Hypericin, a potential new BH3 mimetic.

Many types of cancer such as prostate cancer, myeloid leukemia, breast cancer, glioblastoma display strong chemo resistance, which is supported by enhanced expression of multiple anti-apoptotic Bcl-2, Bcl-XL and Mcl-1 proteins. The viable anti-cancer strategies are based on developing anti-apoptotic Bcl-2 proteins inhibitors, BH3 mimetics. Our focus in past years has been on the investigating a new potential BH3 mimetic, Hypericin (Hyp). Hyp is a naturally occurring photosensitive compound used in photodynamic therapy and diagnosis. We have demonstrated that Hyp can cause substantial effects in cellular ultrastructure, mitochondria function and metabolism, and distribution of Bcl2 proteins in malignant and non-malignant cells. One of the possible mechanisms of Hyp action could be the direct interactions between Bcl-2 proteins and Hyp. We investigated this assumption by in silico computer modelling and in vitro fluorescent spectroscopy experiments with the small Bcl2 peptide segments designed to correspond to Bcl2 BH3 and BH1 domains. We show here that Hyp interacts with BH3 and BH1 peptides in concentration dependent manner, and shows the stronger interactions than known BH3 mimetics, Gossypol (Goss) and ABT-263. In addition, interactions of Hyp, Goss and ABT263, with whole purified proteins Bcl-2 and Mcl-1 by fluorescence spectroscopy show that Hyp interacts stronger with the Bcl-2 and less with Mcl-1 protein than Goss or ABT-263. This suggest that Hyp is comparable to other BH3 mimetics and could be explore as such. Hyp cytotoxicity was low in human U87 MG glioma, similar to that of ABT263, where Goss exerted sufficient cytotoxicity, suggesting that Hyp acts primarily on Bcl-2, but not on Mcl-1 protein. In combination therapy, low doses of Hyp with Goss effectively decreased U87 MG viability, suggesting a possible synergy effect. Overall, we can conclude that Hyp as BH3 mimetic acts primarily on Bcl-2 protein and can be explored to target cells with Bcl-2 over-expression, or in combination with other BH3 mimetics, that target Mcl-1 or Bcl-XL proteins, in dual therapy.

Toxicological evaluation of fluorescent 11-mercaptoundecanoic gold nanoclusters as promising label-free bioimaging probes in different cancer cell lines.

Due to advancement in nanomaterials and increasing use of functionalized gold nanoclusters (AuNCs) in different biomedical applications, better understanding of their potential cytotoxicity is necessary. Interactions of ultra-small fluorescent AuNCs with mammalian cells remains up to this day poorly understood, therefore, cytotoxic evaluation of thoroughly characterized ca. 2.5 nm spherical water-soluble 11-mercaptoundecanoic acid coated AuNCs (AuNC@M) with diverse fluorescent properties in variety of mammalian cancer cell lines was performed. Cell viability was assessed by traditional MTT assay and xCELLigence real time cell analyzer. Cell apoptosis was evaluated via an Annexin V-FITC/propidium iodide (PI) assay. Confocal fluorescence imaging confirmed that tested AuNC@M entered live cells and were homogeneously distributed in their cytoplasm. The results suggested that the cytotoxicity of tested nanoclusters was very low, or near the control level at concentrations 0.1 and 0.5 mg/mL in the cell lines after 24 h exposition. The purity of tested AuNC@M had no relevant effect on cell viability and no differences were observed after 24 h in our study. The low toxicity toward cancer cells further strengthens our view that AuNC@M are promising label-free fluorescent probes for bio-labelling and bio-imaging, or they can even serve as platforms for antitumor drug delivery systems.

Importance of Hypericin-Bcl2 interactions for biological effects at subcellular levels.

Hypericin (Hyp) is a naturally occurring compound used as photosensitizer in photodynamic therapy and diagnosis. Recently, we have shown that Hyp presence alone, without illumination, resulted in substantial biological effects at several sub-cellular levels. Hyp induced changes in cellular ultrastructure, mitochondria function and metabolism, and distribution of Bcl2 proteins in malignant and non-malignant cells. The molecular mechanisms that underlie Hyp light-independent effects are still elusive. We have hypothesized that Bcl2-Hyp interactions might be one possible mechanism. We performed molecular docking studies to determine the Hyp-Bcl2 interaction profile. Based on the interaction profiles small Bcl2 peptide segments were selected for further study. We designed small peptides corresponding to Bcl2 BH3 and BH1 domains and tested the binding of Hyp and Bcl2 known inhibitor, ABT263, to the peptides in computer modeling and in vitro binding studies. We employed endogenous tryptophan and tyrosine in the BH3 and BH1 peptides, respectively, and their fluorescent properties to show interaction with Hyp and ABT263. Overall, our results indicate that Hyp can interact with Bcl2 protein at its BH3-BH1 hydrophobic groove, and this interaction may trigger changes in intracellular distribution of Bcl2 proteins. In addition, our computer modeling results suggest that Hyp also interacts with other anti-apoptotic members of Bcl2 family similar to the known BH3 mimetics. Our findings are novel and might contribute to understanding Hyp light-independent effects. In addition, they may substantiate the therapeutic use of Hyp as a BH3 mimetic molecule to enhance other cancer treatments.

