Pro-Inflammatory Activation Suppresses TRAIL-induced Apoptosis of Acute Myeloid Leukemia Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising agent for treatment of AML due to its specific apoptosis-inducing effect on tumor cells but not normal cells. However, emergence of resistance to TRAIL in the AML cells limits its potential as an antileukemic agent. Previously, we revealed increase in the resistance of the human AML THP-1 cells to the TRAIL-induced death during their LPS-dependent proinflammatory activation and in the in vitro model of LPS-independent proinflammatory activation - in a long-term high-density cell culture. In this study, we investigated mechanisms of this phenomenon using Western blot analysis, caspase 3 enzymatic activity analysis, quantitative reverse transcription-PCR, and flow cytometry. The results showed that the increased resistance to the TRAIL-induced cell death of AML THP-1 cells during their pro-inflammatory activation is associated with the decrease in the surface expression of the proapoptotic receptors TRAIL-R1/DR4 and TRAIL-R2/DR5, as well as with the increased content of members of the IAPs family - Livin and cIAP2. The results of this article open up new insights into the role of inflammation in formation of the resistance of AML cells to the action of mediators of antitumor immunity, in particular TRAIL.

Soft Biomimetic Approach for the Development of Calcinosis-Resistant Glutaraldehyde-Fixed Biomaterials for Cardiovascular Surgery.

Pathological aseptic calcification is the most common form of structural valvular degeneration (SVD), leading to premature failure of heart valve bioprostheses (BHVs). The processing methods used to obtain GA-fixed pericardium-based biomaterials determine the hemodynamic characteristics and durability of BHVs. This article presents a comparative study of the effects of several processing methods on the degree of damage to the ECM of GA-fixed pericardium-based biomaterials as well as on their biostability, biocompatibility, and resistance to calcification. Based on the assumption that preservation of the native ECM structure will enable the creation of calcinosis-resistant materials, this study provides a soft biomimetic approach for the manufacture of GA-fixed biomaterials using gentle decellularization and washing methods. It has been shown that the use of soft methods for preimplantation processing of materials, ensuring maximum preservation of the intactness of the pericardial ECM, radically increases the resistance of biomaterials to calcification. These obtained data are of interest for the development of new calcinosis-resistant biomaterials for the manufacture of BHVs.

Improving the Stability and Effectiveness of Immunotropic Squalene Nanoemulsion by Adding Turpentine Oil.

Turpentine oil, owing to the presence of 7-50 terpenes, has analgesic, anti-inflammatory, immunomodulatory, antibacterial, anticoagulant, antioxidant, and antitumor properties, which are important for medical emulsion preparation. The addition of turpentine oil to squalene emulsions can increase their effectiveness, thereby reducing the concentration of expensive and possibly deficient squalene, and increasing its stability and shelf life. In this study, squalene emulsions were obtained by adding various concentrations of turpentine oil via high-pressure homogenization, and the safety and effectiveness of the obtained emulsions were studied in vitro and in vivo. All emulsions showed high safety profiles, regardless of the concentration of turpentine oil used. However, these emulsions exhibited dose-dependent effects in terms of both efficiency and storage stability, and the squalene emulsion with 1.0% turpentine oil had the most pronounced adjuvant and cytokine-stimulating activity as well as the most pronounced stability indicators when stored at room temperature. Thus, it can be concluded that the squalene emulsion with 1% turpentine oil is a stable, monomodal, and reliably safe ultradispersed emulsion and may have pleiotropic effects with pronounced immunopotentiating properties.

Conjugation of Triterpenic Acids of Ursane and Oleanane Types with Mitochondria-Targeting Cation F16 Synergistically Enhanced Their Cytotoxicity against Tumor Cells.

The present work shows the cytotoxic effects of novel conjugates of ursolic, oleanolic, maslinic, and corosolic acids with the penetrating cation F16 on cancer cells (lung adenocarcinoma A549 and H1299, breast cancer cell lines MCF-7 and BT474) and non-tumor human fibroblasts. It has been established that the conjugates have a significantly enhanced toxicity against tumor-derived cells compared to native acids and also demonstrate selectivity to some cancer cells. The toxic effect of the conjugates is shown to be due to ROS hyperproduction in cells, induced by the effect on mitochondria. The conjugates caused dysfunction of isolated rat liver mitochondria and, in particular, a decrease in the efficiency of oxidative phosphorylation, a decrease in the membrane potential, and also an overproduction of ROS by organelles. The paper discusses how the membranotropic- and mitochondria-targeted effects of the conjugates may be related to their toxic effects.

