AbstractWe have recently demonstrated that riboflavin (Rf) functions as unconventional bioorthogonal photocatalyst for the activation of Pt^IV prodrugs. In this study, we show how the combination of light and Rf with two Pt^IV prodrugs is a feasible strategy for light-mediated pancreatic cancer cell death induction. In Capan-1 cells, which have high tolerance against photodynamic therapy, Rf-mediated activation of the cisplatin and carboplatin prodrugs cis,cis,trans-[Pt(NH₃)₂(Cl)₂(O₂CCH₂CH₂CO₂H)₂] (1) and cis,cis,trans-[Pt(NH₃)₂(CBDCA)(O₂CCH₂CH₂CO₂H)₂] (2, where CBDCA = cyclobutane dicarboxylate) resulted in pronounced reduction of the cell viability, including under hypoxia conditions. Such photoactivation mode occurs to a considerable extent intracellularly, as demonstrated for 1 by uptake and cell viability experiments. ¹⁹⁵Pt NMR, DNA binding studies using circular dichroism, mass spectrometry and immunofluorescence microscopy were performed using the Rf-1 catalyst-substrate pair and indicated that cell death is associated with the efficient light-induced formation of cisplatin. Accordingly, Western blot analysis revealed signs of DNA damage and activation of cell death pathways through Rf-mediated photochemical activation. Phosphorylation of H₂AX as indicator for DNA damage, was detected for Rf-1 in a strictly light-dependent fashion while in case of free cisplatin also in the dark. Photochemical induction of nuclear pH₂AX foci by Rf-1 was confirmed in fluorescence microscopy again proving efficient light-induced cisplatin release from the prodrug system.