Immunochemical Techniques Research Articles

On the Interaction of the Na/K ‐Pump ATPase (NAKPA) with Pro‐Survival and Pro‐Apoptotic Bcl‐2 Proteins

By combining in silico approaches with pharmacological studies on the inhibitory action of quaternary benzenephenanthridine alkaloids (QBA) on Na/K Pump ATPase (NAKPA), we recently proposed that QBA, which binds to specific motifs in Bcl‐2 proteins (J Mol Biol 364: 536‐549, 2006), does so also to analogous regions in the NAKPA protein. Hence by analogy, Bcl‐2 proteins must also interact with and may modulate NAKPA (Cell Physiology and Biochemistry 2013). To prove this new hypothesis, we have recently demonstrated by immunochemical techniques that indeed NAKPA interacts with both pro‐survival BclXl and pro‐apoptotic BAK proteins, and thus may constitute an important sensor complex in life and death decisions of the cell (Am J Cell Physiol 2014, PMID 25318106). Presently, this complex appears to be of quaternary nature, in which Bcl‐2 proteins of opposite action may bind to and modulate directly NAKPA and vice versa because only NAKPA antibodies can pull down both Bcl‐2 proteins but not vice versa. Here we review this new model and present further immunochemical data with additional Bcl‐2 proteins, and with bodipy‐fluorescent ouabain and anti‐Bcl‐2 protein directed antibodies to support or modify this new concept.

The effect of an ActRIIB ligand trapping agent on the number of satellite cells and myonuclei in dystrophic (mdx) muscle fibers

Duchenne muscular dystrophy is characterized by repeated cycles of degeneration and regeneration ultimately leading to respiratory failure and death. Muscle growth is largely under the control of myostatin, which negatively influences fiber growth and regeneration by binding to the ActRIIB receptor. Ligand trapping agents for the ActRIIB receptor (RAP‐435) are therefore currently being tested as therapeutic agents using the mdx mouse model for Duchenne muscular dystrophy. The present study utilized confocal immunochemical techniques to examine the effects of in vivo RAP‐435 treatment on the number of satellite cells and myonuclei associated with isolated mdx costal diaphragm fibers. One month old mdx mice were treated with RAP435 or vehicle for one month. Following treatment, the isolated muscles were exposed to 0.2% collagenase. Satellite cells were identified with a PAX 7 primary and an AlexaFluor 488 secondary antibody. Myonuclei were identified by DAPI. Mdx costal diaphragm fibers exhibited approximately 700 myonuclei and 5 satellite cells per muscle fiber. Experiments comparing specific age‐matched nondystrophic and mdx respiratory muscles will determine if the regeneration characteristic of dystrophic muscle is associated with corresponding increases in the number of myonuclei per fiber. RAP‐435 treatment influenced neither the number of myonuclei nor satellite cells per muscle fiber. Morphometric analyses are being performed in conjunction with these confocal studies to determine if ActRIIB inhibition specifically influences myonuclear domain in promoting fiber growth and regeneration.

MPTP‐induced changes in hippocampal synaptic plasticity and memory are prevented by memantine through the BDNF‐TrkB pathway

Mild cognitive deficit in early Parkinson's disease (PD) has been widely studied. Here we have examined the effects of memantine in preventing memory deficit in experimental PD models and elucidated some of the underlying mechanisms. I.p. injection of 1-methyl-4- phenyl-1,2,3,6-tetrahydro pyridine (MPTP) in C57BL/6 mice was used to produce models of PD. We used behavioural tasks to test memory. In vitro, we used slices of hippocampus, with electrophysiological, Western blotting, real time PCR, elisa and immunochemical techniques. Following MPTP injection, long-term memory was impaired and these changes were prevented by pre-treatment with memantine. In hippocampal slices from MPTP treated mice, long-term potentiation (LTP) -induced by θ burst stimulation (10 bursts, 4 pulses) was decreased, while long-term depression (LTD) induced by low-frequency stimulation (1 Hz, 900 pulses) was enhanced, compared with control values. A single dose of memantine (i.p., 10 mg·kg(-1) ) reversed the decreased LTP and the increased LTD in this PD model. Activity-dependent changes in tyrosine kinase receptor B (TrkB), ERK and brain-derived neurotrophic factor (BDNF) expression were decreased in slices from mice after MPTP treatment. These effects were reversed by pretreatment with memantine. Incubation of slices in vitro with 1-methyl-4-phenylpyridinium (MPP(+) ) decreased depolarization-induced expression of BDNF. This effect was prevented by pretreatment of slices with memantine or with calpain inhibitor III, suggesting the involvement of an overactivated calcium signalling pathway. Memantine should be useful in preventing loss of memory and hippocampal synaptic plasticity in PD models.