The flashlights on a distinct role of protein kinase C δ: Phosphorylation of regulatory and catalytic domain upon oxidative stress in glioma cells.

Glioblastoma multiforme are considered to be aggressive high-grade tumors with poor prognosis for patient survival. Photodynamic therapy is one of the adjuvant therapies which has been used for glioblastoma multiforme during last decade. Hypericin, a photosensitizer, can be employed in this treatment. We have studied the effect of hypericin on PKCδ phosphorylation in U87 MG cells before and after light application. Hypericin increased PKCδ phosphorylation at tyrosine 155 in the regulatory domain and serine 645 in the catalytic domain. However, use of the light resulted in apoptosis, decreased phosphorylation of tyrosine 155 and enhanced serine 645. The PKCδ localization and phosphorylation of regulatory and catalytic domains were shown to play a distinct role in the anti-apoptotic response of glioma cells. We hypothesized that PKCδ phosphorylated at the regulatory domain is primarily present in the cytoplasm and in mitochondria before irradiation, and it may participate in Bcl-2 phosphorylation. After hypericin and light application, PKCδ phosphorylated at a regulatory domain which is in the nucleus. In contrast, PKCδ phosphorylated at the catalytic domain may be mostly active in the nucleus before irradiation, but active in the cytoplasm after the irradiation. In summary, light-induced oxidative stress significantly regulates PKCδ pro-survival and pro-apoptotic activity in glioma cells by its phosphorylation at serine 645 and tyrosine 155.

Assessing light-independent effects of hypericin on cell viability, ultrastructure and metabolism in human glioma and endothelial cells.

Cell exposure to light-independent effects of photosensitizers (PS) used in PDT is clinically relevant when PS affect the pro-apoptotic cascade. In many malignant cells, Hypericin (Hyp) has PS displayed light-dependent anti-proliferative and cytotoxic effects with no cytotoxicity in the dark. Recent studies have shown that Hyp also exhibited light-independent cytotoxic effects in a wide range of concentrations. The molecular mechanisms underlying Hyp light-independent (dark) toxicity may be due to its interaction with different molecules at the Hyp accumulation sites including mitochondria, and these mechanisms are not understood in detail. Here, we demonstrate that in human glioma and endothelial cells, Hyp displayed light-independent effects at several sub-cellular levels (ultrastructure, mitochondria function and metabolism, and protein synthesis). Taking together previously published and our present results, the findings strongly suggest that Hyp light independent effects: (i) depend on the cell type and metabolism; (ii) underlying molecular mechanisms are due to Hyp interaction with the multiple target molecules including Bcl2 family of proteins. In addition, the findings suggest that Hyp without illumination can be explored as an adjuvant therapeutic drug in combination with chemo- or radiation cancer therapy.

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

Photosensitizers (PSs) in photodynamic therapy (PDT) are, in most cases, administered systemically with preferential accumulation in malignant tissues; however, exposure of non-malignant tissues to PS may also be clinically relevant, when PS molecules affect the pro-apoptotic cascade without illumination. Hypericin (Hyp) as PS and its derivatives have long been studied, regarding their photodynamic and photocytotoxic characteristics. Hyp and its derivatives have displayed light-activated antiproliferative and cytotoxic effects in many tumor cell lines without cytotoxicity in the dark. However, light-independent effects of Hyp have emerged. Contrary to the acclaimed Hyp minimal dark cytotoxicity and preferential accumulation in tumor cells, it was recently been shown that non-malignant and malignant cells uptake Hyp at a similar level. In addition, Hyp has displayed light-independent toxicity and anti-proliferative effects in a wide range of concentrations. There are multiple mechanisms underlying Hyp light-independent effects, and we are still missing many details about them. In this paper, we focus on Hyp light-independent effects at several sub-cellular levels-protein distribution and synthesis, organelle ultrastructure and function, and Hyp light-independent effects regarding reactive oxygen species (ROS). We summarize work from our laboratories and that of others to reveal an intricate network of the Hyp light-independent effects. We propose a schematic model of pro- and anti-apoptotic protein dynamics between cell organelles due to Hyp presence without illumination. Based on our model, Hyp can be explored as an adjuvant therapeutic drug in combination with chemo- or radiation cancer therapy.

