[Duodenal bleeding due to metastasis from lung adenocarcinoma controlled by radiotherapy: A case report and literature review]. / Saignement duodénal dû à une métastase d'un cancer pulmonaire contrôlé par radiothérapie : cas clinique et revue de la littérature.

INTRODUCTION: Gastrointestinal (GI) metastases in lung cancer rarely occur. CASE REPORT: We report here the case of a 43-year-old male active smoker who was admitted to our hospital for cough, abdominal pain and melena. Initial investigations revealed poorly differentiated adenocarcinoma of the superior-right lobe of the lung: positive for thyroid transcription factor-1 and negative for protein p40 and for antigen CD56, with peritoneal, adrenal and cerebral metastasis, as well as anemia requiring major transfusion support. Over 50% of cells were positive for PDL-1, and ALK gene rearrangement was detected. GI endoscopy showed a large ulcerated nodular lesion of the genu superius with active intermittent bleeding, as well as an undifferentiated carcinoma with positivity for CK AE1/AE3 and TTF-1, and negativity for CD117, corresponding to metastatic invasion originating from lung carcinoma. Palliative immunotherapy with pembrolizumab was proposed, followed by targeted therapy with brigatinib. Gastrointestinal bleeding was controlled with a single 8Gy dose of haemostatic radiotherapy. CONCLUSION: GI metastases are rare in lung cancer and present nonspecific symptoms and signs but no characteristic endoscopic features. GI bleeding is a common revelatory complication. Pathological and immunohistological findings are critical to diagnosis. Local treatment is usually guided by the occurrence of complications. In addition to surgery and systemic therapies, palliative radiotherapy may contribute to bleeding control. However, it must be used cautiously, given a present-day lack of evidence and the pronounced radiosensitivity of certain gastrointestinal tract segments.

Proliferative and Differentiation Potential of Multipotent Mesenchymal Stem Cells Cultured on Biocompatible Polymer Scaffolds with Various Physicochemical Characteristics.

Biocompatibility of film and fibrous scaffolds from polylactide-based polymers and the relationship between their architecture and the functional characteristics of mesenchymal stem cells were studied. Cell culturing on polylactide-based film and fibrous matrixes did not deteriorate cell morphology and their proliferation and differentiation capacities. The rate of cell proliferation and penetration in microporous 3D matrices with the same porosity parameters and pore size depended on their spatial organization. The above materials can be used as scaffolds for mesenchymal stem cells for creation of tissue engineering implants. The scaffold size and structure should be determined by the defects in the organs in which the regeneration processes have to be stimulated.

Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes.

UNLABELLED: Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak-dependent F-actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F-actin in platelet biogenesis. SUMMARY: Background Blood platelet biogenesis results from the maturation of megakaryocytes (MKs), which involves the development of a complex demarcation membrane system (DMS). Therefore, MK differentiation is an attractive model for studying membrane remodeling. Objectives We sought to investigate the mechanism of DMS structuration in relationship to the cytoskeleton. Results Using three-dimensional (3D) confocal imaging, we have identified consecutive stages of DMS organization that rely on F-actin dynamics to polarize membranes and nuclei territories. Interestingly, microtubules are not involved in this process. We found that the mechanism underlying F-actin-dependent DMS formation required the activation of the guanosine triphosphate hydrolase Cdc42 and its p21-activated kinase effectors (Pak1/2/3). Förster resonance energy transfer demonstrated that active Cdc42 was associated with endomembrane dynamics throughout terminal maturation. Inhibition of Cdc42 or Pak1/2/3 severely destructured the DMS and blocked proplatelet formation. Even though this process does not require containment within the hematopoietic niche, because DMS structuration was observed upon thrombopoietin-treatment in suspension, integrin outside-in signaling was required for Pak activation and probably resulted from secretion of extracellular matrix by MKs. Conclusions These data indicate a functional link, mandatory for MK differentiation, between actin dynamics, regulated by Cdc42/Pak1/2/3, and DMS maturation.

ATP-binding cassette transporter 1 (ABCA1) deficiency decreases platelet reactivity and reduces thromboxane A2 production independently of hematopoietic ABCA1.