Detection of exosomal prions in blood by immunochemistry techniques.

In most forms of prion diseases, blood is infectious, but detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of the exosomes on a sucrose gradient was necessary to remove plasma immunoglobulins, which interfere with PrPTSE, masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.

Immunological appraisal of wheat varieties in relation to chapatti-making characteristics

The current research work was conducted to characterize wheat proteins through immunochemical techniques and to find out their relationship with wheat quality traits. The results revealed that wheat variety AARI-11 possessed higher protein content (11.96%), wet gluten (31.39%), dry gluten (9.66%), Pelshenke value (190.52 min), and SDS-Sedimentation value (28.27 ml) than other tested varieties. The chapattis prepared from the wheat variety AARI-11 got significantly higher sensory scores owing to its higher protein contents. The wheat variety AARI-11 also exhibited significantly the highest antibody response against all the assessed protein fractions. The results of the present study suggest that anti-glutenin and anti-high molecular weight glutenin subunits (HMW-GS) antibody response was found positively correlated to the quality characteristics of flours and chapattis. The present study suggests that the use of antibodies response against glutenin and HMW-GS offers good tool for predicting quality and suitability of wheat to chapatti-making quality.

LA PROBLEMÁTICA DEL ENDOSULFÁN: ASPECTOS QUÍMICOS, ANALÍTICOS Y AMBIENTALES

Introduction. Due to the residual character and high level of toxicity of endosulfan, worldwide concern for its environmental impact has increased. Therefore, the search for analytical methodologies that allow its detection and quantification continues to be a topic of current research. Endosulfan properties and its degradation pattern confer it characteristics that make very complex the problematic while the pesticide and its metabolites are extensively spread throughout the atmosphere and contaminate both lipophilic and hydrophilic matrixes. Objective. To conduct a review on some recent research about the endosulfan problematic including chemical aspects, environmental issues and immunochemical techniques for its analysis, which is an area of current interest in this research group at Universidad de Caldas. Method. This review article analyzes qualitatively scientific literature from Science Direct, PubMed, Scielo databases and official Web pages using key words such as endosulfan, endosulfan immunochemical analysis, endosulfan environmental problematic, endosulfan international regulations, endosulfan current normativity and instrumental methods for the analysis. Results. Relevant information related to the proposed objective was found which is presented in 3 sections: chemical aspects of endosulfan, environmental issues, and immunochemical techniques for its analysis. Conclusions. Endosulfan has been banned by international organizations. However, the pesticide is still being used in some countries and is easily spread to other regions, so that the problem became a global concern. There is some recent research on biological or chemical remediation techniques, but efforts seem insufficient in comparison to the enormous impact of the pesticide. In regard to Endosulfan chemical analyses, during the past 15 years immunochemical hapten-based analysis techniques have been investigated as alternatives to the instrumental chromatographic methods.

Early Detection of <i>Cercospora</i> Species in Soybean Plants: Immunologic and Molecular Methods

Late-cycle diseases (LCD) cause a significant deterioration in quality and reduce yields in soybean crops. In Argentina, in particular, leaf blight and purple seed stain, caused by the agent Cercospora kikuchii, and frog eye spot, caused by C. sojina, are the prevailing sources of diseases. The early, rapid and accurate detection of these phytopathogens becomes essential, and would contribute to preserving both the environment and the health of humans and animals by preventing the wasteful or improper use of chemicals such as pesticides. In order to detect Cercospora species in soybean plants at an early stage, immunochemical and molecular techniques were developed in this work. Strains from the NITE Biological Resource Center collection (Japan): Cercospora kikuchii NBRC 6711 and Cercospora sojina NBRC 6715 and regional isolates of C. kikuchii were used. To develop Dot-Blot and PCR techniques, experiments with plants undergoing different treatments were carried out: those experimentally inoculated with these fungi, those treated with sterile water and healthy plants as well. Both techniques allowed the detection, at early stages, of Cercospora species involved in two of the most frequent LCD in the country, when the cercosporin concentration produced by the fungus was higher than 3.93 ± 0.39 nmol·cyl-1 ±SD. The sensitivity between both techniques was very different. While Dot-Blot allowed the detection of the disease 4 days after inoculation, PCR detected it after 4 hours, even without visible symptoms of the disease.

Truffles contain endocannabinoid metabolic enzymes and anandamide

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography–mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit.Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.

