Modulation and proteomic changes on the heme pathway following treatment with 5-aminolevulinic acid

Abstract

5-ALA-mediated photodynamic therapy (PDT) has been developed around the heme biosynthesis physiological pathway. It is based on the external supplementation of 5 aminolevulinic acid (5-ALA), increasing the activity of the heme pathway and leading to a significant protoporphyrin IX (PpIX) accumulation. Interestingly, this metbolite accumulation is predominant in cancer cells, induced by a highly active metabolism, therefore limiting off-target side effects and increasing therapy specificity. Nevertheless, the intrinsic mechanism responsible of PpIX accumulation on cells following PDT is still unknown, limiting clinical therapy translation. In order to further understand the mechanisms behind 5-ALA-induced PDT, in this study we aimed to evaluate the proteome changes reported on the physiological heme pathway, in response to an external 5-ALA supplementation. We studied two different scenarios following 5-ALA treatment, 5-ALA accumulation (5-ALA metabolization into the heme pathway blocked with inhibitors) and accumulation of PpIX (normal heme pathway with 5-ALA supplementation). Therefore, we were able to characterize enzymatic changes and to describe bottlenecks in the pathway. Following mass spectrometry analysis, we reported significant differences between 5-ALA and PpIX effects on heme biosynthesis and regulation of degradation. 5-ALA accumulation significantly decreased porphobilinogen deaminase (HMBS) expression, while phorphyrins accumulation (PpIX) upregulated heme synthesis, specifically HMBS and uroporphyrinogen decarboxylase (UROD), and enhanced the enzymatic level of the heme degradation pathway, including Heme oxygenase 1 (HMOX1) and biliverdin reductase A (BLVRA). Interestingly, porphyrins induced a significant downregulation effect on oxygen-dependent coproporphyrinogen-III oxidase (CPOX). In conclusion, in this study we demonstrated that porphyrins play the most relevant role in heme biosynthesis modulation, while 5-ALA alone (PDT substrate) is not responsible of the main changes observed in this pathway during PDT treatment. Understanding heme enzyme modulation would help to design a more rational approach for patient treatment in the clinic. AIM: Effect of 5-ALA and porphyrins on the different Heme biosynthesis and degradation enzymes.

Keywords: 5-aminolevulinic acid; Cancer; Detection; Heme biosynthesis; Mass spectrometry; Photodetection; Photodynamic therapy; Proteomic analysis; Protoporphyrin IX; Treatment.