Biomimetic Remineralized Three-Dimensional Collagen Bone Matrices with an Enhanced Osteostimulating Effect.

Bone grafts with a high potential for osseointegration, capable of providing a complete and effective regeneration of bone tissue, remain an urgent and unresolved issue. The presented work proposes an approach to develop composite biomimetic bone material for reconstructive surgery by deposition (remineralization) on the surface of high-purity, demineralized bone collagen matrix calcium phosphate layers. Histological and elemental analysis have shown reproduction of the bone tissue matrix architectonics, and a high-purity degree of the obtained collagen scaffolds; the cell culture and confocal microscopy have demonstrated a high biocompatibility of the materials obtained. Adsorption spectroscopy, scanning electron microscopy, microcomputed tomography (microCT) and infrared spectroscopy, and X-ray diffraction have proven the efficiency of the deposition of calcium phosphates on the surface of bone collagen scaffolds. Cell culture and confocal microscopy methods have shown high biocompatibility of both demineralized and remineralized bone matrices. In the model of heterotopic implantation in rats, at the term of seven weeks, an intensive intratrabecular infiltration of calcium phosphate precipitates, and a pronounced synthetic activity of osteoblast remodeling and rebuilding implanted materials, were revealed in remineralized bone collagen matrices in contrast to demineralized ones. Thus, remineralization of highly purified demineralized bone matrices significantly enhanced their osteostimulating ability. The data obtained are of interest for the creation of new highly effective osteoplastic materials for bone tissue regeneration and augmentation.

Comparative Study of Cytotoxic and Membranotropic Properties of Betulinic Acid-F16 Conjugate on Breast Adenocarcinoma Cells (MCF-7) and Primary Human Fibroblasts.

The present study evaluates the cytotoxicity of a previously synthesized conjugate of betulinic acid (BA) with the penetrating cation F16 on breast adenocarcinoma (MCF-7) and human fibroblast (HF) cell lines, and also shows the mechanism underlying its membranotropic action. It was confirmed that the conjugate exhibits higher cytotoxicity compared to native BA at low doses also blocking the proliferation of both cell lines and causing cell cycle arrest in the G0/G1 phase. We show that the conjugate indeed has a high potential for accumulation in mitochondria, being visualized in these organelles, which is most pronounced in cancer cells. The effect of the conjugate was observed to be accompanied by ROS hyperproduction in both cancerous and healthy cells, despite the lower base level of ROS in the latter. Along with this, using artificial liposomes, we determined that the conjugate is able to influence the phase state of lipid membranes, make them more fluid, and induce nonspecific permeabilization contributing to the overall cytotoxicity of the tested agent. We conclude that the studied BA-F16 conjugate does not have significant selective cytotoxicity, at least against the studied breast cancer cell line MCF-7.

Differences in the Formation of Reactive Oxygen Species and Their Cytotoxicity between Thiols Combined with Aqua- and Cyanocobalamins.

Cobalamin is an essential nutrient required for the normal functioning of cells. Its deficiency can lead to various pathological states. Hydroxocobalamin (HOCbl) and cyanocobalamin (CNCbl) are the forms of vitamin B12 that are most commonly used for supplementation. There is substantial evidence indicating that cobalamins can both suppress and promote oxidative stress; however, the mechanisms underlying these effects are poorly understood. Here, it was shown that the oxidation of thiols catalyzed by HOCbl and CNCbl is accompanied by reactive oxygen species (ROS) production and induces, under certain conditions, oxidative stress and cell death. The form of vitamin B12 and the structure of thiol play a decisive role in these processes. It was found that the mechanisms and kinetics of thiol oxidation catalyzed by HOCbl and CNCbl differ substantially. HOCbl increased the rate of oxidation of thiols to a greater extent than CNCbl, but quenched ROS in combination with certain thiols. Oxidation catalyzed by CNCbl was generally slower. Yet, the absence of ROS quenching resulted in their higher accumulation. The aforementioned results might explain a more pronounced cytotoxicity induced by combinations of thiols with CNCbl. On the whole, the data obtained provide a new insight into the redox processes in which cobalamins are involved. Our results might also be helpful in developing new approaches to the treatment of some cobalamin-responsive disorders in which oxidative stress is an important component.

Disulfiram Oxy-Derivatives Suppress Protein Retrotranslocation across the ER Membrane to the Cytosol and Initiate Paraptosis-like Cell Death.

