R482L Mutation of the LMNA Gene Affects Dynamics of C2C12 Myogenic Differentiation and Stimulates Formation of Intramuscular Lipid Droplets.

Mutations in the LMNA gene resulting in the substitution of the highly conserved arginine 482 residue in the globular C-terminal domain of lamin A/C are associated with the Dunnigan-type familial partial lipodystrophy (FPLD2) often accompanied by impairments in the muscle tissue development. The mechanisms underlying these impairments remain unknown. The purpose of our work was to investigate the effects of the LMNA gene mutation R482L on the muscle differentiation and intramuscular fat accumulation using C2C12 mouse myoblasts transduced with the lentiviral constructs carrying the wild-type human LMNA gene (LMNA-WT) or the LMNA-R482L mutant gene. After stimulation of myogenesis and adipogenesis in the transduced cell, expression of muscle and adipose tissue differentiation markers, morphology of differentiated myotubes, and formation of intramuscular lipid droplets were analyzed. C2C12 cells carrying the LMNA-R482L construct exhibited upregulated desmin expression at all stages of muscle differentiation and transformed into hypertrophied myotubules (in comparison with C2C12 myoblasts transduced with LMNA-WT). Reduced expression levels of the myogenic transcription factor Myf6 in the cells with the LMNA-R482L mutant indicated delayed maturation of muscle fibers. These cells more actively accumulated fat in response to the stimulation of adipose differentiation than myoblasts modified with the wild-type LMNA; they also expressed the markers of lipid droplets, such as FABP4 (fatty acid-binding protein 4), ATGL (adipose triglyceride lipase), and PLIN2 (perilipin 2). Therefore, the R482L mutation of the LMNA gene affects the dynamics of C2C12 myoblast differentiation into myotubules and stimulates formation of fat deposits in the myoblasts and myotubules in a tissue-specific manner.

Skeletal Muscle Resident Progenitor Cells Coexpress Mesenchymal and Myogenic Markers and Are Not Affected by Chronic Heart Failure-Induced Dysregulations.

BACKGROUND AND PURPOSE: In heart failure (HF), metabolic alterations induce skeletal muscle wasting and decrease of exercise capacity and quality of life. The activation of skeletal muscle regeneration potential is a prospective strategy to reduce muscle wasting; therefore, the aim of this project was to determine if functional properties of skeletal muscle mesenchymal progenitor cells (SM-MPC) were affected by HF-induced functional and metabolic dysregulations. METHODS: Gastrocnemius muscle biopsy samples were obtained from 3 healthy donors (HD) and 12 HF patients to purify mRNA for further analysis and to isolate SM-MPC. Cells were expanded in vitro and characterized by immunocytochemistry and flow cytometry for expression of mesenchymal (CD105/CD73/CD166/CD146/CD140b/CD140a/VIM) and myogenic (Myf5/CD56/MyoG) markers. Cells were induced to differentiate and were then analyzed by immunostaining and Q-PCR to verify the efficiency of differentiation. The expression of genes that control muscle metabolism and development was compared for HD/HF patients in both muscle biopsy and in vitro-differentiated myotubes. RESULTS: The upregulation of MYH3/MYH8/Myf6 detected in HF skeletal muscle along with metabolic alterations indicates chronic pathological activation of the muscle developmental program. SM-MPC isolated from HD and HF patients represented a mixed population that coexpresses both mesenchymal and myogenic markers and differs from AD-MMSC, BM-MMSC, and IMF-MSC. The functional properties of SM-MPC did not differ between HD and HF patients. CONCLUSION: In the present work, we demonstrate that the metabolic and functional alterations we detected in skeletal muscle from HF patients do not dramatically affect the functional properties of purified and expanded in vitro SM-MPC. We speculate that skeletal muscle progenitor cells are protected by their niche and under beneficial circumstances could contribute to muscle restoration and prevention and treatment of muscle wasting. The potential new therapeutic strategies of HF-induced skeletal muscle wasting should be targeted on both activation of SM-MPC regeneration potential and improvement of skeletal muscle metabolic status to provide a favorable environment for SM-MPC-driven muscle restoration.

Components of the Hepatocellular Carcinoma Microenvironment and Their Role in Tumor Progression.

This review summarizes recently published data on the mechanisms of tumor cell interaction with the tumor microenvironment. Tumor stroma influences the processes of hepatocarcinogenesis, epithelial-to-mesenchymal transition, invasion, and metastasis. The tumor microenvironment includes both cellular and noncellular components. Main cellular components of hepatocellular carcinoma (HCC) stroma are tumor-associated fibroblasts, hepatic stellate cells, immune cells, and endothelial cells that produce extracellular components of tumor microenvironment such as extracellular matrix, various proteins, proteolytic enzymes, growth factors, and cytokines. The noncellular components of the stroma modulate signaling pathways in tumor cells and stimulate invasion and metastasis. The tumor microenvironment composition and organization can serve as prognostic factors in HCC pathogenesis. Current approaches in HCC targeted therapy are aimed at creating efficient strategies for interrupting tumor interactions with the stroma. Recent data on the composition and role of the microenvironment in HCC pathogenesis, as well as new developments in antitumor drug design are discussed.

[A change in the expression of membrane-associated proteins and cytoplasmic actin isoforms in the progression of human colon tumors]. / Izmenenie ékspressii membranoassotsiirovannykh belkov i tsitoplazmaticheskikh izoform aktina pri progressirovanii opukholei tolstoi kishki cheloveka.

