Accumulation of ß-Amyloid Leads to a Decrease in Lynx1 and Lypd6B Expression in the Hippocampus and Increased Expression of Proinflammatory Cytokines in the Hippocampus and Blood Serum.

Alzheimer's disease is a rapidly progressive neurodegenerative disease, the development of which is associated with the accumulation of ß-amyloid oligomers, dysfunction of the α7-nAChR nicotinic acetylcholine receptor, and activation of inflammation. Previously, we showed that the neuromodulator Lynx1, which belongs to the Ly6/uPAR family, competes with ß-amyloid(1-42) for binding to α7-nAChR. In this work, we studied the expression and localization of Ly6/uPAR family proteins in the hippocampus of 2xTg-AD transgenic mice that model AD and demonstrate increased amyloidosis in the brain. Using real-time PCR, we showed a decrease in the expression of the genes encoding Lynx1, Lypd6b, and the postsynaptic marker PSD95, as well as an increase in the expression of the TNFα gene in the hippocampus of 2xTg-AD mice. Histochemical analysis showed that, in the hippocampus of 2xTg-AD mice, Lynx1 does not colocalize with α7-nAChR, which can lead to the development of pathology when the receptor interacts with oligomeric ß-amyloid. In addition, in 2xTg-AD mice, activation of systemic inflammation was shown, which manifests itself in a decrease in the serum level of SLURP-1, a Ly6/uPAR family protein capable of regulating inflammatory processes, as well as in an increase in the content of proinflammatory cytokines TNFα and TNFß. Thus, α7-nAChR dysfunction and maintenance of the inflammatory microenvironment in the brain in Alzheimer's disease may be associated with a decrease in the expression of Ly6/uPAR family proteins that regulate α7-nAChR activity and inflammation.

Recombinant Analogue of the Human Protein SLURP-1 Inhibits the Growth of U251 MG and A172 Glioma Cells.

The alpha7 nicotinic acetylcholine receptor (α7-nAChR) is considered a promising pharmacological target for the carcinoma therapy. We have previously shown that the recombinant analogue of the human protein SLURP-1 (rSLURP-1) effectively inhibits the growth of carcinomas of various origins via the interaction with α7-nAChR and down-regulation of expression of this receptor. Expression of α7-nAChR is increased in gliomas compared to healthy human brain tissues; however, the role of this receptor in the gliomas development is poorly understood. It was shown for the first time that rSLURP-1 significantly inhibits the growth of glioma model cells U251 MG and A172 up to ∼70%, which is comparable with the effect of α-bungarotoxin, a selective α7-nAChR inhibitor. The half-maximum effective concentrations of rSLURP-1 for U251 MG and A172 cells were 2.82 ± 0.2 and 8.9 ± 0.3 nM, respectively. Coincubation of U251 MG cells with rSLURP-1 and the nAChR inhibitor mecamylamine attenuates the antiproliferative activity of rSLURP-1, indicating nAChR as a molecular target for the rSLURP-1 action in gliomas.

Revealing Color Forces with Transverse Polarized Electron Scattering.

The Spin Asymmetries of the Nucleon Experiment measured two double spin asymmetries using a polarized proton target and polarized electron beam at two beam energies, 4.7 and 5.9 GeV. A large-acceptance open-configuration detector package identified scattered electrons at 40° and covered a wide range in Bjorken x (0.3

Recombinant Analogue of the Human Protein SLURP-1 Inhibits the Growth of Multicellular Spheroids Reconstructed from Carcinoma Cells.

In the present study we showed that the recombinant analogue of the SLURP-1 protein effectively inhibits the growth of a 3D model of tumors-multicellular spheroids reconstructed from human epidermoid carcinoma A431 cells and human lung adenocarcinoma A549 cells. The combined application of rSLURP-1 with gefitinib (inhibitor of epidermal growth factor receptor (EGFR)) leads to the synergistic antiproliferative effect on spheroids from A431 cells. The results obtained suggest the possibility for design of first-in-class anticancer drugs based on recombinant SLURP-1.