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

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.

Endosomes: guardians against [Ru(Phen)3]2+ photo-action in endothelial cells during in vivo pO2 detection?

Phototoxicity is a side-effect of in vitro and in vivo oxygen partial pressure (pO2) detection by luminescence lifetime measurement methods. Dichlorotris(1,10-phenanthroline)-ruthenium(ii) hydrate ([Ru(Phen)3]2+) is a water soluble pO2 probe associated with low phototoxicity, which we investigated in vivo in the chick's chorioallantoic membrane (CAM) after intravenous or topical administration and in vitro in normal human coronary artery endothelial cells (HCAEC). In vivo, the level of intravenously injected [Ru(Phen)3]2+ decreases within several minutes, whereas the maximum of its biodistribution is observed during the first 2 h after topical application. Both routes are followed by convergence to almost identical "intra/extra-vascular" levels of [Ru(Phen)3]2+. In vitro, we observed that [Ru(Phen)3]2+ enters cells via endocytosis and is then redistributed. None of the studied conditions induced modification of lysosomal or mitochondrial membranes without illumination. No nuclear accumulation was observed. Without illumination [Ru(Phen)3]2+ induces changes in endoplasmic reticulum (ER)-to-Golgi transport. The phototoxic effect of [Ru(Phen)3]2+ leads to more marked ultrastructural changes than administration of [Ru(Phen)3]2+ only (in the dark). These could lead to disruption of Ca2+ homeostasis accompanied by mitochondrial changes or to changes in secretory pathways. In conclusion, we have demonstrated that the intravenous injection of [Ru(Phen)3]2+ into the CAM model mostly leads to extracellular localization of [Ru(Phen)3]2+, while its topical application induces intracellular localization. We have shown in vivo that [Ru(Phen)3]2+ induces minimal photo-damage after illumination with light doses larger by two orders of magnitude than those used for pO2 measurements. This low phototoxicity is due to the fact that [Ru(Phen)3]2+ enters endothelial cells via endocytosis and is then redistributed towards peroxisomes and other endosomal and secretory vesicles before it is eliminated via exocytosis. Cellular response to [Ru(Phen)3]2+, survival or death, depends on its intracellular concentration and oxidation-reduction properties.

The role of anti-apoptotic protein kinase Cα in response to hypericin photodynamic therapy in U-87 MG cells.

Hypericin photodynamic therapy (HypPDT) has been found to be an efficient inducer of cell death. However, there are indications that HypPDT also activates rescuing pathways. Cell responses to HypPDT are highly dependent on the Hyp intracellular localization and accumulation. We have shown previously that in U87 MG cells Hyp localizes mostly in ER and partially in mitochondria, lysosomes and Golgi, and that HypPDT resulted primarily in apoptosis via the mitochondrial apoptotic pathway. We have also shown that Hyp co-localizes and interacts with anti-apoptotic PKCα in U87 MG cells. To follow up on our previous work, we investigated how HypPDT influences PKCα in U87 MG cells. Here, we show that majority of PKCα present in U87 MG cells is already in a catalytically competent form phosphorylated at Thr638, and it is a likely Bcl2 kinase. The presence of Hyp itself does not affect PKCα distribution. HypPDT acute effect caused PKCα activation and translocation along the plasma membrane and partially in the nuclei. The prolonged effect of HypPDT, 5 and 24h post PDT, results in PKCα located predominantly in cytosol and nuclei. Moreover, we have shown that phosphorylated catalytically competent PKCα is critical for U87 glioma cell viability in response to HypPDT treatment.

Bcl-2 proapoptotic proteins distribution in U-87 MG glioma cells before and after hypericin photodynamic action.

