Characterization of the interaction of hypericin with protein kinase C in U-87 MG human glioma cells

A fluorescence imaging technique was used to monitor intracellular localization of protein kinase C (PKC) in U-87 MG human glioma cells in the presence of hypericin (Hyp) and phorbol 12-myristate-13-acetate (PMA). It is shown that PKC localization, which reflects its activity, is influenced by Hyp and this influence is different from that observed for PMA which acts as PKC activator. Fluorescence binding experiments were used to determine the binding constants of Hyp to several isoforms of PKC. The obtained values of K(d)s (approximately 100 nM) suggest that Hyp binds with high affinity to PKC. Finally, molecular modeling was used to compare structural models of the interaction of C1B domain of PKC (PKC isoforms alpha, delta, gamma) with Hyp and our previously published model of the (C1B domain PKCgamma)/PMA complex. The influence of Hyp on PKC translocation in U-87 MG cells in comparison with PMA, colocalization fluorescence pattern of Hyp and PKC, the higher binding affinity of Hyp to PKC in comparison with known binding constants of phorbol esters, as well as the binding mode of Hyp and PMA to the C1B domain of PKC suggested by molecular modeling, support the idea that Hyp and PMA might competitively bind to the regulatory domain of PKC.     

Neobavaisoflavone May Modulate the Activity of Topoisomerase Inhibitors towards U-87 MG Cells: An In Vitro Study

Despite many advances in therapy, glioblastoma (GB) is still characterized by its poor prognosis. The main reason for this is unsuccessful treatment, which slightly extends the duration of remission; thus, new regimens are needed. One of many types of chemotherapeutics that are being investigated in this field is topoisomerase inhibitors, mainly in combination therapy with other drugs. On the other hand, the search for new anti-cancer substances continues. Neobavaisoflavone (NBIF) is a natural compound isolated from Psoralea corylifolia L., which possesses anti-oxidant, anti-inflammatory, and anti-cancer properties. The aim of this study was to evaluate the effect of NBIF in human U-87 MG glioblastoma cells in comparison to normal human NHA astrocytes, and to examine if it influences the activity of irinotecan, etoposide, and doxorubicin in this in vitro model. We demonstrated that NBIF decreases U-87 MG cells viability in a dose-dependent manner. Furthermore, we found that it inhibits cell growth and causes glutathione (GSH) depletion more intensely in U-87 MG cells than in astrocytes. This study also provides, for the first time, evidence of the potentialization of the doxorubicin effect by NBIF, which was shown by the reduction in the viability in U-87 MG cells.     

Kinetics of hypericin association with low-density lipoproteins

Steady‐state and time‐resolved fluorescence spectroscopy have been used for the study of the incorporation kinetics of hypericin (Hyp) into low‐density lipoproteins (LDL). Biphasic kinetics of Hyp association with LDL was observed when solutions of Hyp and LDL were mixed at various concentration ratios. The rapid phase of Hyp incorporation is completed within seconds, while the slow phase lasts several minutes. The relative contributions of the individual phases show that a higher amount of Hyp molecules (65%) are incorporated into LDL in the second phase. The kinetics of the incorporation of Hyp into LDL particles preloaded with Hyp (Hyp/LDL = 25:1) was also investigated. The decreased intensity of Hyp fluorescence is a sign of the formation of Hyp aggregates after penetration of additional Hyp molecules into Hyp/LDL = 25:1 complex. The time dependence of Hyp fluorescence was measured after mixing the complex Hyp/LDL = 200:1 with appropriate amounts of free LDL molecules. For each final Hyp/LDL ratio, an increase in the intensity and lifetime of Hyp fluorescence was observed, suggesting a monomerization of Hyp aggregates. The half‐time of Hyp transfer from Hyp/LDL complex to LDL particles is similar to the half‐time of the slow phase of Hyp incorporation into free LDL particles.     