Three-Finger Proteins from the Ly6/uPAR Family: Functional Diversity within One Structural Motif.

The discovery in higher animals of proteins from the Ly6/uPAR family, which have structural homology with snake "three-finger" neurotoxins, has generated great interest in these molecules and their role in the functioning of the organism. These proteins have been found in the nervous, immune, endocrine, and reproductive systems of mammals. There are two types of the Ly6/uPAR proteins: those associated with the cell membrane by GPI-anchor and secreted ones. For some of them (Lynx1, SLURP-1, SLURP-2, Lypd6), as well as for snake α-neurotoxins, the target of action is nicotinic acetylcholine receptors, which are widely represented in the central and peripheral nervous systems, and in many other tissues, including epithelial cells and the immune system. However, the targets of most proteins from the Ly6/uPAR family and the mechanism of their action remain unknown. This review presents data on the structural and functional properties of the Ly6/uPAR proteins, which reveal a variety of functions within a single structural motif.

The effect of cisplatin on cytotoxicity of anticancer cytokine TRAIL and its receptor-selective mutant variant DR5-B1.

Cytokine TRAIL selectively induces apoptosis in vitro and in vivo in tumor cells without affecting normal cells, but its therapeutic application is limited, since many primary tumors are insensitive to TRAIL. To improve the efficiency of TRAIL, we have previously developed TRAIL mutant variant DR5-B, which binds the apoptosis-inducing death receptor DR5 as efficiently as wild type TRAIL, but shows almost no affinity to other receptors. In this study, we investigated the effect of the chemotherapeutic agent cisplatin on the cytotoxicity of TRAIL variants in 12 tumor cell lines of various origin. Cisplatin effectively enhances the cytotoxic activity of TRAIL preparations. The synergistic effect is most pronounced in the prostate cancer cell lines, where the combined effect exceeds the sum of the separate effects by more than 2 times. The cytotoxicity of DR5-B variant is significantly higher compared to wild-type TRAIL in combination with cisplatin in 9 of 12 tumor cell lines.

Mutations Enhancing Selectivity of Antitumor Cytokine TRAIL to DR5 Receptor Increase Its Cytotoxicity against Tumor Cells.

Tumor necrosis factor superfamily cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in tumor cells by binding to death receptors DR4 and DR5 without affecting normal cells. However, the therapeutic use of TRAIL is limited, because many tumor cells are resistant to it. The resistance is partially related to interaction of TRAIL with the decoy receptors DcR1 and DcR2, which do not trigger the apoptotic signal and inhibit signaling of death receptors. Previously, we designed a unique DR5-specific TRAIL mutant variant DR5-B, which binds to DR5 receptor as effectively as the original cytokine, but has practically no interaction with DR4 and DcR1 receptors, and its affinity for DcR2 is reduced 400-fold. In the present work, the cytotoxity of TRAIL and DR5-B was analyzed on 12 different tumor cell lines and two types of normal cells. In nine of 12 tumor cell lines, DR5-B killed 1.5-5.0 times more tumor cells than TRAIL, and it did not exhibit toxicity towards normal cells. Chemotherapeutic drugs such as doxorubicin, paclitaxel, and bortezomib augmented the effect of both TRAIL variants, and the enhancing effect was more pronounced for DR5-B. Half-maximal effective concentrations (EC50) for DR5-B in combination with chemotherapeutic agents were 1.5-10.0 times lower than for wild-type TRAIL. Thus, DR5-B is a promising candidate both for monotherapy and in combination with chemotherapy for treatment of TRAIL-resistant tumors.

[Gastric juice calcium content in patients with gastroesophageal reflux disease].