Carrier priming with CRM197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: Biophysical and immunochemical interpretation

Glycoconjugate vaccines play an enormous role in preventing infectious diseases. The main carrier proteins used in commercial conjugate vaccines are the non-toxic mutant of diphtheria toxin (CRM197), diphtheria toxoid (DT) and tetanus toxoid (TT). Modern childhood routine vaccination schedules include the administration of several vaccines simultaneously or in close sequence, increasing the concern that the repeated exposure to conjugates based on these carrier proteins might interfere with the anti-polysaccharide response. Extending previous observations we show here that priming mice with CRM197 or DT does not suppress the response to the carbohydrate moiety of CRM197 meningococcal serogroup A (MenA) conjugates, while priming with DT can suppress the response to DT-MenA conjugates. To explain these findings we made use of biophysical and immunochemical techniques applied mainly to MenA conjugates. Differential scanning calorimetry and circular dichroism data revealed that the CRM197 structure was altered by the chemical conjugation, while DT and the formaldehyde-treated form of CRM197 were less impacted, depending on the degree of glycosylation. Investigating the binding and avidity properties of IgGs induced in mice by non-conjugated carriers, we found that CRM197 induced low levels of anti-carrier antibodies, with decreased avidity for its MenA conjugates and poor binding to DT and respective MenA conjugates. In contrast, DT induced high antibody titers able to bind with comparable avidity both the protein and its conjugates but showing very low avidity for CRM197 and related conjugates. The low intrinsic immunogenicity of CRM197 as compared to DT, the structural modifications induced by glycoconjugation and detoxification processes, resulting in conformational changes in CRM197 and DT epitopes with consequent alteration of the antibody recognition and avidity, might explain the different behavior of CRM197 and DT in a carrier priming context.

Role of AMP-activated protein kinase in cross-talk between apoptosis and autophagy in human colon cancer.

Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay between autophagy and apoptosis.

Oxygen transport in a cross section of the rat inner medulla: Impact of heterogeneous distribution of nephrons and vessels

We have developed a highly detailed mathematical model of oxygen transport in a cross section of the upper inner medulla of the rat kidney. The model is used to study the impact of the structured organization of nephrons and vessels revealed in anatomic studies, in which descending vasa recta are found to lie distant from clusters of collecting ducts. Specifically, we formulated a two-dimensional oxygen transport model, in which the positions and physical dimensions of renal tubules and vessels are based on an image obtained by immunochemical techniques (T. Pannabecker and W. Dantzler, Three-dimensional architecture of inner medullary vasa recta, Am. J. Physiol. Renal Physiol. 290 (2006) F1355–F1366). The model represents oxygen diffusion through interstitium and other renal structures, oxygen consumption by the Na+/K+-ATPase activities of the collecting ducts, and basal metabolic consumption. Model simulations yield marked variations in interstitial PO2, which can be attributed, in large part, to the heterogeneities in the position and physical dimensions of the collecting ducts. Further, results of a sensitivity study suggest that medullary oxygenation is highly sensitive to medullary blood flow, and that, at high active consumption rates, localized patches of tissue may be vulnerable to hypoxic injury.

Structural diversity and biological significance of glycosphingolipids in pathogenic and opportunistic fungi.

Glycosphingolipids (GSLs) are ubiquitous membrane components and have key roles in biological systems, acting as second messengers or modulators of signal transduction by affecting several events, ranging from cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle. Over the last 20 years our laboratory and other research groups determined the glycan and ceramide structures of more than 20 GSLs from several pathogenic/opportunistic fungi, using a combination of gas chromatography, mass spectrometry, nuclear magnetic resonance as well as other immunochemical and biochemical techniques. Fungal GSLs can be divided in two major classes: neutral GSLs, galactosyl- and glucosylceramide (GlcCer), and acidic GSLs, the glycosylinositol-phosphorylceramides (GIPCs). Glycosyl structures in fungal GIPCs exhibited significant structural diversity and distinct composition when compared to mammalian GSLs, e.g., the expression of inositol-mannose and inositol-glucosamine cores and the terminal residue of β-D-galactofuranose which are absent in mammalian cells. Studies performed by our group demonstrated that GIPC (Galfβ 6[Manα3]Manα2InsPCer) elicited in patients with paracoccidioidomycosis an immune response with production of antibodies directed to the terminal residue of β-D-galactofuranose. Further studies also showed that inhibition of GlcCer biosynthetic pathways affects fungal colony formation, spore germination and hyphal growth, indicating that enzymes involved in GlcCer biosynthesis may represent promising targets for the therapy of fungal infections. Recently, it was shown that GlcCer and GIPCs are preferentially localized in membrane microdomains and monoclonal antibodies directed to these GSLs interfere in several fungal biological processes such as growth and morphological transition. This review focuses on glycan structures carried on sphingolipids of pathogenic/opportunistic fungi, and aspects of their biological significance are discussed.

