe oxidative stress.
Reactive oxygen species (ROS) are constantly produced in cells and play important roles in both
cancer progression and
cancer suppression. ROS levels can be higher in
tumor compared to surrounding healthy tissue but ROS-induced specific protein carbonylation and its unique role in
cancer progression or suppression is poorly understood. In this study, we utilized previously validated ELISA and western blot methods to analyze the total and specific protein carbonylation in flash-frozen human
breast cancer and matched adjacent healthy tissue to compare relative total, and specific protein carbonylation. Mass spectrometry, two-color western, and immunoprecipitation methods were used to identify and confirm the specifically carbonylated
proteins in
breast tumor tissue.
Superoxide dismutase (SOD) activity was measured as an
indicator of
antioxidant activity, and LC3-II
protein level was analyzed for autophagy by western blot. Findings were further confirmed using the immortalized MDA-MB-231 and MDA-MB-468
breast cancer and MCF-12A noncancerous human epithelial breast cell lines. Our results indicate that
tumor tissue has greater total protein carbonylation, lower SOD1 and
SOD2 protein levels, lower total SOD activity, and higher LC3-II levels compared to adjacent healthy tissue. We identified and confirmed three specific
proteins of interest;
filamin A,
heat shock protein 90β (HSP90β), and bifunctional
glutamate/
proline-
tRNA ligase (EPRS), that were selectively carbonylated in
tumor tissue compared to matched adjacent healthy tissue. Correspondingly, compared to noncancerous MCF-12A epithelial cells, MDA-MB-231
cancer cells exhibited an increase in
filamin A and EPRS protein carbonylation, decreased total SOD activity, and increased autophagy, but not increased HSP90β protein carbonylation. Identification of selectively carbonylated
proteins and defining their roles in
cancer progression may promote the development of targeted therapeutic approaches toward mitigating oxidative damage of these
proteins.