UNLABELLED: ESSENTIALS: The role of ATP-binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized. We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and two Tangier patients. ABCA1-deficient platelets exhibit reduced positive feedback loop mechanisms. This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1. BACKGROUND: The ATP-binding cassette transporter ABCA1 is required for the conversion of apolipoprotein A-1 to high-density lipoprotein (HDL), and its defect causes Tangier disease, a rare disorder characterized by an absence of HDL and accumulation of cholesterol in peripheral tissues. The role of ABCA1 in platelet functions remains poorly characterized. OBJECTIVE: To determine the role of ABCA1 in platelet functions and to clarify controversies concerning its implication in processes as fundamental as platelet phosphatidylserine exposure and control of platelet membrane lipid composition. METHODS AND RESULTS: We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and in two Tangier patients. We show that platelets in ABCA1-deficient mice are slightly larger in size and exhibit aggregation and secretion defects in response to low concentrations of thrombin and collagen. These platelets have normal cholesterol and major phospholipid composition, granule morphology, or calcium-induced phosphatidylserine exposure. Interestingly, ABCA1-deficient platelets display a reduction in positive feedback loop mechanisms, particularly in thromboxane A2 (TXA2) production. Hematopoietic chimera mice demonstrated that defective eicosanoids production, particularly TXA2, was primarily dependent on external environment and not on the hematopoietic ABCA1. Decreased aggregation and production of TXA2 and eicosanoids were also observed in platelets from Tangier patients. CONCLUSIONS: Absence of ABCA1 and low HDL level induce reduction of platelet reactivity by decreasing positive feedback loops, particularly TXA2 production through a hematopoietic ABCA1-independent mechanism.

[Molecular mechanisms of niclosamide antitumor activity].

In this review the recent data regarding the antitumor activity of niclosamide and the molecular mechanisms of its antitumor activity are presented. Niclosamide has been used in the clinic for the treatment of intestinal parasite infections. In recent years in several screening investigations of various drugs and chemical compounds niclosamide was identified as a potential anticancer agent. Niclosamide not only inhibits the Wnt/ß-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways, but also targets mitochondria in cancer cells to induce growth inhibition and apoptosis. A number of studies have established the anticancer activity of niclosamide in both in vitro and in vivo in xenotransplantation models using human tumors and immunodeficient mice. It is important that niclosamide is active not only against tumor cells but also cancer stem cells. Normal cells are resistant to niclosamide. The accumulated experimental data suggest niclosamide is a promising drug for the treatment of various types of cancer.

Telomerase as a tumor marker in diagnosis of prostatic intraepithelial neoplasia and prostate cancer.

BACKGROUND: Early diagnosis of prostate cancer (CaP) can be addressed by studying prostatic intraepithelial neoplasia (PIN) as precancer (high-grade PIN or HGPIN). This article attempts to analyze the diagnostic role of telomerase as an early marker of carcinogenesis. METHODS: Complex urological patient evaluation and assessment of telomerase activity. RESULTS: Out of 92 patients 44% were diagnosed with CaP, 49% with low-grade PIN (LGPIN) in association with benign prostatic hyperplasia (BPH), and 7% with HGPIN in association with BPH. Active telomerase (AT) in prostate biopsy specimens was detected in 98% of patients with CaP, in 33% of patients with HGPIN, and in 20% of patients with LGPIN. In the event of simultaneous detection of AT and PIN in initial prostate biopsy specimens, further monitoring for 0.5-4.0 years revealed CaP development in 50-56% of cases. Further follow-up of patients with PIN and absent telomerase activity in initial biopsy specimens did not demonstrate the development of CaP. The PSA level was significantly higher in patients with active telomerase in the prostate tissue than in telomerase negative patients. CONCLUSIONS: Telomerase activity in the prostate tissue increases the risk of CaP development in patients with PIN. Detection of telomerase activity in prostate biopsy specimens from patients with PIN enables selection of a group of patients with high risk of CaP development and reduction of the number of prostate biopsies performed in other patients.

Effect of anthralin on cell viability in human prostate adenocarcinoma.