Immunochemical Techniques for the Detection of the Parasiticide Dicyclanil in Ovine Tissue

Dicyclanil is a parasiticide used to combat myiasis (flystrike), the infectious infestation of sheep with fly larvae. The European Union has established maximum residue limits (MRLs) for the drug in sheep tissue of 200 μg kg−1 for muscle, 150 μg kg−1 for fat and 400 μg kg−1 for kidney and liver. It is therefore a requirement for regulatory laboratories to have reliable analytical methods at their disposal for the detection of the target analytes, dicyclanil and its major metabolite, 2,4,6-triamino-pyrimidine-5-carbonitrile (TPC). While physicochemical methods have been employed previously, this is the first report of an immunochemically based assay for dicyclanil and TPC. A structural analogue, 2-chloro-4, 6-diamino-5-cyanopyrimidine (CDC), was chosen as a hapten with potential to produce an antibody capable of binding both dicyclanil and TPC. Using homologous ELISA and biosensor assays (in buffer), superior sensitivity was displayed towards dicyclanil (IC50 = 2.1 and 5.0 ng mL−1 respectively) compared to TPC (IC50 = 34.3 and 48.7 ng mL−1, respectively). However, switching to a heterologous format (using a further analogue) for both technologies produced IC50 values of 1.5 ng mL−1 for both analytes by ELISA and 2.8 ng mL−1 (dicyclanil) and 3.7 ng mL−1 (TPC) by biosensor. The heterologous formats were employed to develop ELISA and biosensor methods for the analyses of ovine muscle samples using a simple acetonitrile extraction. Validation of both methods found them to be sufficiently reproducible and sensitive for the purpose of monitoring sheep muscle samples for the presence of non-compliant concentrations of dicyclanil. The detection capability of both methods was found to be less than 100 μg kg−1.

Novel multiplex fluorescent immunoassays based on quantum dot nanolabels for mycotoxins determination

The aim of this manuscript was the development of easy-to-operate quantum dots (QDs)-based immunochemical techniques for simultaneous screening of several mycotoxins in cereals. Two different approaches for multiplex fluorescent immunosorbent assay (FLISA) were employed. In the first approach a multiwell plate in which the different wells express a different mycotoxin (deoxynivalenol, zearalenone, aflatoxin B1, T2-toxin and fumonisin B1) was considered as a multiplex because each sample was pretreated once and then will be distributed over a series of wells within the same plate (single-analyte multiplex, SAM). The entire assay allows the simultaneous determination of all compounds. For the double-analyte multiplex (DAM) two different specific antibodies were co-immobilized in one single well. Zearalenone and aflatoxin B1 were simultaneously determined, provided their conjugates are labeled with QDs which are fluorescent in different parts of the spectrum, by scanning the assay outcome at two different wavelengths. The limits of detection (LOD) for the simultaneous determination of deoxynivalenol, zearalenone, aflatoxin B1, T2-toxin and fumonisin B1 by SAM FLISA were 3.2, 0.6, 0.2, 10 and 0.4µgkg−1, respectively, while for the DAM FLISA they were 1.8 and 1µgkg−1 for zearalenone and aflatoxin B1, respectively. SAM FLISA principle was also presented in a qualitative on-site format and tested for on-site multiplex determination of four mycotoxins in cereals. The achieved cut-off values of 500, 100, 2 and 100µgkg−1 for deoxynivalenol, zearalenone, aflatoxin B1 and T2-toxin respectively. For simplification of multiassay results' evaluation the conjugates with QDs of different colors were used.

Dormancy alleviation by NO or HCN leading to decline of protein carbonylation levels in apple (Malus domestica Borkh.) embryos

Deep dormancy of apple (Malus domestica Borkh.) embryos can be overcome by short-term pre-treatment with nitric oxide (NO) or hydrogen cyanide (HCN). Dormancy alleviation of embryos modulated by NO or HCN and the first step of germination depend on temporary increased production of reactive oxygen species (ROS). Direct oxidative attack on some amino acid residues or secondary reactions via reactive carbohydrates and lipids can lead to the formation of protein carbonyl derivatives. Protein carbonylation is a widely accepted covalent and irreversible modification resulting in inhibition or alteration of enzyme/protein activities. It also increases the susceptibility of proteins to proteolytic degradation. The aim of this work was to investigate protein carbonylation in germinating apple embryos, the dormancy of which was removed by pre-treatment with NO or HCN donors. It was performed using a quantitative spectrophotometric method, while patterns of carbonylated protein in embryo axes were analyzed by immunochemical techniques. The highest concentration of protein carbonyl groups was observed in dormant embryos. It declined in germinating embryos pre-treated with NO or HCN, suggesting elevated degradation of modified proteins during seedling formation. A decrease in the concentration of carbonylated proteins was accompanied by modification in proteolytic activity in germinating apple embryos. A strict correlation between the level of protein carbonyl groups and cotyledon growth and greening was detected. Moreover, direct in vitro carbonylation of BSA treated with NO or HCN donors was analyzed, showing action of both signaling molecules as protein oxidation agents.