The purpose of this study was to investigate free calcium content of gastric juice in patients with gastroesophageal reflux disease (GERD). After analyzing the calcium levels of gastric juice in patients with GERD and healthy people we found that calcium levels in the gastric juice of patients with GERD was significantly lower than normal. It was also analyzes the relationship between free calcium content of saliva and the same index in the gastric juice. It was revealed increasing the ratio of free calcium saliva and gastric juice in patients with GERD. This ratio increases with the progression of the disease, which may indicate on deep motility disorders of gastro-esophageal connection.

Selective targeting of α7 nicotinic acetylcholine receptor by synthetic peptide mimicking loop I of human SLURP-1 provides efficient and prolonged therapy of epidermoid carcinoma in vivo.

α7-Type nicotinic acetylcholine receptor (α7-nAChR) promotes the growth and metastasis of solid tumors. Secreted Ly6/uPAR-Related Protein 1 (SLURP-1) is a specific negative modulator of α7-nAChR produced by epithelial cells. Here, we investigated mechanisms of antiproliferative activity of recombinant SLURP-1 in epidermoid carcinoma A431 cells and activity of SLURP-1 and synthetic 21 a.a. peptide mimicking its loop I (Oncotag) in a xenograft mice model of epidermoid carcinoma. SLURP-1 inhibited the mitogenic pathways and transcription factors in A431 cells, and its antiproliferative activity depended on α7-nAChR. Intravenous treatment of mice with SLURP-1 or Oncotag for 10 days suppressed the tumor growth and metastasis and induced sustained changes in gene and microRNA expression in the tumors. Both SLURP-1 and Oncotag demonstrated no acute toxicity. Surprisingly, Oncotag led to a longer suppression of pro-oncogenic signaling and downregulated expression of pro-oncogenic miR-221 and upregulated expression of KLF4 protein responsible for control of cell differentiation. Affinity purification revealed SLURP-1 interactions with both α7-nAChR and EGFR and selective Oncotag interaction with α7-nAChR. Thus, the selective inhibition of α7-nAChRs by drugs based on Oncotag may be a promising strategy for cancer therapy.

Extracellular Vesicles Derived from Metastatic Melanoma Cells Transfer α7-nAChR mRNA, Thus Increasing the Surface Expression of the Receptor and Stimulating the Growth of Normal Keratinocytes.

We have previously shown that extracellular vesicles secreted by metastatic melanoma cells stimulate the growth, migration, and stemness of normal keratinocytes. This study showed for the first time that extracellular vesicles secreted by the metastatic melanoma cell lines mel H, mel Kor, and mel P contain, both at the mRNA and protein levels, the α7-type nicotinic acetylcholine receptor (α7-nAChR), which is involved in the regulation of the oncogenic signaling pathways in epithelial cells. Incubation with the vesicles secreted by mel H cells and containing the highest amount of mRNA coding α7-nAChR increased the surface expression of α7-nAChR in normal Het-1A keratinocytes and stimulated their growth. Meanwhile, both of these effects disappeared in the presence of α-bungarotoxin, an α7-nAChR inhibitor. A bioinformatic analysis revealed a correlation between the increased expression of the CHRNA7 gene coding α7-nAChR in patients with metastatic melanoma and a poor survival prognosis. Therefore, extracellular vesicles derived from metastatic melanoma cells can transfer mRNA coding α7-nAChR, thus enhancing the surface expression of this receptor and stimulating the growth of normal keratinocytes. Targeting of α7-nAChR may become a new strategy for controlling the malignant transformation of keratinocytes.

ASIC1a Inhibitor mambalgin-2 Suppresses the Growth of Leukemia Cells by Cell Cycle Arrest.