Tumor progression is a complex process that also involves the restructuring of the actin cytoskeleton and the weakening of intercellular adhesive contacts due to the tumor cells that pass through the epithelial-mesenchymal transition (EMT). AIM: Тo identify correlations between clinical features, risk of progression and/or recurrence of human colon adenocarcinomas (CAC), and EMT-related tumor markers. MATERIAL AND METHODS: Descending colon and sigmoid colon adenocarcinoma samples were examined immunohistochemically. Formalin-fixed paraffin-embedded tissue sections were incubated with antigen-specific antibodies, then secondary antibodies labeled with fluorochromes, and the fluorescence intensity of microscopy images was analyzed. RESULTS: The cells of a tumor compared to those of intact colon tissue showed a weak staining of E-cadherin in the cell-cell contact areas. The reduced membrane staining and nuclear localization of ß-catenin were detected in moderately (G2) and poorly (G3) differentiated tumors. There were substantially decreased ß-actin levels in almost all tumor samples and increased γ-actin ones, mainly in the samples belonging to stage IV disease. CONCLUSION: A correlation was found between stage, tumor differentiation grade, risk for relapse or progression of disease, and the impaired expression of different EMT markers: total or partial loss of E-cadherin expression, ß-catenin reorganization in cell-cell contacts, and a change in the ratio of cytoplasmic actin isoforms in the late stages of CAC development. We believe that these molecular markers may have a prognostic potential.

E-Cadherin repression increases amount of cancer stem cells in human A549 lung adenocarcinoma and stimulates tumor growth.

Here we show that cancer stem cells amount in human lung adenocarcinoma cell line A549 depends on E-cadherin expression. In fact, downregulation of E-cadherin expression enhanced expression of pluripotent genes (c-MYC, NESTIN, OCT3/4 and SOX2) and enriched cell population with the cells possessing the properties of so-called 'cancer stem cells' via activation of Wnt/ß-catenin signaling. Repression of E-cadherin also stimulated cell proliferation and migration in vitro, decreased cell amount essential for xenografts formation in nude mice, increased tumors vascularization and growth. On the other hand, E-cadherin upregulation caused opposite effects i.e. diminished the number of cancer stem cells, decreased xenograft vascularization and decelerated tumor growth. Therefore, agents restoring E-cadherin expression may be useful in anticancer therapy.

Downregulation of VEGF-C expression in lung and colon cancer cells decelerates tumor growth and inhibits metastasis via multiple mechanisms.

Experimental and clinical studies positively correlate expression of vascular endothelial growth factor (VEGF)-C in cancer cells with accelerated tumor progression and/or unfavorable clinical outcome. However, many aspects of tumor-promoting activity of VEGF-C and consequences of its downregulation for tumor progression remain poorly understood. To clarify these points, we created a set of VEGF receptor 3-positive lung carcinoma A549 and colon carcinoma HCT116 cell sublines with stable repression of VEGF-C synthesis. Analysis of the behavior of these cells revealed multiple effects of VEGF-C downregulation, which, in addition to deceleration of cell proliferation and invasion in vitro and inhibition of lymphangiogenesis in tumor and surrounding tissues observed earlier, included previously undescribed effects, in particular, partial restoration of epithelial phenotype, reduction in the percentage of tumor-initiating cells (cancer stem cells) in the cell population and inhibition of metastasis of orthotopic lung cancer xenografts to other lung lobes. These results are consistent with the idea of high potentiality of VEGF-C as a cancer drug target.

p53 hot-spot mutants increase tumor vascularization via ROS-mediated activation of the HIF1/VEGF-A pathway.

The function of p53 tumor suppressor is often altered in various human tumors predominantly through missense-mutations resulting in accumulation of mutant proteins. We revealed that expression of p53 proteins with amino-acid substitutions at codons 175 (R175H), 248 (R248W), and 273 (R273H), representing the hot-spots of mutations in various human tumors, increased the number of vessels in HCT116 human colon carcinoma xenografts and, as a result, accelerated their growth. Stimulation of tumor angiogenesis was connected with about 2-fold increase in intracellular level of reactive oxygen species (ROS). Antioxidant N-acetyl-l-aspartate (NAC) decreased vessels number in tumors formed by cells with inactivated p53 and inhibited their growth. Effect of ROS on angiogenesis in tumors expressing hot-spot p53 mutants was correlated with their ability to increase a content of HIF1 transcriptional factor responsible for up-regulation of VEGF-A mRNAs.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells.

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53(-/-) mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53(-/-) xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53(-/-) and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53(-/-) tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

[New nucleoside conjugates with phospholipids: synthesis of anti-HIV activity of rac-1-hexadecyl-2-acyl-sn-glycero-3-phosphonucleosides]. / Novye kon"iugaty nukleozidov i anti-VICH-aktivnost' rac-1-geksadetsil-2-atsil-sn-glitsero-3-fosfonuckleozidov.

New nucleoside-phospholipid conjugates were synthesized based on 1,2-disubstituted glycerides and nucleosides. These contain rac-1-hexadecyl-2-palmitoyl(or 2-methylcarbamoyl)-sn-glycero-3-phosphate as the phospholipid component and 2',3'-didehydro-3'-deoxythymidine, 1-(Z-5-hydroxypentene-2-yl)thymine, or 2',3'-isopropylideneuridine as a nucleoside component. The conjugates were synthesized by three different ways: from rac-1-hexadecyl-2-acyl-sn-glycero-3-phospodichlorides, -3-phosphatidic acids, or -3-H-phosphonates. When subjected to mild alkaline hydrolysis, conjugates containing a 2-palmitoyl group formed conjugates with the lysophospholipid component that had not yet been described. All the conjugates obtained were amorphous compounds stable at room temperature. Their hemolytic and anti-HIV activities were determined. Some conjugates were found to completely inhibit in vitro HIV-1 reproduction in lower doses than the corresponding nucleosides.