Disulfiram (DSF) and its derivatives were here investigated as antineoplastic agents, and their important feature is the ability to influence the UPS. We have recently shown that hydroxocobalamin catalyzes the aerobic oxidation of diethyldithiocarbamate to form disulfiram and its oxy-derivatives (DSFoxy; i.e., sulfones and sulfoxides), which induce cytoplasm vacuolization and paraptosis-like cancer cell death. We used LC-MS/MS and bioinformatics analysis to determine the key points in these processes. DSFoxy was found to induce an increase in the number of ubiquitinated proteins, including oxidized ones, and a decrease in the monomeric ubiquitin. Enhanced ubiquitination was revealed for proteins involved in the response to exogenous stress, regulation of apoptosis, autophagy, DNA damage/repair, transcription and translation, folding and ubiquitination, retrograde transport, the MAPK cascade, and some other functions. The results obtained indicate that DSF oxy-derivatives enhance the oxidation and ubiquitination of many proteins regulating proteostasis (including E3 ligases and deubiquitinases), which leads to inhibition of protein retrotranslocation across the ER membrane into the cytosol and accumulation of misfolded proteins in the ER followed by ER swelling and initiates paraptosis-like cell death. Our results provide new insight into the role of protein ubiquitination/deubiquitination in regulating protein retrotranslocation across the ER membrane into the cytosol and paraptosis-like cell death.

The Increase in the Drug Resistance of Acute Myeloid Leukemia THP-1 Cells in High-Density Cell Culture Is Associated with Inflammatory-like Activation and Anti-Apoptotic Bcl-2 Proteins.

It is known that cell culture density can modulate the drug resistance of acute myeloid leukemia (AML) cells. In this work, we studied the drug sensitivity of AML cells in high-density cell cultures (cell lines THP-1, HL-60, MV4-11, and U937). It was shown that the AML cells in high-density cell cultures in vitro were significantly more resistant to DNA-damaging drugs and recombinant ligand izTRAIL than those in low-density cell cultures. To elucidate the mechanism of the increased drug resistance of AML cells in high-density cell cultures, we studied the activation of Bcl-2, Hif-1alpha, and NF-kB proteins, as well as cytokine secretion, the inflammatory immunophenotype, and the transcriptome for THP-1 cells in the low-density and high-density cultures. The results indicated that the increase in the drug resistance of proliferating THP-1 cells in high-density cell cultures was associated with the accumulation of inflammatory cytokines in extracellular medium, and the formation of NF-kB-dependent inflammatory-like cell activation with the anti-apoptotic proteins Bcl-2 and Bcl-xl. The increased drug resistance of THP-1 cells in high-density cultures can be reduced by ABT-737, an inhibitor of Bcl-2 family proteins, and by inhibitors of NF-kB. The results suggest a mechanism for increasing the drug resistance of AML cells in the bone marrow and are of interest for developing a strategy to suppress this resistance.

Disulfiram oxy-derivatives induce entosis or paraptosis-like death in breast cancer MCF-7 cells depending on the duration of treatment.

BACKGROUND: Dithiocarbamates and derivatives (including disulfiram, DSF) are currently investigated as antineoplastic agents. We have revealed earlier the ability of hydroxocobalamin (vitamin В12b) combined with diethyldithiocarbamate (DDC) to catalyze the formation of highly cytotoxic oxidized derivatives of DSF (DSFoxy, sulfones and sulfoxides). METHODS: Electron and fluorescent confocal microscopy, molecular biology and conventional biochemical techniques were used to study the morphological and functional responses of MCF-7 human breast cancer cells to treatment with DDC and B12b alone or in combination. RESULTS: DDC induces unfolded protein response in MCF-7 cells. The combined use of DDC and B12b causes MCF-7 cell death. Electron microscopy revealed the separation of ER and nuclear membranes, leading to the formation of both cytoplasmic and perinuclear vacuoles, with many fibers inside. The process of vacuolization coincided with the appearance of ER stress markers, a marked damage to mitochondria, a significant inhibition of 20S proteasome, and actin depolimerization at later stages. Specific inhibitors of apoptosis, necroptosis, autophagy, and ferroptosis did not prevent cell death. A short- time (6-h) exposure to DSFoxy caused a significant increase in the number of entotic cells. CONCLUSIONS: These observations indicate that MCF-7 cells treated with a mixture of DDC and B12b die by the mechanism of paraptosis. A short- time exposure to DSFoxy caused, along with paraptosis, a significant activation of the entosis and its final stage, lysosomal cell death. GENERAL SIGNIFICANCE: The results obtained open up opportunities for the development of new approaches to induce non-apoptotic death of cancer cells by dithiocarbamates.

