Anti- and Pro-apoptotic Bcl2 Proteins Distribution and Metabolic Profile in Human Coronary Aorta Endothelial Cells Before and After HypPDT

Abstract

Understanding apoptosis regulatory mechanisms in endothelial cells (ECs) has great importance for the development of novel therapy strategies for cancer and cardiovascular pathologies. An oxidative stress with the generation of reactive oxygen species (ROS) is a common mechanism causing ECs' dysfunction and apoptosis. The generation of ROS can be triggered by various stimuli including photodynamic therapy (PDT). In most PDT treatments, photosensitizer (PS) is administered systemically, and thus, possibility of high exposure to PS in the ECs remains high. PS accumulation in ECs may be clinically relevant even without PDT, if PS molecules affect the pro-apoptotic cascade without illumination. In the present work, we focused on Hypericin (Hyp) and HypPDT effects on the cell viability, oxidative stress, and the distribution of Bcl2 family members in human coronary artery endothelial (HCAEC) cells. Our findings show that the presence of Hyp itself has an effect on cell viability, oxidative stress, and the distribution of Bcl2 family members, without affecting the mitochondria function. In contrast, HypPDT resulted in mitochondria dysfunction, further increase of oxidative stress and effect on the distribution of Bcl2 family members, and in primarily necrotic type of death in HCAEC cells.

Hypericin in the Dark: Foe or Ally in Photodynamic Therapy?

“…They play a key role in mitochondria apoptotic pathway and were shown to regulate mitochondria morphogenesis (fission/fusion) [19,20,33,34]. We studied the Hyp light-independent effects on members’ distribution of the Bcl2 family in malignant U87 MG and non-malignant HCAEC cells [29,30]. The U87 MG is a commercially available grade IV human glioma cell line, which was used for various research purposes over four decades.…”

“…Incubation with Hyp changed Bcl2 distribution (Figure 1A). The Bcl2 signal apparently decreased in foci outside the nucleus, and there was noticeable Bcl2 translocation into nuclei in U87 MG (Figure 1) and HAEC cells [30]. Bcl2 translocation into nuclei can enable the on-set of apoptosis via the association with nuclear receptor Nur77, which plays a role in regulation of differentiation, proliferation, apoptosis, and survival in different cell types [35].…”

“…Hyp presence resulted in distinctive changes in Bax distribution. In U87 MG (Figure 1A) and HCAEC [30], there was significant Bax translocation into distinct foci throughout the cell. In HCAEC, there was also a substantial Bax translocation into the nuclei.…”

“…The intracellular Hyp concentration depends on the final concentration of Hyp in the cell media, incubation times and on the cell uptake rate [38,39]. Bcl2 and Bax translocation patterns in U87 MG and HCAEC cells due to Hyp in the dark may underlie a different cell response to Hyp in viability assays measured via flow cytometry with Annexin V and propidium iodide [30,39]. In U87 MG, Hyp did not affect cell viability [25,29], and in the HCAEC resulted in a significant decrease in viability from 90% to 50% [30].…”

“…Up to this date, findings regarding Hyp light-independent effects are as follows. (I) Hyp colocalizes with PKCα in U87 MG cells, and Hyp binding assays and molecular modeling indicate direct interactions between Hyp and PKCα [26,27]; (II) in U87 MG cells, the majority of phosphorylated Bcl2 co-localizes with PKCα [25]; (III) Hyp in the dark follows the ceramide pathway, translocates, and increases PKCδ autophosphorylation at Ser645 in the GA and nucleus of U87 MG cells [28]; (IV) Hyp affects distribution of pro-apoptotic proteins Bak and Bax, and anti-apoptotic Bcl2 in human glioma U87 MG and endothelial HCAEC cells [29,30]; (V) Hyp affects cell viability in metabolically distinct cell phenotypes differently [4,21,22,25,30]. In addition, our newest work suggests that Hyp causes the light-independent effects in bioenergetics, increases oxidative stress, and results in adaptive changes in ultrastructure.…”