Time-resolved Luminescence and Singlet Oxygen Formation After Illumination of the Hypericin–Low-density Lipoprotein Complex

Time-resolved fluorescence and phosphorescence study of hypericin (Hyp) in complex with low-density lipoproteins (LDL) as well as the evolution of singlet oxygen formation and annihilation after illumination of Hyp/LDL complexes at room temperature are presented in this work. The observed shortening of the fluorescence lifetime of Hyp at high Hyp/LDL molar ratios (>25:1) proves the self-quenching of the excited singlet state of monomeric Hyp at these concentration ratios. The very short lifetime (∼0.5 ns) of Hyp fluorescence at very high Hyp/LDL ratios (>150:1) suggests that at high local Hyp concentration inside LDL molecules fast and ultrafast nonradiative decay processes from excited singlet state of Hyp become more important. Contrary to the lifetime of the singlet excited state, the lifetime (its shorter component) of Hyp phosphorescence is not dependent on Hyp/LDL ratio in the studied concentration range. The amount of singlet oxygen produced as well as the integral intensity of Hyp phosphorescence after illumination of Hyp/LDL complexes resemble the dependence of the concentration of molecules of Hyp in monomeric state on Hyp/LDL until a concentration ratio of 60:1. This fact confirms that only monomeric Hyp is able to produce the excited triplet state of Hyp, which in aerobic conditions leads to singlet oxygen production. The value of singlet oxygen lifetime (∼8 μs) after its formation from the excited triplet state of Hyp in LDL proves that molecules of singlet oxygen remain for a certain period of time inside LDL particles and are not immediately released to the aqueous surrounding. That Hyp exists in the complex with LDL in the monodeprotonated state is also demonstrated.     

Fluorescence spectroscopic study of hypericin-photosensitized oxidation of low-density lipoproteins

By means of UV-VIS absorption and fluorescence spectroscopy, we demonstrate that the photosensitizer hypericin (Hyp) interacts nonspecifically with low-density lipoproteins (LDL), most probably with the lipid fraction of LDL. The molar ratio of monomeric Hyp binding to nonoxidized LDL and mildly oxidized LDL is 30:1. Increasing the Hyp concentration further leads to the formation of Hyp aggregates inside the LDL molecule. We also demonstrate that photoactivated Hyp oxidizes LDL in a light dose and excitation wavelength dependent manner. The level of oxidation of LDL depends on the amount of Hyp inside the LDL molecule. The maximum of the photosensitized oxidation of the LDL by Hyp is achieved for a 30:1 molar ratio, which corresponds to the maximum concentration of monomeric form of Hyp in LDL.     

Green synthesis of Zn nanoparticles and in situ hybridized with BSA nanoparticles for Baicalein targeted delivery mediated with glutamate receptors to U87-MG cancer cell lines

Glioblastoma multiforme (GBM) is the most aggressive malignant tumor of the brain. It has different glutamate receptor types. So, these receptors can be a suitable target for GBM treatment. The current study investigated the anticancer effects of bovine serum albumin (BSA)-Baicalein @Zn-Glu nanostructure mediated-GluRs in human glioblastoma U87 cells. BSA-Ba@Zn-Glu hybrid nanoparticles (NPs) were set and considered transporters for Baicalein (Ba) active compound delivery. BSA-Ba@Zn-Glu NPs were synthesized by a single-step reduction process. The successful production was confirmed through transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), and hemolysis test. The cytotoxic efficacy and apoptosis rate of the nanostructures on U87 glioblastoma cells were investigated by 3-(4,5-dimethylthialzol-a-yl)-2,5 diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. The synthesized BSA-Ba@Zn-Glu nanostructures with a diameter of 142.40 ± 1.91 to 177.10 ± 1.87 nm and zeta potential of −10.57 ± 0.71 to −35.77 ± 0.60 mV are suitable for extravasation into tumor cells. The drug release from the BSA-Ba@Zn NPs showed controlled and pH-dependent behavior. In vitro results indicated that the BSA-Ba@Zn-Glu NPs significantly reduce cell viability and promote apoptosis of U87 cancer cells. It revealed the cytotoxic effect of the Baicalein and an increase in cellular uptake of nanoparticles by Glu receptors. Zn NPs were synthesized based on a green synthesis method. BSA NPs were used as a nano-platform for Glu conjugation and Ba drug delivery. BSA-Ba@Zn-Glu NPs induce cytotoxicity and apoptosis in human brain cancer cells (U87) in a dose-dependent manner. Finally, this nanostructure could be served in targeted drug delivery in vivo studies and applied along with other strategies such as X-ray irradiation as combinational therapies in future studies.     

