Nitric Oxide-Releasing Gemcitabine Derivatives Induce Apoptotic Cancer Cell Death

Background/Aim: Poor tumor perfusion can limit drug delivery to solid tumors and reduce the therapeutic efficacy of anticancer agents. Nitric oxide (NO) is a bioactive molecule that can modulate tumor vasculature. In this study, we synthesized two NO-releasing gemcitabine derivatives, GEM-NPB and GEM-NO, to explore whether NO-releasing modification preserves the anticancer activity of gemcitabine while providing NO-donor properties. Materials and Methods: The NO-donor properties of GEM-NPB and GEM-NO were evaluated by measuring NOx formation and compared with those of the previously developed NO donor NPB. The antiproliferative effects of gemcitabine, GEM-NPB, GEM-NO, and NPB were examined in C26 cancer cells using an MTS assay. Cell death was assessed by an Annexin V assay. Apoptotic signaling was evaluated by western blotting for poly(ADP-ribose) polymerase (PARP) cleavage. The effects of these compounds were also examined using C26 spheroids. Results: GEM-NPB and GEM-NO showed time-dependent NOx formation, indicating that both compounds retained NO-donor properties. In C26 cells, both derivatives inhibited cell proliferation in a concentration-dependent manner, although their effects were weaker than those of gemcitabine. The Annexin V assay showed that GEM-NPB and GEM-NO induced cell death; however, at 50 μM, GEM-NPB induced significantly less cell death than gemcitabine. Gemcitabine, GEM-NPB and GEM-NO induced PARP cleavage, whereas NPB alone did not. In spheroids, gemcitabine, GEM-NPB, and GEM-NO caused surface disruption suggestive of cell damage or cell death, whereas NPB alone did not induce apparent surface disruption. Conclusion: GEM-NPB and GEM-NO retain gemcitabine-like apoptosis-inducing activity while acquiring NO-donor properties. These findings provide a basis for the further development of NO-releasing gemcitabine derivatives as candidate anticancer agents for poorly perfused solid tumors.