Macrophage-like THP-1 Cells Derived from High-Density Cell Culture Are Resistant to TRAIL-Induced Cell Death via Down-Regulation of Death-Receptors DR4 and DR5.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a highly selective and promising anticancer agent due to its specific apoptosis-inducing effect on tumor cells, rather than most normal cells. TRAIL is currently under investigation for use in the treatment of leukemia. However, the resistance of leukemic cells to TRAIL-induced apoptosis may limit its efficacy. The mechanisms of leukemic cell resistance to antitumor immunity remains a topical issue. In this work, we have found an increase in the resistance to TRAIL-induced cell death in human leukemia THP-1 cells, which was caused by differentiation into a macrophage-like phenotype in high-density culture in vitro. Stressful conditions, manifested by the inhibition of cell growth and the activation of cell death in high-density culture of THP-1 cells, induced the appearance of cells adhered to culture dishes. The THP-1ad cell line was derived by selection of these adhered cells. The genetic study, using STR and aCGH assays, has shown that THP-1ad cells were derived from THP-1 cells due to mutagenesis. The THP-1ad cells possessed high proliferative potential and a macrophage-like immunophenotype. The adhesion of THP-1ad cells to the extracellular matrix was mediated by αVß5 integrin. The cytokine production, as well as the rise of intracellular ROS and NO activities by LPS in THP-1ad cell culture, were characteristic of macrophage-like cells. The THP-1ad cells were found to appear to increase in resistance to TRAIL-induced cell death in comparison with THP-1 cells. The mechanism of the increase in TRAIL-resistance can be related to a decrease in the expression of death receptors DR4 and DR5 on the THP-1ad cells. Thus, the macrophage-like phenotype formation with the maintenance of a high proliferative potential of leukemic cells, caused by stress conditions in high-density cell cultures in vitro, can induce an increase in resistance to TRAIL-induced cell death due to the loss of DR4 and DR5 receptors. The possible realization of these events in vivo may be the reason for tumor progression.

Improved Anticancer Effect of Recombinant Protein izTRAIL Combined with Sorafenib and Peptide iRGD.

One of the main problems in oncology is the development of drugs that cause the death of cancer cells without damaging normal cells. Another key problem to be solved is to suppress the drug resistance of cancer cells. The third important issue is to provide effective penetration of drug molecules to cancer cells. TRAIL (TNFα-related apoptosis inducing ligand)/Apo2L is a highly selective anticancer agent. However, the recombinant TRAIL protein having high efficiency against cancer cells in vitro was not effective in clinical trials. Recently we have discovered an acquisition of TRAIL resistance by cancer cells in confluent cultures, which is apparently a manifestation of the general phenomenon of multicellular resistance. The aim of this study was to evaluate whether the anticancer effect of the recombinant protein TRAIL in vivo can be improved by the suppression of multicellular TRAIL-resistance using sorafenib and a tumor-penetrating peptide iRGD, c(CRGDKGPDC). The results testified a great increase in the resistance of human fibrosarcoma HT-1080 cells to izTRAIL both in confluent cultures and in spheroids. Sorafenib administered at nontoxic concentration effectively suppressed confluent- or spheroid-mediated TRAIL-resistance of HT-1080 cells in vitro. Sorafenib combined with iRGD significantly improved the anticancer effect of the recombinant protein izTRAIL in HT-1080 human fibrosarcoma grafts in BALB/c nude mice. Consistent with this finding, multicellular TRAIL-resistance may be a reason of inefficacy of izTRAIL alone in vivo. The anticancer effect of the recombinant protein izTRAIL in vivo may be improved in combination with sorafenib, an inhibitor of multicellular TRAIL resistance and iRGD, the tumor-penetrating peptide.

Possible Involvement of 2',3'-Cyclic Nucleotide-3'-Phosphodiesterase in the Protein Phosphorylation-Mediated Regulation of the Permeability Transition Pore.