Although tyrosine kinase inhibitors have brought significant success in the treatment of chronic myelogenous leukemia, the search for novel molecular targets for the treatment of this disease remains relevant. Earlier, expression of acid-sensing ion channels, ASIC1a, was demonstrated in the chronic myelogenous leukemia K562 cells. Three-finger toxins from the black mamba (Dendroaspis polylepis) venom, mambalgins, have been shown to efficiently inhibit homo- and heteromeric channels containing the ASIC1a subunit; however, their use as possible antitumor agents had not been examined. In this work, using the patch-clamp technique, we detected, for the first time, an activation of ASIC1a channels in the leukemia K562 cells in response to an extracellular pH decrease. Recombinant mambalgin-2 was shown to inhibit ASIC1a activity and suppress the proliferation of the K562 cells with a half-maximal effective concentration (EC50) ~ 0.2 µM. Maximum mambalgin-2 inhibitory effect is achieved after 72 h of incubation with cells and when the pH of the cell medium reaches ~ 6.6. In the K562 cells, mambalgin-2 caused arrest of the cell cycle in the G1 phase and reduced the phosphorylation of G1 cell cycle phase regulators: cyclin D1 and cyclin-dependent kinase CDK4, without affecting the activity of CDK6 kinase. Thus, recombinant mambalgin-2 can be considered a prototype of a new type of drugs for the treatment of chronic myelogenous leukemia.

Lynx1 Prevents Long-Term Potentiation Blockade and Reduction of Neuromodulator Expression Caused by Aß1-42 and JNK Activation.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. Many neurodegenerative diseases are accompanied by cognitive impairment associated with the dysfunction of nAChRs. The human membrane-tethered prototoxin Lynx1 modulates nAChR function in the brain areas responsible for learning and memory. In this study, we have demonstrated for the first time that the ß-amyloid peptide Aß1-42 decreases Lynx1 mRNA expression in rat primary cortical neurons, and that this decrease is associated with the activation of c-Jun N-terminal kinase (JNK). In addition, we have demonstrated that the Lynx1 expression decrease, as well as the blockade of the long-term potentiation underlying learning and memory, caused by Aß1-42, may be prevented by incubation with a water-soluble Lynx1 analogue. Our findings suggest that the water-soluble Lynx1 analogue may be a promising agent for the improvement of cognitive deficits in neurodegenerative diseases.

Human secreted proteins SLURP-1 and SLURP-2 control the growth of epithelial cancer cells via interactions with nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (nAChRs) are a promising target for development of new anticancer therapies. Here we have investigated the effects of the endogenous human proteins SLURP-1 and SLURP-2, antagonists of nAChRs, on human epithelial cancer cells. EXPERIMENTAL APPROACH: Growth of epithelial cancer cells (A431, SKBR3, MCF-7, A549, HT-29) exposed to SLURP-1, SLURP-2, mecamylamine, atropine, timolol and gefitinib was measured by the WST-1 test. Expression levels of SLURP-1, α7-nAChR and EGF receptors and their distribution in cancer cells were studied by confocal microscopy and flow cytometry. Secretion of endogenous SLURP-1 by A431 cells under treatment with recombinant SLURP-1 was analysed by Western-blotting. KEY RESULTS: SLURP-1 and SLURP-2 significantly inhibited growth of A431, SKBR3, MCF-7 and HT-29 cells at concentrations above 1 nM, to 40-70% of the control, in 24 h. Proliferation of A549 cells was inhibited only by SLURP-1. The anti-proliferative activity of SLURPs on A431 cells was associated with nAChRs, but not with ß-adrenoceptors or EGF receptors. Action of gefitinib and SLURPs was additive and resulted almost complete inhibition of A431 cell proliferation during 24 h. Exposure of A431 cells to recombinant SLURP-1 down-regulated α7-nAChR expression and induced secretion of endogenous SLURP-1 from intracellular depots, increasing its concentration in the extracellular media. CONCLUSIONS AND IMPLICATIONS: SLURPs inhibit growth of epithelial cancer cells in vitro and merit further investigation as potential agents for anticancer therapy. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors.

Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3ß2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved ß-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, ß2, and ß4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4ß2 and α3ß2-nAChRs (IC50 ~0.17 and >3 µM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 µM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3ß2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3ß2-nAChRs.

[Chemotherapy of malignant tumors]. / Khimioterapiia zlokachestvennykh opukholei.

