Nowadays, cancer is a well-recognized and prevalent cause of death worldwide, accounting for nearly 10 million deaths in 2020. Following its relevant socioeconomic consequences, significant efforts were focused on developing therapies to confront and diagnose this pathology. However, to date, cancer incidence and mortality remain high. Consequently, new approaches have been developed. Among them, photodynamic therapy (PDT) has shown significant potential for the treatment and detection of cancer. This technique is based on the administration of a molecule (photosensitizer), and then, after accumulation, light is applied, which will activate the photosensitizer, producing fluorescence. In addition, in the presence of oxygen, this process could lead to the accumulation of reactive oxygen species, leading to cellular death. Among the most promising prodrugs employed nowadays, 5-ALA mediated PpIX gathers major recognition and promise. This molecule (5-ALA) is preferentially metabolized in cancer cells. Aiming to further characterize and refine 5-ALA-mediated PpIX, for the first time, we evaluated the consequences of this treatment in cancer cells to identify significant changes in protein expression, either implicated in 5-ALA metabolism or constituting interesting targets.
Along the present thesis, we compared bladder cancer cells (T24) treated with 5-ALA, SA (inhibitor of HMBS) to non-treated cells, employing proteomic mass spectrometry analysis. Interestingly, our results demonstrated that either porphyrin upregulation (PpIX accumulation) or 5-ALA accumulation (5-ALA + SA treatment) induced a significant change in several enzymes implicated in the metabolism of this pro-drug. More interestingly, each condition opposite changes on protein expression, either upregulating or downregulating those enzymes involved in heme degradation and synthesis pathway.
In parallel to the significant changes of the 5-ALA metabolism-related enzymes, we also identified several promising targets overexpressed after 5-ALA treatment. Indeed, OR1B1, AGRN, NCSTN, TMEM, and SLC44A1 were significantly upregulated after 5-ALA supply and without light activation. Mass spectrometry data was then further validated by immunofluorescence in T24, A549, and PC3 cells. Importantly, the transmembrane nature of these proteins is a relevant property, facilitating the detection and targeting of cancer cells with intact cellular membranes.
In conclusion, our results demonstrate for the first time a potential regulatory mechanism modulating both the synthesis and degradation of heme, preventing the accumulation of toxic intermediates (ie., PpIX, heme, or PBG). Indeed, to our knowledge, this is the first time this mechanism has been endorsed with significant experimental data. Consequently, we hypothesized that upon 5-ALA exogenous supply, the described changes in protein expression support cell viability. Along the second aim, we also demonstrated that 5-ALA treatment regulates the expression of several proteins non-related to the porphyrin pathway. These can be used to develop a new therapy independent of light stimulation further.
