Comparison of Cellular Death Pathways after mTHPC-mediated Photodynamic Therapy (PDT) in Five Human Cancer Cell Lines.

Carsten Lange,Martin Mahler,Christiane Lehmann,Patrick J Bednarski

doi:10.3390/cancers11050702

Abstract

One of the most promising photosensitizers (PS) used in photodynamic therapy (PDT) is the porphyrin derivative 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC, temoporfin), marketed in Europe under the trade name Foscan®. A set of five human cancer cell lines from head and neck and other PDT-relevant tissues was used to investigate oxidative stress and underlying cell death mechanisms of mTHPC-mediated PDT in vitro. Cells were treated with mTHPC in equitoxic concentrations and illuminated with light doses of 1.8–7.0 J/cm2 and harvested immediately, 6, 24, or 48 h post illumination for analyses. Our results confirm the induction of oxidative stress after mTHPC-based PDT by detecting a total loss of mitochondrial membrane potential (Δψm) and increased formation of ROS. However, lipid peroxidation (LPO) and loss of cell membrane integrity play only a minor role in cell death in most cell lines. Based on our results, apoptosis is the predominant death mechanism following mTHPC-mediated PDT. Autophagy can occur in parallel to apoptosis or the former can be dominant first, yet ultimately leading to autophagy-associated apoptosis. The death of the cells is in some cases accompanied by DNA fragmentation and a G2/M phase arrest. In general, the overall phototoxic effects and the concentrations as well as the time to establish these effects varies between cell lines, suggesting that the cancer cells are not all dying by one defined mechanism, but rather succumb to an individual interplay of different cell death mechanisms. Besides the evaluation of the underlying cell death mechanisms, we focused on the comparison of results in a set of five identically treated cell lines in this study. Although cells were treated under equitoxic conditions and PDT acts via a rather unspecific ROS formation, very heterogeneous results were obtained with different cell lines. This study shows that general conclusions after PDT in vitro require testing on several cell lines to be reliable, which has too often been ignored in the past.

Highlights

Photodynamic therapy (PDT) is a method for the medical treatment of solid tumors based on the generation of reactive oxygen species (ROS) after photoactivation of a photosensitizer (PS)
The overall phototoxic effects and the concentrations as well as the time to establish these effects varies between cell lines, suggesting that the cancer cells are not all dying by one defined mechanism, but rather succumb to an individual interplay of different cell death mechanisms
After treatment with mTHPC and subsequent application of light, a decrease in red fluorescence was observed in all cell lines. These results indicate that mTHPC-PDT leads to a depolarization of the ∆ψm and a loss of mitochondrial activity, which is a sign of early apoptosis

Summary

Introduction

Photodynamic therapy (PDT) is a method for the medical treatment of solid tumors based on the generation of reactive oxygen species (ROS) after photoactivation of a photosensitizer (PS). The PS is excited by light of a certain wavelength and transfers its energy to triplet oxygen to form highly reactive singlet oxygen (1 O2 ) or generates other ROS via a direct electron transfer to biological substrates, e.g., components of the cellular membrane. The ROS formed lead to oxidative stress within the cells of the treated tumor and eventually cause cellular death [1,2]. Further advantages over established cancer therapies include nontoxicity in the absence of light, the possibility of repeated treatments without cumulative toxicity, no carcinogenicity, and the fact, that PDT is a non- or only minimally-invasive approach [1,3]. One of the most promising PS used in PDT is the porphyrin derivative 5,10,15,20-tetra(mhydroxyphenyl)chlorin (mTHPC, temoporfin), which was developed in 1989 [4] and approved under the trade name Foscan® in 2001 in Europe for the treatment of head and neck cancers [5,6]

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Discussion

Conclusion