Calcium as a secondary messenger regulates the phosphorylation of several membrane-bound proteins in brain and liver mitochondria. Regulation of the activity of different protein kinases and phosphatases by Ca2+ occurs through its binding with calmodulin. The protein phosphorylation is strongly dependent on the Ca2+-induced mitochondrial permeability transition pore (mPTP) opening. 2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNPase) was phosphorylated by protein kinases A and C. CNPase and melatonin (MEL) might interact with calmodulin. The effects of the calmodulin antagonist calmidazolium and the inhibitor of protein kinase A H89 on mPTP opening in rat brain mitochondria of male Wistar rats were investigated. In addition, the role of CNPase, serine/threonine kinases, and MEL in the mPTP opening was examined. The anti-CNPase antibody added to rat brain mitochondria (RBM) reduced the content of CNPase in mitochondria. The threshold [Ca2+] decreased, and mitochondrial swelling was accelerated in the presence of the anti-CNPase antibody. H89 enhanced the effect of anti-CNPase antibody and accelerated the swelling of mitochondria, while CmZ abolished the effect of anti-CNPase antibody under mPTP opening. The levels of phospho-Akt and phospho-GSK3ß increased, while the MEL content did not change. It can be assumed that CNPase may be involved in the regulation of these kinases, which in turn plays an important role in mPTP functioning.

Melatonin Can Strengthen the Effect of Retinoic Acid in HL-60 Cells.

Melatonin is produced by the pineal gland. It can be regarded as an anticancer agent and used for combined therapy, owing to its oncostatic, antioxidant, and immunoregulatory activities. Retinoic acid is widely used for the treatment of acute promyelocytic leukemia; however, it has adverse effects on the human organism. We investigated the effect of melatonin and reduced concentrations of retinoic acid on the activation of proliferation in acute promyelocytic leukemiaon a cell model HL-60. The combined effect of these compounds leads to a reduction in the number of cells by 70% and the index of mitotic activity by 64%. Combined treatment with melatonin and retinoic acid decreased the expression of the Bcl-2. The mitochondrial isoform VDAC1 can be a target in the treatment of different tumors. The combined effect of and retinoic acid at a low concentration (10 nM) decreased VDAC1 expression. Melatonin in combination with retinoic acid produced a similar effect on the expression of the translocator protein. The coprecipitation of VDAC with 2',3'-cyclonucleotide-3'-phosphodiesterase implies a possible role of its in cancer development. The combined effect of retinoic acid and melatonin decreased the activity of the electron transport chain complexes. The changes in the activation of proliferation in HL-60 cells, the mitotic index, and Bcl-2 expression under combined effect of retinoic acid (10 nM) with melatonin (1 mM) are similar to changes that are induced by 1 µM retinoic acid. Our results suggest that MEL is able to improve the action the other chemotherapeutic agent.

Composite tendon implant based on nanofibrillar polyhydroxybutyrate and polyamide filaments.

The composite material based on reinforcement of polyamide filaments enclosed by a nonwoven matrix of nanoscaled bioresorbable poly(3-hydroxybutyrate) fibers was developed for application as an artificial ligament implant. The aim of this study was to investigate biodegradability and biocompatibility of the developed implant, as well as its stress-strain properties. The study results show the polyamide core of the implant has stress-strain properties comparable with a natural ligament. Simultaneously, the polyhydroxybutyrate external layer provides high biocompatibility and bioresorbability of the developed implant. The material has proven to be effective under in vivo tests with experimental rats as a ligament replacement for damaged Achilles tendons. Due to cell attachment and growth on the fibrous matrix during 5 weeks postsurgery, regenerated connective tissue was formed substituting for the polymeric implant, which confirmed its efficiency in contrast to the polyamide filament implant with a much longer resorption time. The results obtained indicate application prospects of polyamide-polyhydroxybutyrate implants for reconstructive surgery. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2708-2713, 2018.

Effect of Melatonin on Rat Heart Mitochondria in Acute Heart Failure in Aged Rats.

Excessive generation of reactive oxygen species (ROS) in mitochondria and the opening of the nonselective mitochondrial permeability transition pore are important factors that promote cardiac pathologies and dysfunction. The hormone melatonin (MEL) is known to improve the functional state of mitochondria via an antioxidant effect. Here, the effect of MEL administration on heart mitochondria from aged rats with acute cardiac failure caused by isoprenaline hydrochloride (ISO) was studied. A histological analysis revealed that chronic intake of MEL diminished the age-dependent changes in the structure of muscle fibers of the left ventricle, muscle fiber swelling, and injury zones characteristic of acute cardiac failure caused by ISO. In acute heart failure, the respiratory control index (RCI) and the Ca2+ retention capacity in isolated rat heart mitochondria (RHM) were reduced by 30% and 40%, respectively, and mitochondrial swelling increased by 34%. MEL administration abolished the effect of ISO. MEL partially prevented ISO-induced changes at the subunit level of respiratory complexes III and V and drastically decreased the expression of complex I subunit NDUFB8 both in control RHM and in RHM treated with ISO, which led to the inhibition of ROS production. MEL prevents the mitochondrial dysfunction associated with heart failure caused by ISO. It was shown that the level of 2',3'-cyclicnucleotide-3'-phosphodiasterase (CNPase), which is capable of protecting cells in aging, increased in acute heart failure. MEL also retained the CNPase content in RHM both in control experiments and after ISO-induced heart damage. We concluded that an increase in the CNPase level promotes cardioprotection.