The study revealed the key role of serine protease hepsin activity in transition of in situ prostate adenocarcinoma into the metastasizing form. Inhibition of hepsin activity suppresses the invasive growth of the tumor. Hepsin is an convenient target for pharmacological agents, so the study of its inhibitory mechanisms is a promising avenue in drug development. Assay of proteolytic activity in various tumor cell lines in vitro showed that this activity in prostate adenocarcinoma cells significantly surpasses proteolytic activity in other examined tumor cell lines. Selective cytotoxic action of anthralin, an inhibitor of hepsin activity, on human adenocarcinoma cells was demonstrated in comparison with other tumor cell lines.

[The distribution of iodine-125 labeled alpha-fetoprotein in the animal organism and its accumulation in the tumor].

The distribution of iodine-125 labeled human alpha-fetoprotein in mice was studied after its intravenous injection. The maximal accumulation of alpha-fetoprotein in different tissues and organs of animals was observed mainly 5 hours after injection. Then the protein was gradually eliminated from the body. In the liver, intestine and blood of intact animals 125I-alpha-fetoprotein persists for at least three days. Accumulation of alpha-fetoprotein in various tissues and organs may determine the different biological effects of this protein. In the mice with transplanted lymphatic leukemia cells P388 the high level of alpha-fetoprotein accumulation was detected in the tumor tissue, reaching 6% of the injected amount per 1 g of tissue. This allows considering the radionuclide-labeled alpha-fetoprotein as a promising medical radionuclide marker for the radiological detection of malignant tumors.

Distinct and overlapping functional roles of Src family kinases in mouse platelets.

BACKGROUND AND OBJECTIVES: Src family kinases (SFKs) play a critical role in initiating and propagating signals in platelets. The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbß3, and in vivo. METHODS AND RESULTS: Mouse platelets express four SFKs, Fgr, Fyn, Lyn and Src, with Lyn expressed at a considerably higher level than the others. Using mutant mouse models, we demonstrate that platelet activation by collagen-related peptide (CRP) is delayed and then potentiated in the absence of Lyn, but only marginally reduced in the absence of Fyn or Fgr, and unaltered in the absence of Src. Compound deletions of Lyn/Src or Fyn/Lyn, but not of Fyn/Src or Fgr/Lyn, exhibit a greater delay in activation relative to Lyn-deficient platelets. Fibrinogen-adherent platelets show reduced spreading in the absence of Src, potentiation in the absence of Lyn, but no change in the absence of Fyn or Fgr. In mice double-deficient in Lyn/Src or Fgr/Lyn, the inhibitory role of Lyn on spreading on fibrinogen is lost. Lyn is the major SFK-mediating platelet aggregation on collagen at arterial shear and its absence leads to a reduction in thrombus size in a laser injury model. CONCLUSION: These results demonstrate that SFKs share individual and overlapping roles in regulating platelet activation, with Lyn having a dual role in regulating GPVI signaling and an inhibitory role downstream of αIIbß3, which requires prior signaling through Src.

[Analysis of complex formation of human recombinant HSP70 with tumor-associated peptides].

Molecular chaperones of HSP70 family assists presentation of exogenous antigenic peptides by antigen-presenting cells (APC). HSP70-peptide complexes are powerful immunotherapeutic agents, which enhance cross-presentation of captured antigen in dendritic cells and macrophages. Several clinical trials have shown that HSP-based cancer vaccines possess good efficacy and safety. However, sometime it is impossible to isolate sufficient amount of vaccine. These make us to pay attention for recombinant HSP70-based vaccines and to optimize in vitro complex formation mechanism. Here we have investigated two human recombinant proteins HSP70(HYB) and HSC70. Optimal values of ADP concentration, pH, temperature and peptides excess are determined in this work. We have also shown that proposed complex formation method enriches eluted from HSP70-complexes peptide repertoire compared to in vivo assembled ones.

[Characterization of recombinant human HSP70's domains functions and interdomain interactions].

We investigated ATP-ase and peptide-binding activity of recombinant human heat shock protein HSP70(A1B), HSC70, and two hybrid proteins derived from those. The UV-spectral recorded data was used to characterize conformational rearrangements, which were induced by domain replacement or HSP70-peptide interaction. We have shown that N-terminal domain dramatically affect substrate specificity of C-terminal peptide-binding domain. This proposes new hypothesis for HSP70 chaperone machinery. The linear dependence between ATP-ase activity and peptide complex ratio was found. This relationship could be used for unlabeled peptide-HSP70 complex quantification.

[High-efficient renaturation of immobilized recombinant C-terminal fragment of human alpha-fetoprotein].

C-terminal fragment of a human oncofetal alpha-fetoprotein (AFP) may be used in targeted cytostatics delivery to malignant cells of many tumors. AFP fragment (from 404 to 595 amino acids residues of a full-sized protein) was cloned and produced in Escherichia coli cells, BL21 strain (DE3) in the form of inclusion bodies. To obtain a functionally active protein, is it necessary to renature the protein. The renaturation procedure of the AFP third domain (rAFP3D) is considerably complicated by the fact that the protein is hydrophobic and contains a large number of S-S bonds. A renaturation technique of rAFP3D immobilized on silicic metal chelate resin has been developed. The yield of renatured C-terminal fragment was no less than 60% with purity on the order of 98%. The developed technique has been applied for the first time for hydrophobic protein with a large number of S-S bonds. The approach can be applied for efficient renaturation of other hydrophobic proteins with a large number of disulfide bonds for scientific and practical purposes.

Src family kinases are essential for primary aggregation by G(i) -coupled receptors.

INTRODUCTION AND BACKGROUND: Adrenaline stimulates biphasic aggregation in plasma through the G(i) -coupled α(2A) -adrenoreceptor. In the present study, we demonstrate that both primary and secondary wave aggregation induced by adrenaline in plasma is blocked by two structurally distinct inhibitors of Src family kinases, dasatinib and PD0173952. METHODS AND RESULTS: In contrast, primary aggregation is partially inhibited or unaffected in the presence of inhibitors of cyclo-oxygenase, phosphoinositide (PI) 3-kinases, and P2Y(1) and P2Y(12) ADP receptors, although secondary aggregation is abolished. The ability of adrenaline to inhibit adenylyl cyclase and to synergize with platelet agonists in mediating platelet activation in plasma is retained in the presence of Src family kinase inhibition. Moreover, adrenaline does not activate Src family kinases, as determined by western blotting of their regulatory tyrosines, suggesting that constitutive signaling from Src family kinases may underlie their role in activation. Adrenaline is widely used in clinical laboratories for investigation of patients with suspected bleeding disorders. In a group of 90 unrelated patients with a clinically diagnosed platelet bleeding disorder, we identified four who did not exhibit primary wave aggregation in response to adrenaline, although the catecholamine potentiated the response to other agonists, and five who failed to undergo secondary wave aggregation. In contrast, adrenaline stimulated biphasic aggregation in 60 controls. All of the patients with a defective response to adrenaline had impaired ADP-induced platelet activation. CONCLUSIONS: The present results indicate a previously unappreciated role for Src family kinases in mediating G(i) signaling in plasma, and demonstrate heterogeneity in response to adrenaline in patients with a clinically diagnosed platelet disorder.

Isolation, purification, and study of properties of recombinant hepsin from Escherichia coli.

A recombinant hepsin-producing strain of Escherichia coli was obtained and the conditions for hepsin expression in a bacterial system were optimized. To study the physicochemical properties of the enzyme, a procedure for purification of active recombinant hepsin using metal-chelate affinity chromatography and ion-exchange chromatography was developed. The interaction of recombinant hepsin with various peptide substrates is characterized. The dose-dependent inhibition of the recombinant hepsin enzyme activity by anthralin in vitro and an increase in the hepsin enzymatic activity in the presence of resveratrol were revealed.

Targeted delivery of anti-cancer growth inhibitory peptides derived from human alpha-fetoprotein: review of an International Multi-Center Collaborative Study.

The alpha-fetoprotein derived growth inhibitory peptide (GIP) is a 34-amino acid peptide composed of three biologically active subfragments. GIP-34 and its three constituent segments have been synthesized, purified, and studied for biological activity. The GIP-34 and GIP-8 have been characterized as anticancer therapeutic peptides. An multicenter study was initiated to elucidate the means by which these peptide drugs could be targeted to tumor cells. The study first established which cancer types were specifically targeted by the GIP peptides in both in vitro and in vivo investigations. It was next demonstrated that radiolabeled peptide ((125)I GIP-34) is specifically localized to rodent breast tumors at 24 h post-injection. The radionuclide studies also provided evidence for a proposed cell surface receptor; this was confirmed in a further study using fluorescent-labeled GIP-nanobeads which localized at the plasma membrane of MCF-7 breast cancer cells. Finally, it was readily demonstrated that GIP conjugated to either fluorescein or doxorubicin (DOX) underwent tumor cell uptake; subsequently, DOX-GIP conjugates induced cytotoxic cell destruction indicating the utility of GIP segments as cancer therapeutic agents. Following a discussion of the preceding results, a candidate cell surface receptor family was proposed which correlated with previous published reports for a putative AFP/GIP receptor.

[Purification and characterization of recombinant human alpha-fetoprotein fragment, corresponding to the C-terminal structural domain].

Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.

[Regulation of human dendrite cells' physiological functions with the recombinant heat shock protein Hsp70].

Reference literature review. The dendrite cells (DC) are the most important antigen-representing cells of the organism. They are the target for various vaccines, and on the basis of the DC cellular anti-tumour and anti-viral vaccines (DC vaccines) have been developed. At the same time, the DC can be a convenient model for studying activity and action mechanisms of different immune-therapeutic preparations. One of the aspects of the DC use optimization for induction of antigen-specific immune response might involve use of the heat shock proteins (Hsp), the Hsp70 in particular. This protein can be used for administration of protein antigens into the DC and for regulation of the DC activity. Knowledge of the DC physiology and specifics of interrelationship among the Hsp70 and its complexes with the DC antigens of different differentiation degree is an important aspect for implementation of these ideas. Human Hsp70 has been shown both to bring antigens to the DC and to regulate activity of the DC as well as to optimize induction of antigen-specific cellular immune response.

[Live xenogenic vaccine in the prevention of metastases of uveal melanoma].

By the results of experimental studies and by being guided by Protocol No. 22 dated January 22, 2004, by the Ministry of Health of the Russian Federation, the authors have conducted clinical trials of biotherapy used in oncological care. They describe the first experience in using xenovaccination in 35 patients with uveal melanoma to prevent hematogenic metastases. The follow-ups of the patients lasted within 14-49 months after initiation of the vaccination. No recurrences were observed in 30 patients during follow-ups whose durations averaged 41.3 months. The authors consider that this is reason to regard xenovaccination as an available method for the prevention of metastases of uveal melanoma.

Proteomic analysis of integrin alphaIIbbeta3 outside-in signaling reveals Src-kinase-independent phosphorylation of Dok-1 and Dok-3 leading to SHIP-1 interactions.

BACKGROUND AND OBJECTIVES: Outside-in integrin alphaIIbbeta3 signaling involves a series of tyrosine kinase reactions that culminate in platelet spreading on fibrinogen. The aim of this study was to identify novel tyrosine phosphorylated signaling proteins downstream of alphaIIbbeta3, and explore their role in platelet signaling. METHODS AND RESULTS: Utilizing proteomics to search for novel platelet proteins that contribute to outside-in signaling by the integrin alphaIIbbeta3, we identified 27 proteins, 17 of which were not previously shown to be part of a tyrosine phosphorylation-based signaling complex downstream of alphaIIbbeta3. The proteins identified include the novel immunoreceptors G6f and G6b-B, and two members of the Dok family of adapters, Dok-1 and Dok-3, which underwent increased tyrosine phosphorylation following platelet spreading on fibrinogen. Dok-3 was also inducibly phosphorylated in response to the GPVI-specific agonist collagen-related peptide (CRP) and the PAR-1 and -4 agonist thrombin, independently of the integrin alphaIIbbeta3. Tyrosine phosphorylation of Dok-1 and Dok-3 was primarily Src kinase-independent downstream of the integrin, whereas it was Src kinase-dependent downstream of GPVI. Moreover, both proteins inducibly interacted with Grb-2 and SHIP-1 in fibrinogen-spread platelets. CONCLUSIONS: This study provides new insights into the molecular mechanism regulating alphaIIbbeta3-mediated platelet spreading on fibrinogen. The novel platelet adapter Dok-3 and the structurally related Dok-1 are tyrosine phosphorylated in an Src kinase-independent manner downstream of alphaIIbbeta3 in human platelets, leading to an interaction with Grb2 and SHIP-1.