Investigation of therapeutic efficiency of phenytoin (PHT) labeled with radioactive 131I in the cancer cell lines

The aim of this study is to determine the incorporations of PHT radiolabeled with ¹³¹I (¹³¹I-PHT) on U-87 MG, Daoy and A549 cancerous cell lines. For this, cold and radio-labeling studies were carried out. The radio-labeling yield of ¹³¹I-PHT was obtained about 95 %. Subsequently, cell culture studies were carried out and radio-labeling yields of ¹³¹I, ¹³¹I-PHT on U-87 MG, Daoy and A549 cancerous cells were investigated. Cell culture studies demonstrated that the incorporation values of ¹³¹I-PHT on the three cell lines decreased with increasing radioactivity. Consequently, ¹³¹I-PHT may be a good radiopharmaceutical for targeting radionuclide therapy of Central Nervous System Tumors.     

Catanionic solid lipid nanoparticles carrying doxorubicin for inhibiting the growth of U87MG cells

Catanionic solid lipid nanoparticles (CASLNs), loaded with doxorubicin (Dox) and grafted with anti-epithelial growth factor receptor (EGFR) (anti-EGFR/Dox-CASLNs), were applied to suppressing propagation of malignant U87MG cells. U87MG cells were cultured with anti-EGFR/Dox-CASLNs for assessing the cell viability and EGFR expression. When the concentration of catanionic surfactants, containing hexadecyltrimethylammonium bromide and sodium anionic sodium dodecylsulfate, was 1mM, CASLNs entrapped the largest quantity of Dox. The order of cacao butter (CB) in the entrapment efficiency of Dox was 50% CB>0% CB>100% CB. In addition, the release rate of Dox and the antiproliferative effect on U87MG cells were in the following order: 100% CB>0% CB>50% CB. A high level of CB in anti-EGFR/Dox-CASLNs reduced the cytotoxicity to human brain-microvascular endothelial cells. The immunochemical staining revealed that the crosslinked anti-EGFR on the surface of Dox-CASLNs preserved a high specificity in recognizing EGFR on U87MG cells and inducing growth-inhibition effect. The innovated anti-EGFR/Dox-CASLNs can be an effective delivery system with high targeting efficacy against the growth of brain glioblastomas carcinoma.     

Cell-based assays and molecular simulation reveal that the anti-cancer harmine is a specific matrix metalloproteinase-3 (MMP-3) inhibitor

The biological activities of harmine have been a much clearer picture in recent years, which include anti-tumor, anti-inflammation and cytotoxic properties. Numerous in vitro and in vivo animal models have confirmed its activities, but its mode of action remains a relative unsolved issue. We therefore investigated harmine for its effects on MMP-3 and the molecular interaction was also simulated. The human glioma cancer cell line, U-87 MG cells, was subjected to different concentrations (1–10 μM) of harmine for 24 h. Methylthiazol tetrazolium (MTT) test, half maximal inhibitory concentration (IC50), western blot analysis, enzyme-linked immunosorbent assay and molecular docking through BIOVIA DiscoveryStudio™ were performed. These results showed that although harmine stimulation in vitro has very little or no effects on MMP-3 expression by U-87 MG cells, the treatment of harmine decreases MMP-3 activity in a dose dependent manner. It was further calculated that 7.9 μM is the IC50 towards MMP-3. Using a molecular dynamic simulation approach, we identified the N2, methyl of C1 and benzene ring of harmine interact with Zn²⁺ (2.4 Å), His205 (2.4 Å) and His211 (2.4 Å) as well as Val163 (2.7 Å) at the active site of MMP-3, respectively, and thus conferred a striking specific binding advantage. Taken altogether, the present study evidences that harmine acts as an MMP-3 inhibitor specially targeting the enzymatic active site and possibly efficiently ameliorates MMP-3-driven malignant and inflammatory diseases.     

Uptake and trafficking of mildly oxidized LDL and acetylated LDL in THP-1 cells does not explain the differences in lysosomal metabolism of these two lipoproteins.

