Fluorescence lifetime imaging unravels the pathway of glioma cell death upon hypericin-induced photodynamic therapy

Abstract

Malignant primary brain tumors are a group of highly aggressive and often infiltrating tumors that lack adequate therapeutic treatments to achieve long time survival. Complete tumor removal is one precondition to reach this goal. A promising approach to optimize resection margins and eliminate remaining infiltrative so-called guerilla cells is photodynamic therapy (PDT) using organic photosensitizers that can pass the disrupted blood-brain-barrier and selectively accumulate in tumor tissue. Hypericin fulfills these conditions and additionally offers outstanding photophysical properties, making it an excellent choice as a photosensitizing molecule for PDT. However, the actual hypericin-induced PDT cell death mechanism is still under debate. In this work, hypericin-induced PDT was investigated by employing the three distinct fluorescent probes hypericin, resorufin and propidium iodide (PI) in fluorescence-lifetime imaging microscopy (FLIM). This approach enables visualizing the PDT-induced photodamaging and dying of single, living glioma cells, as an in vitro tumor model for glioblastoma. Hypericin PDT and FLIM image acquisition were simultaneously induced by 405 nm laser irradiation and sequences of FLIM images and fluorescence spectra were recorded to analyze the PDT progression. The reproducibly observed cellular changes provide insight into the mechanism of cell death during PDT and suggest that apoptosis is the initial mechanism followed by necrosis after continued irradiation. These new insights into the mechanism of hypericin PDT of single glioma cells may help to adjust irradiation doses and improve the implementation as a therapy for primary brain tumors.

New Avenues and Major Achievements in Phytocompounds Research for Glioblastoma Therapy

“…On the other hand, hypericin is regarded as an excellent photosensitizer and undergoes intensive evaluation in the context of PDT of GBM. Recently, the mechanism of hypericininduced PDT was investigated in single glioma cells [80]. A sequence of steps that cause cell damage was revealed by the spatially resolved fluorescence lifetime and spectroscopic examination of hypericin-induced PDT on glioma cells.…”

“…A sequence of steps that cause cell damage was revealed by the spatially resolved fluorescence lifetime and spectroscopic examination of hypericin-induced PDT on glioma cells. These steps include membrane bleb formation, ER degradation, cell shrinkage and rounding, and breakdown of membrane stability, which leads to flooding of the cell with the surrounding medium [80]. Therefore, it was discovered that apoptosis followed by necrosis were the main causes of cell death in hypericin-induced PDT on glioma cells [80].…”

“…These steps include membrane bleb formation, ER degradation, cell shrinkage and rounding, and breakdown of membrane stability, which leads to flooding of the cell with the surrounding medium [80]. Therefore, it was discovered that apoptosis followed by necrosis were the main causes of cell death in hypericin-induced PDT on glioma cells [80].…”