Apoptosis is a key process in the development and maintenance of tissue homeostasis. This process of controlled cell death is tightly regulated by a balance between cell survival and damage signals. We focused our attention towards one apoptotic pathway, the intrinsic mitochondrial one where Bcl-2 family of proteins plays the major role. We are particularly interested in two pro-apoptotic players Bak and Bax from this family. Here we investigated their role in apoptosis triggered by photodynamic action. Targeted photodynamic therapy (PDT) is a promising approach to diagnose and treat different types of cancer. We show the localization of Bax and Bak in U-87 MG human glioma cells incubated with photosensitizer hypericin (Hyp) before and after photodynamic action. Apoptotic stimulus by Hyp photodynamic action causes Bax translocation into mitochondria. However our results suggest that under these conditions there are two populations of mitochondria: one which contains Bax and Bak simultaneously, and is almost exclusively localized near the plasma membrane; the other which contains Bax only and is distributed throughout the cell. The different protein content and spatial distribution of these two populations suggest that they can play different roles in response to apoptotic stimuli.

The IQ motif is crucial for Cav1.1 function.

Ca(2+)-dependent modulation via calmodulin, with consensus CaM-binding IQ motif playing a key role, has been documented for most high-voltage-activated Ca(2+) channels. The skeletal muscle Ca(v)1.1 also exhibits Ca(2+)-/CaM-dependent modulation. Here, whole-cell Ca(2+) current, Ca(2+) transient, and maximal, immobilization-resistant charge movement (Q(max)) recordings were obtained from cultured mouse myotubes, to test a role of IQ motif in function of Ca(v)1.1. The effect of introducing mutation (IQ to AA) of IQ motif into Ca(v)1.1 was examined. In dysgenic myotubes expressing YFP-Ca(v)1.1(AA), neither Ca(2+) currents nor evoked Ca(2+) transients were detectable. The loss of Ca(2+) current and excitation-contraction coupling did not appear to be a consequence of defective trafficking to the sarcolemma. The Q(max) in dysgenic myotubes expressing YFP-Ca(v)1.1(AA) was similar to that of normal myotubes. These findings suggest that the IQ motif of the Ca(v)1.1 may be an unrecognized site of structural and functional coupling between DHPR and RyR.

Metabolic phenotype and adipose tissue inflammation in patients with chronic obstructive pulmonary disease.

Potential links between metabolic derangements and adipose tissue (AT) inflammation in patients with chronic obstructive pulmonary disease (COPD) are unexplored. We investigated AT expressions of interleukin (IL)-6, tumor necrosis factor (TNF)-α, CD68 (macrophage cell surface receptor), caspase-3, and Bax, and their relationships to the metabolic phenotype in nine cachectic, 12 normal-weight, 12 overweight, and 11 obese patients with COPD (age 62.3 ± 7.2 years). With increasing body mass index, increases in AT expressions of IL-6, TNF-α, and CD68 were observed (P < .001; P = .005; P < .001, resp.), in association with reduced insulin sensitivity (P < .001). No differences were observed between cachectic and normal-weight patients in AT expressions of inflammatory or proapoptotic markers. Adipose tissue CD68 and TNF-α expressions predicted insulin sensitivity independently of known confounders (P = .005; P = .025; R(2) = 0.840). Our results suggest that AT inflammation in obese COPD patients relates to insulin resistance. Cachectic patients remain insulin sensitive, with no AT upregulation of inflammatory or proapoptotic markers.

Ca2+/CaM-dependent inactivation of the skeletal muscle L-type Ca2+ channel (Cav1.1).

Ca2+-dependent modulation via calmodulin (CaM) has been documented for most high-voltage-activated Ca2+ channels, but whether the skeletal muscle L-type channel (Cav1.1) exhibits this property has been unknown. In this paper, whole-cell current and fluorescent resonance energy transfer (FRET) recordings were obtained from cultured mouse myotubes to test for potential involvement of CaM in function of Cav1.1. When prolonged depolarization (800 ms) was used to evoke Cav1.1 currents in normal myotubes, the fraction of current remaining at the end of the pulse displayed classic signs of Ca2+-dependent inactivation (CDI), including U-shaped voltage dependence, maximal inactivation (approximately 30%) at potentials eliciting maximal inward current, and virtual elimination of inactivation when Ba2+ replaced external Ca2+ or when 10 mM BAPTA was included in the pipette solution. Furthermore, CDI was virtually eliminated (from 30 to 8%) in normal myotubes overexpressing mutant CaM (CaM1234) that does not bind Ca2+, whereas CDI was unaltered in myotubes overexpressing wild-type CaM (CaMwt). In addition, a significant FRET signal (E=4.06%) was detected between fluorescently tagged Cav1.1 and CaMwt coexpressed in dysgenic myotubes, demonstrating for the first time that these two proteins associate in vivo. These findings show that CaM associates with and modulates Cav1.1.