Foam cells in the atherosclerotic lesion have substantial cholesterol stores within large, swollen lysosomes. This feature is mimicked by incubating THP-1 macrophages with mildly oxidized low density lipoprotein (LDL). Incubation of THP-1 cells with acetylated LDL produces cytoplasmic cholesteryl ester accumulation rather than lysosomal storage. The differences could be due to differences in uptake and delivery of lipoprotein to lysosomes or to lysosomal and post-lysosomal processing events. We compared uptake and lysosomal trafficking of acetylated and oxidized LDL using colloidal gold-labeled lipoproteins. Labeling did not alter cellular cholesterol accumulation. We found that uptake and delivery to lysosomes are not different for acetylated and oxidized LDL. In fact, both oxidized and acetylated LDL can be delivered to the same lysosomes. Sequential incubation with oxidized LDL followed by acetylated LDL showed that the lipid-engorged lysosomes are long-lived structures, continuously accepting newly ingested lipoprotein. Comparison of acetylated and oxidized LDL in mouse peritoneal macrophages, a cell which does not accumulate substantial lysosomal lipid, also revealed no differences in uptake. This indicates that in THP-1 cells, the differences in metabolism of oxidized and acetylated LDL are due to cell-specific lysosomal or post-lysosomal events not present in B6C3F1 mouse macrophages.     

Synthesis and cytotoxicity of novel cholesterol–cobalt bis(dicarbollide) conjugates

The novel conjugates of cholesterol with cobalt – bis(dicarbollide) were synthesized by the ring-opening reactions of the cyclic oxonium derivatives of [3,3′-Co(C₂B₉H₁₁)₂]^– with the OH group of cholesterol 2-hydroxyethyl ether. The compounds obtained were tested for toxicity to glioblastoma U-87 MG cells and human embryo fibroblasts FECH-15 cells     

Design, synthesis and biological evaluation of choline based SPECT imaging agent: Ga(III)-DO3A-EA-Choline

The enhanced choline uptake and phosphorylation in tumor cells has motivated the development of radiolabeled choline derivatives as diagnostic markers for imaging cell membrane proliferation and noninvasive detection of prostate, brain and breast tumors. In the present work, we report a facile strategy for the synthesis of choline functionalized macrocyclic chelating agent (DO3A-EA-choline) and its radiocomplexation with (67)Ga for potential tumor imaging applications. The synthesis of the desired compound featured quaternization of N,N-dimethylaminoethanol with 1,2-dibromoethane followed by subsequent alkylation with trisubstituted cyclen (DO3A). All intermediates and final compounds have been fully characterized by spectroscopic techniques, namely, (1)H, (13)C NMR and mass spectroscopy. The compound has been successively labeled with (67)Ga-citrate in ammonium acetate buffer (pH 6.5) at 80 °C. MTT assays have been performed on the HEK cell line to determine the cytotoxicity of the compound. Cell uptake studies carried out on the U-87 MG cell line exhibited saturable binding of the radioconjugate in picomolar range with a K(d) value of 0.528 pM. The in vivo biodistribution and blood kinetics studies exhibited rapid clearance of the radiolabeled complex and excretion through the renal and hepatobiliary route. The present studies demonstrate the potential applications of (67)Ga-DO3A-EA-choline as a radiopharmaceutical for molecular imaging using ((67/68)Ga) SPECT and PET modalities.     

Mechanisms controlling the cellular accumulation of copper bis(thiosemicarbazonato) complexes

