Design of fluorescent probes for bioorthogonal labeling of carbonylation in live cancer cells

Oxidative stress is known as the steady state level of oxidative damage in live cell, tissue, or organ, caused by the reactive oxygen species (ROS) such as free radicals. Along with neurodegenerative diseases and diabetes, high levels of reactive oxygen species have been noticed in almost all cancers, where they promote many aspects of tumor development and progression. Bioorthogonal labeling of oxidative stress-induced carbonylation of biomolecules have a critical role in the identification of cancer aggressiveness. Hydrazine or amine-based fluorophores act as strong nucleophiles that react rapidly with carbonyl sections in live cells. In this study, hydrazine and amine derivatives were synthesized in order to detect ROS induced protein carbonylation in various cancer cell lines. 2-Hydrazine-5-nitrophenol was utilized as a novel bioorthogonal fluorescent probes to demonstrate labeling of carbonylated biomolecules. Reaction between carbonyl group and hydrazine containing fluorophore yielded in hydrazone which maintained a spectroscopic alteration. Microscopic and fluorometric analyses were used to distinguish the exogenous and endogenous ROS induced carbonylation profile in A498 primary site and ACHN metastatic site renal cell carcinoma (RRC) cell lines. When cells were treated with H2O2 as an exogenous ROS inducer, A498 cell line demonstrated higher carbonylation level. On the other hand, serum starvation as an endogenous ROS inducer resulted in higher carbonylation level in ACHN metastatic site RCC cell line. The probe developed here may be used as a small molecule in the development of new diagnostics approach in the molecular staging of cancer.