Abstract 2845: PpIX synthesis increases PDT resistant cells due to the modulating effect of EGCG on the heme synthesis pathway

Abstract

Abstract Introduction: Photodynamic therapy (PDT) is a treatment for non melanoma skin cancer. PDT kills cells through reactive oxygen species (ROS), produced by interaction between cellular O2 and an excited photosensitizer (PS) by a specific light. One of the most used PS in dermatology is Protoporphyrin IX (PpIX) and its precursor methyl aminolevulinate (MAL). MAL enters into cells and is converted to PpIX by enzymes of the heme group synthesis pathway. The insertion of ferrous iron into PpIX forms a heme molecule, which has no PS action. A previous study shows that efficacy of PDT, in resistant cells, can be improved by addition of epigallocatechin gallate (EGCG), a green tea polyphenol. Therefore, the aim of this work is to evaluate the rol of EGCG on PPIX synthesis. Material and methods: It was used a A-431 cell line from skin squamous cell carcinoma. The EC50 of EGCG was determined by MTT assay after 24 hours. Clonogenic assay was carried out using EGCG 10, 20 and 40 μM, for 4 and 24 hours of incubation. To evaluate PpIX and ROS, A-431 cells were exposed to EGCG (10, 20, 40 μM) with or without MAL 2 mM. After 4 hours, some plates were irradiated with red light to generate ROS. PpIX and ROS were detected by flow cytometry. Gene expression of enzymes of heme synthesis was analyzed by RT-qPCR. Intracellular iron was measured using a commercial kit.  Results: EC50 determined a concentration of 50 μM of EGCG at 24 hours. EGCG concentrations from 10 μM reduced cell clonogenic capacity, even with only 4 hours of incubation. A-431 cells show PDT resistance with MAL 2 mM and a fluence of 4 J/cm2. The addition of EGCG to MAL-PDT showed a decrement of cell viability with an increase of PpIX and ROS. Besides, incubation with EGCG for 4 hours, triggered an increment of HMBS enzyme and decrease of FECH. In addition, EGCG reduced intracellular iron concentrations. Conclusion: These findings show that EGCG has a PDT enhancing effect, decreasing intracellular iron levels and modulating gene expression of heme synthesis enzymes, which are directly related with high levels of PpIX production and ROS levels, causing the death of resistant cells.Acknowledgements: Postdoctoral FONDECYT Nº 3210618, IDeA I+D FONDEF ID21I10027 Citation Format: Daniela León, Álvaro Gutiérrez, Helga Weber, Ramón Silva, María Elena Reyes, Tamara Viscarra, Kurt Buchegger, Carmen Ili, Priscilla Brebi. PpIX synthesis increases PDT resistant cells due to the modulating effect of EGCG on the heme synthesis pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2845.