2',3'-Cyclic nucleotide 3'-phosphodiesterase as a messenger of protection of the mitochondrial function during melatonin treatment in aging.

The process of aging is considered to be tightly related to mitochondrial dysfunction. One of the causes of aging is an increased sensitivity to the induction of mitochondrial permeability transition pore (mPTP) opening in the inner membrane of mitochondria. Melatonin, a natural antioxidant, is a hormone produced by the pineal gland. The role of melatonin whose level decreases with aging is well understood. In the present study, we demonstrated that long-term treatment of aged rats with melatonin improved the functional state of mitochondria; thus, the Ca2+ capacity was enhanced and mitochondrial swelling was deaccelerated in mitochondria. Melatonin prevented mPTP and impaired the release of cytochrome c and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) from mitochondria of both young and aged rats. Our data suggest that melatonin retains СNPase inside mitochondria, thereby providing the protection of the protein against deleterious effects of 2',3'-cAMP in aging.

Effect of the CRAC Peptide, VLNYYVW, on mPTP Opening in Rat Brain and Liver Mitochondria.

The translocator protein (TSPO; 18 kDa) is a high-affinity cholesterol-binding protein located in the outer membrane of mitochondria. A domain in the C-terminus of TSPO was characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide may participate in the regulation of mitochondrial membrane permeability. Herein, we report the effect of the synthetic CRAC peptide, VLNYYVW, on mitochondrial permeability transition pore (mPTP) opening. It was found that the CRAC peptide alone prevents the mPTP from opening, as well as the release of apoptotic factors (cytochrome c, AIF, and EndoG) in rat brain mitochondria (RBM). Co-incubation of CRAC, together with the TSPO drug ligand, PK 11195, resulted in the acceleration of mPTP opening and in the increase of apoptotic factor release. VLNYYVW did not induce swelling in rat liver mitochondria (RLM). 3,17,19-androsten-5-triol (19-Atriol; an inhibitor of the cholesterol-binding activity of the CRAC peptide) alone and in combination with the peptide was able to stimulate RLM swelling, which was Ca2+- and CsA-sensitive. Additionally, a combination of 19-Atriol with 100 nM PK 11195 or with 100 µM PK 11195 displayed the opposite effect: namely, the addition of 19-Atriol with 100 µM PK 11195 in a suspension of RLM suppressed the Ca2+-induced swelling of RLM by 40%, while the presence of 100 nM PK 11195 with 19-Atriol enhanced the swelling of RLM by 60%. Taken together, these data suggest the participation of the TSPO's CRAC domain in the regulation of permeability transition.

Oleic acid is a key cytotoxic component of HAMLET-like complexes.

HAMLET is a complex of α-lactalbumin (α-LA) with oleic acid (OA) that selectively kills tumor cells and Streptococcus pneumoniae. To assess the contribution of the proteinaceous component to cytotoxicity of HAMLET, OA complexes with proteins structurally and functionally distinct from α-LA were prepared. Similar to HAMLET, the OA complexes with bovine ß-lactoglobulin (bLG) and pike parvalbumin (pPA) (bLG-OA-45 and pPA-OA-45, respectively) induced S. pneumoniae D39 cell death. The activation mechanisms of S. pneumoniae death for these complexes were analogous to those for HAMLET, and the cytotoxicity of the complexes increased with OA content in the preparations. The half-maximal inhibitory concentration for HEp-2 cells linearly decreased with rise in OA content in the preparations, and OA concentration in the preparations causing HEp-2 cell death was close to the cytotoxicity of OA alone. Hence, the cytotoxic action of these complexes against HEp-2 cells is induced mostly by OA. Thermal stabilization of bLG upon association with OA implies that cytotoxicity of bLG-OA-45 complex cannot be ascribed to molten globule-like conformation of the protein component. Overall, the proteinaceous component of HAMLET-like complexes studied is not a prerequisite for their activity; the cytotoxicity of these complexes is mostly due to the action of OA.

A novel method for preparation of HAMLET-like protein complexes.

Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca(2+) transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties.