EGFR Suppression Inhibits the Sphere Formation of MCF7 Cells Overexpressing EGFR.

The epidermal growth factor receptor (EGFR) is an oncogenic tyrosine kinase that is involved in tumor initiation and progression, making EGFR inhibitors and monoclonal antibodies to this receptor essential for anti-tumor therapy. We have previously shown that EGFR transgene expression in the human breast adenocarcinoma cell line MCF7 (MCF7-EGFR) stimulates the 3D spheroid-like growth. The primary focus of our present work was to investigate whether EGFR inhibition could affect the assembly of spheroids or lead to the destruction of pre-existing spheroids. We compared the effects of anti-EGFR siRNA, the anti-EGFR monoclonal antibody cetuximab, and the tyrosine kinase inhibitor AG1478 on dissociated and spheroid MCF7-EGFR cells. MCF7-EGFR cells were found to have a 2.5-fold higher sensitivity towards the cytotoxic effects of cetuximab and AG1478 compared with the parental MCF7 cell line. The suppression of EGFR mRNA with siRNA was found to reduce the sphere formation, whereas treating the pre-existing spheroids had no such effect. Treatment of dissociated spheroids with cetuximab and AG1478 was also found to inhibit the MCF7-EGFR sphere formation. We suggest that EGFR expression is important, at least, during the spheroid formation stage. The transition of a MCF7wt adherent cell culture to MCF7-EGFR spheroids was accompanied by a considerable increase in N-cadherin adhesion proteins. The level of N-cadherin decreased when MCF7-EGFR cells were treated with siRNA and cetuximab. Thus, we have demonstrated that N-cadherin is involved in the EGFR-dependent formation of MCF7-EGFR spheroids. Accordingly, MCF7-EGFR spheroids can be considered a suitable model for studying aggressive hormone-positive breast tumors.

Immunogenic Cell Death in Cancer Therapy.

Apoptosis plays a crucial role in chemotherapy-induced cell death. The conventional theory holding that apoptosis needs to be immunologically silent has recently been revised, and the concept of immunogenic cell death (ICD) has been proposed. This review describes the main features of ICD induction. These ICD markers are important for the effectiveness of anticancer therapy, as well as for basic research into cell death regulation. The mechanism of the "vaccination effect" of dying cancer cells undergoing ICD has been fully described, including the activation of specific antitumor response after re-challenge by the same living tumor cells. This review also discusses the whole set of molecular events attributing cell death to immunogenic type: the exposure of calreticulin and the heat shock protein HSP70 to the outer surface of the cell membrane and the release of the nuclear protein HMGB1 and ATP into the extracellular space. ICD inducers of various nature (chemotherapy drugs, cytotoxic proteins, and oncolytic viruses), as well as physical methods, are classified in the current review.

Cancer-associated fibroblasts and their role in tumor progression.

The stromal elements of a malignant tumor can promote cancer progression and metastasis. The structure of the tumor stroma includes connective tissue elements, blood vessels, nerves, and extracellular matrix (ECM). Some of the cellular elements of the tumor stroma are cancer-associated f ibroblasts (CAFs). The origin and function of CAFs have been actively studied over the past thirty years. CAFs produce collagen, the main scaffold protein of the extracellular matrix. Collagen in the tumor stroma stimulates f ibrosis, enhances the rigidity of tumor tissue, and disrupts the transmission of proliferation and differentiation signaling pathways. CAFs control tumor angiogenesis, cell motility, tumor immunogenic properties, and the development of resistance to chemo- and immunotherapy. As a result of metabolic adaptation of rapidly growing tumor tissue to the nutrients and oxygen deprivation, the main type of energy production in cells changes from oxidative phosphorylation to anaerobic glycolysis. These changes lead to sequential molecular alterations, including the induction of specif ied transcriptional factors that result in the CAFs activation. The molecular phenotype of activated CAFs is similar to f ibroblasts activated during inf lammation. In activated CAFs, alpha-smooth muscle actin (α-SMA) is synthetized de novo and various proteases and f ibronectin are produced. Since CAFs are found in all types of carcinomas, these cells are potential targets for the development of new approaches for anticancer therapy. Some CAFs originate from resident f ibroblasts of the organs invaded by the tumor, while others originate from epithelial tumor cells, which are undergoing an epithelial-mesenchymal transition (EMT). To date, many molecular and metabolic inducers of the EMT have been discovered including the transforming growth factor-beta (TGF-ß), hypoxia, and inf lammation. This review classif ies modern concepts of molecular markers of CAFs, their functional features, and discusses the stages of epithelial- mesenchymal transition, and the potential of CAFs as a target for antitumor therapy.

shp-2 gene knockout upregulates CAR-driven cytotoxicity of YT NK cells.

In Russia, cancer is the second leading cause of death following cardiovascular diseases. Adoptive transfer of NK cells is a promising approach to fight cancer; however, for their successful use in cancer treatment, it is necessary to ensure their robust accumulation at tumor foci, provide resistance to the immunosuppressive tumor microenvironment, and to engineer them with higher cytotoxic activity. NK lymphocytes are known to kill cancer cells expressing a number of stress ligands; and the balance of signals from inhibitory and activating receptors on the surface of the NK cell determines whether a cytotoxic reaction is triggered. We hypothesized that stronger cytotoxicity of NK cells could be achieved via gene editing aimed at enhancing the activating signaling cascades and/or weakening the inhibitory ones, thereby shifting the balance of signals towards NK cell activation and target cell lysis. Here, we took advantage of the CRISPR/Cas9 system to introduce mutations in the coding sequence of the shp-2 (PTPN11) gene encoding the signaling molecule of inhibitory pathways in NK cells. These shp-2 knock-out NK cells were additionally transduced to express a chimeric antigen receptor (CAR) that selectively recognized the antigen of interest on the target cell surface and generated an activating signal. We demonstrate that the combination of shp-2 gene knockout and CAR expression increases the cytotoxicity of effector NK-like YT cells against human prostate cancer cell line Du-145 with ectopic expression of PSMA protein, which is specifically targeted by the CAR.

Cold Physical Plasma Decreases the Viability of Lung Adenocarcinoma Cells.

The high mortality rate that accompanies cancer spurs the search for new methods that can be used to treat malignant neoplasms. In addition to chemotherapy, electrophysical techniques for tumor treatment appear rather promising. The results of in vitro exposure of A549 human lung adenocarcinoma cells to cold atmospheric plasma (CAP) are hereby presented. A gas-discharge device that generates a sequence of streamers propagating along a stream of inert gas in the ambient air was used. In the zone where the plasma jet came into contact with the target object, there were high-intensity electric fields and high plasma concentrations, while the gas temperature changed by less than a degree. In this study, we compared the cytotoxic effect of CAP in helium and argon. Direct irradiation of cells by CAP with U = 4.2 kV for 30-120 s was shown to reduce cell viability by 25%. Variation of the amplitude of the AC voltage in the plasma device in argon within a range of 3.8-5.6 kV did not significantly alter the cell death rate. Further optimization of the modes of CAP generation in gas-discharge devices with various geometries for the trea.

Effect of benzo[a]pyrene on the expression of miR-126, miR-190a and their target genes EGFL7, TP53INP1 and PHLPP1 in primary endometrial cells.

The main topic of this study was to investigate the effect of benzo[a]pyrene (BP) on microRNAs and their target genes expression levels in primary cell cultures from normal and malignant endometrial tissue. MicroRNA-126 (miR-126) and miR-190a were most sensitive to BP treatment. The treatment of both cultures with BP was accompanied by a decrease of miR-126 level and an increase of EGFL7 gene expression level. BP-induced upregulation of miR-190a was detected only in normal cells and it was accompanied with decrease of mRNA levels of TP53INP1 and PHLPP1 genes. Taking into account that BP promoted the proliferation of normal cells and amplified apoptosis of cancer cells, it is possible that miR-190a is involved in general cellular response to BP. The findings of this study indicate that miR-190a and its target genes may be involved in the regulation of cell fate under BP treatment.

[Ischaemia of a femoral stump in short- and long-term periods after limb amputation]. / Ishemiia kul'ti bedra v rannem i pozdnem periodakh posle amputatsii.

The authors retrospectively analysed medical case histories of 287 patients subjected to femoral amputations over the period from January 1, 1998 to December 31, 2013. The studied parameters were as follows: the frequency of and risk factors for femoral stump ischaemia, as well as the effect on patients' survival after femoral amputation. Amongst 156 patients having endured transfemoral truncation of the limb performed as the first amputation, early femoral stump ischaemia (EFSI) within 3 postoperative months was found to have occurred in 43 (27.6%) patients, whereas amongst 127 patients first subjected to amputation of the crus and then to femoral truncation it occurred in 15 (13.2%) cases; p<0.05. The incidence rate of late femoral stump ischaemia (LFSI) was virtually similar in both groups, amounting to 5.8% (9 of 156) and 5.5% (7 of 127), respectively; p>0.05. The survival rate for patients without stump ischaemia at 12 months after amputation amounted to 79.4%, for those with EFSI to 50.0% (p=0.00928), and for those with LFSI to 71.4% (p=0.22576), whereas by the end of a 5-year follow up period these values appeared to equal 49.2%, 32.1% (p=0.13225) and 7.1% (p=0.01385), respectively. The obtained findings demonstrated that the risk factors for EFSI were as follows: the presence of a femoral stump on the contralateral side, grade III ischaemia, and proximal localization of the lesion of the arterial bed (odds ratio 3.3, 2.7 and 3.8, respectively); a risk factor for LFSI was the presence of a femoral stump on the contralateral side (odds ratio 6.0).

Antimetastatic Effect of Liposomal Recombinant Lactaptin.

Antimetastatic effect of the liposomal form of recombinant lactaptin RL2 (a proteolytic fragment of human breast milk κ-casein; 8.6 kDa) was studied in A/Sn mice after intravenous transplantation of GA-1 tumor with high rate of liver metastases. Tumor growth in the liver was found in all mice. In animals dying early, the tumors were presented by multiple nodes of about the same size; in mice dying later, the tumors in the liver were presented by just few large nodes formed by cells that survived chemotherapy. A single intravenous injection of RL2 lactaptin in liposomes prolonged lifespan of animals with liver metastases of GA-1 tumor by 1.5 times in comparison with that in untreated animals.

[Immunogenicity of recombinant analog of antitumor protein lactaptin].

Therapeutic monoclonal antibodies and recombinant proteins including cytokines are commonly used in the treatment of cancer and inflammatory diseases. In most cases, these protein-based drugs exhibit a high therapeutic efficacy, which is unfortunately frequently associated with a variety of side effects. We have investigated the in vitro and in vivo immunogenicity of recombinant antitumor protein lactaptin (RL2). Based on the qRT-PCR analysis, we have shown that, in MDA-MB-231 human breast adenocarcinoma cells, RL2 suppresses the NF-kB signaling cascade that regulates the reactions of innate immunity. RL2 inhibits the expression of the CXCL1 protein and apoptosis inhibitor A20 and enhances expression of IkB, NF-kB repressor. The ELISA method has been used to evaluate the antibody titer in the blood of mice, which received single and triple intravenous or intraperitoneal injections of RL2. The multiplex immunoassay of 23 cytokines in the mice blood has shown that the RL2 injections lead to a slight increase in the levels of systemic pro-inflammatory cytokine interleukin-5 (IL-5) and keratinocyte chemoattractant (KC), a homologue of human macrophage inflammatory protein-1 (MIP-1). These observations indicate the low immunogenicity of the recombinant lactaptin analog, which can be considered to be a potential molecular drug candidate for further clinical development.

Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues.

Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans.

[Semiclosed endarterectomy from iliac arteries under duplex scanning].

The investigation included 149 cases of semiclosed endarterectomy from iliac arteries in 134 patients. Intraoperative duplex scanning were used in 32 patients as a deobliteration control. Other 117 cases were performed by using of traditional method. Specific deobliteration complications such as thrombosis of operated segment and embolism of operated and contralateral limbs arteries with atherogenic masses were observed 10 patients. Intraoperative control was not performed in these cases. Arterial wall perforations with loop tool were observed in two groups, but the frequency was statistically significant lower in the group with duplex scanning. Mortality rates did not differ. Amputation number of lower limbs was statistically lower in case of deobliteration control using.

