Photodynamic therapy photosensitizers and photoactivated chemotherapeutics exhibit distinct bioenergetic profiles to impact ATP metabolism

Abstract

Energy is essential for all life, and mammalian cells generate and store energy in the form of ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. These processes can now be evaluated by extracellular flux analysis (EFA), which has proven to be an indispensable tool in cell biology, providing previously inaccessible information regarding the bioenergetic landscape of cell lines, complex tissues, and in vivo models. Recently, EFA demonstrated its utility as a screening tool in drug development, both by providing insights into small molecule–organelle interactions, and by revealing the peripheral and potentially undesired off-target effects small molecules have within cells. Surprisingly, technologies to quantify cellular bioenergetics have not been systematically applied in phototherapy development, leaving open several questions about how the mechanism of action of a compound can impact essential cellular functions. Here, we utilized the Seahorse analyzer to address this question for photosensitizers (PSs) for photodynamic therapy (PDT) and contrast these systems to molecules that photo-release a ligand and thus act as photocages or photoactivated chemotherapeutics (PACT), intending to understand the influence these two classes of compounds have on cellular bioenergetics. EFA results show that acute treatment of A549 lung adenocarcinoma cells with PDT agents induces a quiescent bioenergetic response as a result of mitochondrial respiration shutdown. The loss of oxidative phosphorylation is followed by disruption of glycolysis, which occurs after an initial increase in glycolytic respiration is unable to compensate for the interruption of the electron transport chain (ETC). In contrast, the PACT agents tested had little impact on cellular respiration, and the minor inhibition of these metabolic processes was not related to the mechanism of action, as reflected by a lack of correlation with photoejection efficiency. Notably, a system capable of both generating ¹O₂ and photo-releasing a ligand exhibited the dominant profile of a PDT agent and induced the quiescent bioenergetic state, indicating potential implications on cellular bioenergetics for so-called dual-action agents. These findings are presented with the aim to provide the necessary groundwork for expanding the application and utility of EFA to phototherapeutics and to highlight the role of metabolic alterations in PDT.