Copper (Cu) bis(thiosemicarbazonato) metal complexes [Cu(II)(btsc)s] have unique tumor-imaging and treatment properties and more recently have revealed potent neuroprotective actions in animal and cell models of neurodegeneration. However, despite the continued development of Cu(II)(btsc)s as potential therapeutics or diagnostic agents, little is known of the mechanisms involved in cell uptake, subcellular trafficking, and efflux of this family of compounds. Because of their high lipophilicity, it has been assumed that cellular accumulation is through passive diffusion, although this has not been analyzed in detail. The role of efflux pathways in cell homeostasis of the complexes is also largely unknown. In the present study, we investigated the cellular accumulation of the Cu(II)(btsc) complexes Cu(II)(gtsm) and Cu(II)(atsm) in human neuronal (M17) and glial (U87MG) cell lines under a range of conditions. Collectively, the data strongly suggested that Cu(II)(gtsm) and Cu(II)(atsm) may be taken into these cells by combined passive and facilitated (protein-carrier-mediated) mechanisms. This was supported by strong temperature-dependent changes to the uptake of the complexes and the influence of the cell surface protein on Cu accumulation. We found no evidence to support a role for copper-transporter 1 in accumulation of the compounds. Importantly, our findings also demonstrated that Cu from both Cu(II)(gtsm) and Cu(II)(atsm) was rapidly effluxed from the cells through active mechanisms. Whether this was in the form of released ionic Cu or as an intact metal complex is not known. However, this finding highlighted the difficulty of trying to determine the uptake mechanism of metal complexes when efflux is occurring concomitantly. These findings are the first detailed exploration of the cellular accumulation mechanisms of Cu(II)(btsc)s. The study delineates strategies to investigate the uptake and efflux mechanisms of metal complexes in cells, while highlighting specific difficulties and challenges that need to be considered before drawing definitive conclusions.     

Effect of increased hydrophobicity on the binding of two model amphiphilic chlorin drugs for photodynamic therapy with blood plasma and its components

The binding of serum albumin and lipoprotein with chlorin p(6) and purpurin 18, two structurally related chlorins, has been studied to understand the role for these proteins as endogenous carriers for these drugs. As a drug carrier a protein may aid in selective delivery of a drug to a tumor region. Binding with serum albumin may result in accumulation of the drug in the stroma of the tumor cell and lead to a reduction of cellular uptake of photosensitizers. However, it is possible that this factor may not be a problem for cellular uptake of chlorin p(6) and purpurin 18 by the tumor tissues, since it binds more efficiently with low-density lipoprotein when it become more lipophilic, indicating that the principal carriers for these molecules are lipoproteins. Since the tumor tissues contain numerous lipoprotein receptors, chlorin p(6) and purpurin 18 could be internalized more efficiently in tumor cells.     

Modulation of rat Kupffer cells on high density lipoprotein receptors on hepatocytes

The present study found that conditioned media from Kupffer cells preincubated with acetylated LDL or acetylated LDL and zymosan increased the number of HDL receptors on hepatocytes, using the method of conditioned media transfer. This indicated that the transferable factors produced by Kupffer cells modulate HDL receptors on hepatocytes.     

An in vitro comparison of the effects of the iron-chelating agents, CP94 and dexrazoxane, on protoporphyrin IX accumulation for photodynamic therapy and/or fluorescence guided resection

Photodynamic therapy (PDT) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. Maximizing the accumulation of the photosensitizer protoporphyrin IX (PpIX) within different cell types would be clinically useful. Dermatological PpIX-induced PDT regimes produce good clinical outcomes but this currently only applies when the lesion remains superficial. Also, as an adjuvant therapy for the treatment of primary brain tumors, fluorescence guided resection (FGR) and PDT can be used to highlight and destroy tumor cells unreachable by surgical resection. By employing iron chelators PpIX accumulation can be enhanced. Two iron-chelating agents, 1,2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) and dexrazoxane, were individually combined with the porphyrin precursors aminolevulinic acid (ALA), methyl aminolevulinate (MAL) and hexyl aminolevulinate (HAL). Efficacies of the iron-chelating agents were compared by recording the PpIX fluorescence in human squamous epithelial carcinoma cells (A431) and human glioma cells (U-87 MG) every hour for up to 6 h. Coincubation of ALA/MAL/HAL with CP94 resulted in a greater accumulation of PpIX compared to that produced by coincubation of these congeners with dexrazoxane. Therefore the clinical employment of iron chelation, particularly with CP94 could potentially increase and/or accelerate the accumulation of ALA/MAL/HAL-induced PpIX for PDT or FGR.     

THE EFFECTS OF LOW DENSITY LIPOPROTEINS ON UPTAKE OF PHOTOFRIN II

The cellular uptake of Photofrin II (PII) was studied using fluorescence imaging and chemical extraction. The influence of serum and low density lipoproteins (LDL) was examined under a variety of experimental conditions employing cultured human cells of different origins as well as a subcutaneously SMT-F tumor implanted in mice. Results showed that serum inhibited PII uptake. In general, LDL also inhibits PII uptake with the exception of an initial increase in the first 10-30 min when the cellular concentration of PII was measured by fluorescence imaging instead of chemical extraction. Our results suggest a possible de-aggregation process occurring upon internalization or binding of PII to LDL.     