Potentialities and principles of cancer chemotherapy, the approaches to the search of new cytostatics are shown. Modern chemotherapy is, as a rule, a combination chemotherapy with simultaneous use of 3-4 cytostatics with different mechanism of action and various toxicity. Chemotherapy is a method of choice not only in hemoblastosis, but in such solid tumours as uterine chorionepithelioma, testicular seminoma, Wilms tumour, lung small cell carcinoma, etc. In case of responsive tumours chemotherapy may result in complete recovery of 8 to 80% of patients.

[Delagil treatment of tumorous pleurisy]. / Lechenie opukholevykh plevritom delagilom.

A review of the literature is given on the efficacy of intrapleural use of different drugs (embichine, thiophosphamide, 5-fluorouracil, etc. and also acrichin (chinacrin) for malignant pleural involvement with the presence of exudate in the pleural cavity. Under study was the efficacy of intrapleural injection of delagil in 28 patients with pulmonary cancer, breast cancer and other malignant tumors with the presence of exudate in the pleural cavities. The effect, manifested in the cessation of the fluid accumulation, its encapsulation, was observed in 60 per cent of patients, an average duration of the remission being 8.3 months. In highly sensitive tumors (cancer of ovaries, breast cancer) the treatment should be initiated with intrapleural injection of thiophosphamide or embichine, and in case of failure delagil is to be employed intrapleurally. As a rule, side-effects, manifested in chest pains and temperature rise up to 38.5 degrees are of a short duration and require no special therapy.

[Chemotherapy and combined chemo- and radiotherapy of small cell lung cancer]. / Khimioterapiia i khimioluchevaia terapiia melkokletochnogo raka legkogo.

Objective response of small cell lung cancer to different modalities of chemo-and radiation chemotherapy was recorded in 68-94% while complete regression of tumor--in 22-50% of a total of 166 cases. Complete regression was registered in approximately 33% of cases of localized tumor process and in 16% of cases of expanded process. The survival median for different treatment modalities was 13.7-22.5 months in cases of complete regression and 8.7-12.4 months for partial regression. The cyclophosphamide + adriamycin + methotrexate treatment scheme proved effective in 87% of localized tumor (complete regression--27%). As a result of treatment with the said drugs plus radiation, objective response was obtained in 95%, with complete regression being registered in 68% of the latter group. Therefore, radiation should be an indispensable component of treatment for small cell lung cancer. A two year relapse- and metastasis--free period was observed in 18% cases of localized process and a five year period--in 9%.

[Combined chemo-radiotherapy of small-cell cancer of the lung]. / Kombinirovannaia khimioluchevaia terapiia melkokletochnogo raka legkikh.

The authors report the results of the combined treatment of 32 patients with small-cell cancer of the lung by irradiation in a dose of 5000 rad and chemotherapy: cyclophosphan, methotrexate, nitrosoethylurea. An immediate objective improvement was noted in 90 per cent of the patients, an average survival being 10 months, while without the treatment the survival was no longer than 5 months.

[The effectiveness of 2 systems of chemoradiation treatment in inoperable small-cell lung cancer]. / Effektivnost' dvukh sistem khimioluchevogo lecheniia pri neoperabel'nom melkokletochnom rake legkogo.

A comparative evaluation of treatment results was carried out in two groups of patients with small-cell lung cancer; in group 1, patients received radiation and chemotherapy simultaneously (68 cases) while in group 2 chemotherapy was followed by radiation (67 cases). Complete remission was observed in 62% in group 1. In group 2, it was 7% immediately after chemotherapy to soar up to 46% following radiation treatment. Radiation-induced pneumosclerosis was roentgenologically detected in all those followed up and presented with symptoms in 12.5% and 27.6% in groups 1 and 2, respectively. The frequency of distant metastasis in patients with complete and partial remission was identical in both groups. Metastasis-free period was significantly longer in complete remission patients as compared to those with partial remission; it was still longer in chemotherapy responders in group 2. The procedure involving sequential chemo- and radiation treatment should be preferred.