[The influence of recombinant nucleophosmin 1 on artificial RNA internalization into human adenocarcinoma MCF-7 cells].

In this study we obtained and characterized the recombinant analogue of multifunctional nucleolar phosphoprotein nucleophosmin 1 (NPM1) involved in crucial cellular processes such as transcription, reparation and mitosis. The influence ofnucleophosmin 1 on extrcellular RNAs accumulation in human adenocarcinoma cells MCF-7 was analyzed. It was found that incubation of AluY RNA (n > 300 nt), U24 snoRNA analogues (n ~ 80 nt) with Npm1-His6 resulted in RNA-protein non-covalent complexes formation, but not in case of the short oligoribonucleotide (n = 22 nt). It was shown that interaction of AluY RNA analogue with Npm1-His6 significantly increases transfection efficacy of the RNA into MCF-7 human cells. Altogether, these data allow us to conclude, that nucleophosmin 1 not only binds RNA with complex secondary structure, but also promotes uptake and internalization of such RNA by human cells.

Alu- and 7SL RNA Analogues Suppress MCF-7 Cell Viability through Modulating the Transcription of Endoplasmic Reticulum Stress Response Genes.

11% of the human genome is composed of Alu-retrotransposons, whose transcription by RNA polymerase III (Pol III) leads to the accumulation of several hundreds to thousands of Alu-RNA copies in the cytoplasm. Expression of Alu-RNA Pol III is significantly increased at various levels of stress, and the increase in the Alu-RNA level is accompanied by a suppression of proliferation, a decrease in viability, and induction of apoptotic processes in human cells. However, the question about the biological functions of Pol III Alu-transcripts, as well as their mechanism of action, remains open. In this work, analogues of Alu-RNA and its evolutionary ancestor, 7SL RNA, were synthesized. Transfection of human breast adenocarcinoma MCF-7 cells with the Alu-RNA and 7SL RNA analogues is accompanied by a decrease in viability and by induction of proapoptotic changes in these cells. The analysis of the combined action of these analogues and actinomycin D or tamoxifen revealed that the decreased viability of MCF-7 cells transfected with Alu-RNA and 7SL RNA was due to the modulation of transcription. A whole transcriptome analysis of gene expression revealed that increased gene expression of the transcription regulator NUPR1 (p8), as well as the transcription factor DDIT3 (CHOP), occurs under the action of both the Alu- and 7SL RNA analogues on MCF-7 cells. It has been concluded that induction of proapoptotic changes in human cells under the influence of the Alu-RNA and 7SL RNA analogues is related to the transcriptional activation of the genes of cellular stress factors, including the endoplasmic reticulum stress response factors.

[The analysis of biochemical markers of MCF-7 cells apoptosis induced by recombinant analog of lactaptin].

Earlier we isolated and characterized human milk pro-apoptotic peptide - lactaptin and generated its engineered analog - RL2. It was shown that both lactaptin and RL2 are capable to induce apoptotic death of MCF-7 cells. In this report we have analyzed biochemical markers of RL2 induced MCF-7 apoptosis. The activation of initiator and effector caspases as well as mitochondrial membrane potential and cytoplasm membrane changes were analyzed using flow cytometry and Western-blot methods. We have found that RL2 induced apoptotic death of MCF-7 cells was accompanied by PS exposure on the plasma membrane surface. It also was shown that RL2 has induced dissipation of mitochondrial membrane potential and resulted in activation of initiator caspases 8, 9 and effector caspase 7.

Analogues of Artificial Human Box C/D Small Nucleolar RNA As Regulators of Alternative Splicing of a pre-mRNA Target.

Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA (rRNA) biogenesis. Box C/D snoRNAs guide the site-specific 2'-O-ribose methylation of nucleotides in rRNAs and small nuclear RNAs (snRNAs). A number of box C/D snoRNAs and their fragments have recently been reported to regulate post-transcriptional modifications and the alternative splicing of pre-mRNA. Artificial analogues of U24 snoRNAs directed to nucleotides in 28S and 18S rRNAs, as well as pre-mRNAs and mature mRNAs of human heat shock cognate protein (hsc70), were designed and synthesized in this study. It was found that after the transfection of MCF-7 human cells with artificial box C/D RNAs in complex with lipofectamine, snoRNA analogues penetrated into cells and accumulated in the cytoplasm and nucleus. It was demonstrated that the transfection of cultured human cells with artificial box C/D snoRNA targeted to pre-mRNAs induce partial splicing impairments. It was found that transfection with artificial snoRNAs directed to 18S and 28S rRNA nucleotides, significant for ribosome functioning, induce a decrease in MCF-7 cell viability.

[Dynamics of the incidence of major amputations and mortality rates in peripheral arterial diseases over 1993-2007: findings of a population-based study].

Analysed were a total of 17,695 cases of hospital admission of patients presenting with arterial insufficiency of extremities, complications of diabetes mellitus, ulcers and necroses of the lower limbs, including 11,773 cases of hospitalization of patients diagnosed with peripheral artery diseases according to ICD-9 (till 1998) or ICD-10 (commencing from 1999) over the periods from 1993-1997 (period 1), 1998-2002 (period 2) and 2003-2007 (period 3). The correlation dependence was studied using the Pearson criterion, with the statistical significance of differences between the relative incidence of the parameters being calculated by means of the chi-square (chi2) test. The obtained findings showed a decrease in the number of amputees, the incidence of lethal outcomes and mortality rates, being most significant in the group of patients under 60 years of age. There was an inverse correlation between the above measures and the indices of the number of operations on the arteries performed in grade IV limb ischaemia.

[Comparative assessment of methods used for predicting efficiency of lumbar sympathectomy in patients with lower-limb critical ischaemia].

The article deals with the findings of examination and treatment of patients presenting with lesions of the distal-bed arteries and critical lower-limb ischaemia who were subjected to lumbar sympathectomy (LSE). The methods aimed at predicting efficacy of LSE in seventy-five patiens included Doppler ultrasonography with the nitroglycerine test and with the epidural blockade. Of these forty-five patients were subjected to laser Doppler flowmetry (LDF) and measurement of the transcutaneous oxygen tension in tissues before and after the epidural blockade. Presented herein are remote results of LPE obtained at terms up to 36 months for sixty-nine patients (92%) and analyzed depending on the parameters of the functional tests. Our findings suggest that the most statistically significant methods of study for predicting the outcome of lumbar sympathectomy turned out to be LDF and Doppler ultrasonography performed before and after the epidural blockade.

Specific complications of semi-closed endarterectomy from the iliac arteries.

Altogether 104 patients were examined. Semi-closed endarterectomy from the iliac arteries was attempted in 110 limbs of these patients. In 13.6% of cases, operation was unsuccessful. In 3.6% of cases, the failure was caused by arterial perforation when applying a loop-shaped tool. Ninety-five patients underwent deobliteration of the iliac arteries. In the short-term postoperative period, 2 (2.1%) patients developed reocclusion of the operated segment and 5 (5.3%) had acute ischemia of the contralateral limb. The causes and the clinical importance of errors and complications from semi-closed endarterectomy are discussed.

Oxidative DNA cleavage by free and oligonucleotide-conjugated O-bromobenzoic acid in the presence of copper(II) ions.

o-Bromobenzoic acid was found to promote copper-dependent reactive oxygen species formation from molecular oxygen, resulting in DNA base modification and backbone cleavage. The oligonucleotide conjugate bearing 5-(4'-aminopropyl-sulfomoyl)-2-bromobenzoic acid as a reactive group was synthesized and DNA cleavage activity of this oligonucleotide conjugate was tested on a model deoxyoligonucleotide.

New reagent for protein-DNA contacts footprinting.

We have found, that the reaction of o-bromobenzoic acid with Cu2+ ions can be used as a source of activated oxygen species capable of cleaving DNA. Possibility to apply this reaction for footprinting the nucleosome core in the reconstituted chromatin was demonstrated.