State of the art in the delivery of photosensitizers for photodynamic therapy

In photodynamic therapy, one of the problems limiting the use of many photosensitizers (PS) is the difficulty in preparing pharmaceutical formulations that enable their parenteral administration. Due to their low water solubility, the hydrophobic PS cannot be simply injected intravenously. Different strategies, including polymer-PS conjugation or encapsulation of the drug in colloidal carriers such as oil-dispersions, liposomes and polymeric particles, have been investigated. Although these colloidal carriers tend to accumulate selectively in tumour tissues, they are rapidly taken up by the mononuclear phagocytic system. In order to reduce this undesirable uptake by phagocytic cells, long-circulating carriers that consist of surface modified carriers have been developed. Moreover, considerable effort has been directed towards using other types of carriers to improve tumour targeting and to minimize the side effects. One of the approaches is to entrap PS into the lipophilic core of low-density lipoproteins (LDL) without altering their biological properties. The LDL receptor pathway is an important factor in the selective accumulation of PS in tumour tissue owing to the increased number of LDL receptors on the proliferating cell surface. Specific targeting can also be achieved by binding of monoclonal antibodies or specific tumour-seeking molecules to PS or by the coating of PS loaded carriers.     

Comparative analysis of foetal calf and human low density lipoprotein: relevance for pharmacodynamics of photosensitizers

The effects of differences in lipoprotein content on the distribution of the novel hydrophobic photosensitizer n-butyl-3-[18-(2-butylcarbamoyl-ethyl)-3,7,12,17-tetramethyl-18,13-divinyl-22,24-dihydro-porphin-2-yl]propionamide (PP-N-3) and haematoporphyrin ester (HpE), a relatively hydrophilic photosensitizer, in human (HS) and foetal calf sera (FCS), were investigated. The binding characteristics of human and foetal calf low-density lipoprotein (LDL) were characterised using a human fibroblast line (Vag 12). The uptake into cells of HpE and PP-N-3 was also examined. A comparison of the lipoprotein content, composition and receptor-binding characteristics of foetal calf and human serum was also carried out. LDL content was measured directly using sequential ultracentrifugation to isolate LDL. In our study, we found haematoporphyrin ester to bind to human very low-density lipoprotein (VLDL), LDL and high-density lipoprotein (HDL) in the ratio 2:31:65. In the case of PP-N-3 this ratio was 56:10:33. As VLDL was not detected in foetal calf serum, only binding to LDL and HDL was observed. Using the sequential ultracentrifugation technique, foetal calf serum was found to contain LDL which in turn did bind to human LDL receptors. The uptake of PP-N-3 and HpE in the presence of low density lipoprotein from foetal calf serum (FC-LDL) was not significantly different to values observed in the presence of human serum low density lipoprotein (HS-LDL).     

Isoginkgetin—A Natural Compound to Control U87MG Glioblastoma Cell Growth and Migration Activating Apoptosis and Autophagy

Isoginkgetin (Iso) is a natural bioflavonoid isolated from the leaves of Ginkgo biloba, this natural substance exhibits many healing properties, among which the antitumor effect stands out. Here we tested the effect of Iso on the growth of U87MG glioblastoma cells. Growth curves and MTT toxicity assays showed time and dose-dependent growth inhibition of U87MG after treatment with Iso (15/25 µM) for 1, 2, and 3 days. The cell growth block of U87MG was further investigated with the colony formation test, which showed that iso treatment for 24 h reduced colony formation. The present study also aimed to evaluate the effect of Iso on U87MG glioblastoma cell migration. The FACS analysis, on the other hand, showed that treatment with Iso 15 µM determines a blockage of the cell cycle in the S1 phase. Further investigation shows that Iso treatment of U87MG altered the protein pathways of homeostasis including autophagy and apoptosis. The present study demonstrated, for the first time, that Iso could represent an excellent adjuvant drug for the treatment of glioblastoma by simultaneously activating multiple mechanisms that control the growth and migration of neoplastic cells.