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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Doroshenko, Tomkova, Kozar and Stroffekova.)

Cancer cell metabolism is a very attractive target for anticancer treatments. This work focuses on protein kinase C (PKC) signaling in the U87 MG glioma. By means of western blot, fluorescence and time-resolved fluorescence microscopy the correlation between the Golgi apparatus (GA), lysosomes and mitochondria were evaluated. The known regulators of PKC were applied to cancer cells. Phorbol myristate acetate (PMA) was chosen as the activator of PKC. Gö6976, hypericin and rottlerin, the inhibitors of PKCα and PKCδ were selected as well. Stabilization, destabilization processes occurring in cells allow classification of observations into several groups. Multiple versions of hierarchical cluster analysis have been applied and similarities have been found between organelles and PKC regulators. The method identified GA as an extraordinary organelle whose functionality is significantly influenced by PKC regulators as well as oxidative stress. Therefore, combination therapy has been designed according to the results of the cluster analysis. Furthermore, the efficacy of photodynamic therapy mediated by hypericin, and the consequent apoptosis, was significantly increased during the treatment. To our knowledge, this is the first demonstration of the effectiveness of the clustering in the given area., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Natural polyphenols, curcumin, rottlerin and EGCG were selected for initial computational modeling of protein-ligand interaction patterns. The docking calculations demonstrated that these polyphenols can easily adjust their conformational shape to fit well into the binding sites of amyloidogenic proteins. The experimental part of the study focused on the effect of rottlerin on fibrillation of three distinct amyloidogenic proteins, namely insulin, lysozyme and Aβ 1-40 peptide. Different experimental protocols such as fluorescence spectroscopy, circular dichroism and atomic force microscopy, demonstrated that amyloid fibril formation of any of the three proteins is inhibited by low micromolar rottlerin concentrations. Most likely, the inhibition of amyloid formation proceeded via interaction of rottlerin with amyloidogenic regions of the studied proteins. Moreover, rottlerin was also effective in pre-formed fibrils disassembly, suggesting that interactions of rottlerin with fibrils were capable to interrupt the fibril-stabilizing bonds of β-sheets. The apparent IC 50 and DC 50 values were calculated in the range of 1.3-36.4 μM and 15.6-25.8 μM, respectively. The strongest inhibiting/disassembling effect of rottlerin was observed on Aβ 1-40 peptide. The cytotoxicity assay performed on the Neuro 2a cells indicated time-dependent cell morphology changes but rottlerin affected the cell viability only at concentration above 50 μM. The results of this study suggest that chemical modifications on rottlerin could be tested in the future as a promising strategy for the modulation of amyloidogenic proteins aggregation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker™ Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Prenatal stress and drug exposure induce permanent alterations of the brain. Even though different brain structures are involved, alterations almost always refer to the hippocampus. The aim of this study was to investigate the excitability of hippocampal slices in low-magnesium epilepsy model of prenatally methamphetamine (MA, 5mg/kg sc.) or saline (sc., stress model) exposed animals in adult male rats. The second aim was to investigate, if a low dose of MA (1ml/kgs.c.) administered in adulthood changes the hippocampal activity of these animals. Adult Wistar male rats were divided into groups according to their prenatal treatment (C - naïve control; Sa - saline; MA - MA administration). One half of the animals was treated with a challenge dose of MA (1mg/kg sc.) 45min before hippocampal slices were cut. The activity of 350μ thick transversal slices of CA1 hippocampi was recorded (latencies of the first epileptiform discharge and the regular epileptiform activity) and evaluated in ACSF with low-magnesium concentration. Effects of prenatal exposure: The highest excitability was found in the Sa (prenatally stressed) group in respect to C and MA groups. This group developed also the highest number of seizure-like events. In addition, the prenatally MA treated group had also higher excitability than C group. Effects of the MA challenge dose: The challenge dose decreased the excitability of prenatally SA- exposed group. To conclude, even a mild prenatal stress significantly increases hippocampal excitability in adulthood and a challenge dose of MA is able to dampen it., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Growth hormone deficiency (GHD) is associated with reduced bone mineral content and increased risk of osteoporotic fractures. Reduced peak bone mass might explain the low bone mineral density (BMD) among patients with childhood onset GHD (CO-GHD) whilst the cause of osteopenia in adult-onset GHD (AO-GHD) is not fully understood., Objectives: Prospective multicentric study to asses bone status in GHD adults after two years of recombinant growth hormone replacement treatment., Methods: In 94 GHD adults (49 men; Ø 34.5 yrs) we have measured BMD and bone markers (CTX, osteocalcin) during two years of rhGH treatment (at baseline, after 3 and 6 months, and after 1 and 2 years). Patients were adequately substituted for GHD and other pituitary deficiencies., Results: We have observed an increase in BMD-lumbar spine: n=42, 0.8155 →0.9418 g/cm2, p<0.0001; femoral neck n=41; 0.8468 →0.9031; p= 0.0004; BMD-whole body 1.0179 →1.0774; p=0.0003. We have compared gender difference: BMD-L-spine by 15.8 % in men (n=21) and by 5.6 % in women (n=19) (p= 0.008); BMD-femoral neck increased by 11.03 % in men and by about 3.0 % in women (p=0.032). In women, the initial decrease in BMD was recorded after 3 months. CO-GHD adults yielded a higher increase in BMD -L-spine (16.6 %, p=0.022). A correlation exists between IGF-I levels and BMD in lumbar spine (1st year: R=0.348, p=0.026; 2nd year: R= 0.33, p=0.0081) and between IGF-I and osteocalcin (1st year: R=0.383; p=0.0038)., Conclusion: Two-year therapy with recombinant human growth hormone improved bone status. IGF-I appears to be a good indicator of rhGH effect on bone (Tab. 3, Fig. 9, Ref. 36). Text in PDF www.elis.sk., (growth hormone deficiency, bone mineral density, bone markers, IGF-I.)