The ubiquitin ligase Deltex‐3L regulates endosomal sorting of the G protein‐coupled receptor CXCR4 (1066.18)

G protein‐coupled receptor (GPCR) sorting into the degradative pathway is important for attenuating signaling. Upon agonist activation of the chemokine receptor CXCR4, a GPCR, it is rapidly ubiquitinated, internalized to endosomes and sorted to lysosomes via the endosomal sorting complex required for transport (ESCRT) pathway. ESCRT‐0 ubiquitination is linked to the efficiency with which CXCR4 is sorted for lysosomal degradation, however mechanistic insight is lacking. Here, we show for the first time that the RING‐domain E3 ubiquitin ligase Deltex‐3‐like (DTX3L) mediates CXCR4 sorting from early endosomes to lysosomes. Using several biochemical and immunochemical techniques including fixed cell confocal immunofluoresence microscopy, co‐immunoprecipitation and in vitro ubiquitination assays, we show that DTX3L localizes to early endosomes upon CXCR4 activation and interacts directly with and inhibits the activity of the E3 ubiquitin ligase atrophin‐1 interacting protein 4 (AIP4). This serves to limit the extent to which ESCRT‐0 is ubiquitinated and is able to sort CXCR4 for lysosomal degradation. Therefore we have defined a novel role for DTX3L in GPCR endosomal sorting and propose that DTX3L may play a broad role in endosomal sorting. In addition, our data reveal an unprecedented link between two distinct E3 ubiquitin ligases to control the activity of the ESCRT machinery.Grant Funding Source: GM075159

Perianal alveolar soft-part sarcoma: a rare case

Abstract

1D4: a versatile epitope tag for the purification and characterization of expressed membrane and soluble proteins.

Incorporation of short epitope tags into proteins for recognition by commercially available monoclonal or polyclonal antibodies has greatly facilitated the detection, characterization, localization, and purification of heterologously expressed proteins for structure-function studies. A number of tags have been developed, but many epitope-antibody combinations do not work effectively for all immunochemical techniques due to the nature of the tag and the specificity of the antibodies. A highly versatile, multipurpose epitope tag is the 9 amino acid C-terminal 1D4 peptide. This peptide tag together with the Rho1D4 monoclonal antibody can be used to detect proteins in complex mixtures by western blotting and ELISA assays, localize proteins in cells by immunofluorescence and immunoelectron microscopic labeling techniques, identify subunits and interacting proteins by co-immunoprecipitation, and purify functionally active proteins including membrane proteins by immunoaffinity chromatography. In this chapter we describe various immunochemical procedures which can be used for the detection, purification and localization of 1D4-tagged proteins for structure-function studies.

Practical effect of introduction of colorimetric and immuno chemical techniques at the establishment of presence of sperm on material evidences

The authors analyze the experience of both domestic and of foreign authors in application of the methods for detection of rough and demonstrative presence of the sperm in the stains on the material evidences. Colorimetric method for definition of the acid phosphotase presence and the ELISA technique for definition of PSA are compared with other modern techniques applied in Russia. Sensitivity, objectivity, actuality, economyand other potentialities and advantages of both methods are more pronounced in forensic medicine practice.

Expression of voltage-gated sodium channel in primary tumors and corresponding metastases in the Copenhagen rat model of prostate cancer

Objective: Increasing evidence indicates that prostate cancer (PCa) cells possess voltage-gated sodium channels (VGSCs) and these channels contribute to the metastatic progression of the disease. In the present study, it was aimed to investigate the heterotopic expression of VGSCs in carcinogenesis and metastasis. It was evaluated whether the expression of VGSC in strongly metastatic rat Mat-LyLu PCa cells observed in vitro is also conserved when these cells form primary tumors and metastasize to the lungs upon their subcutaneous injection in rats. Methods: PCa model was induced in male Copenhagen rats by subcutaneous implantation of strongly metastatic Mat-LyLu cells. The expression of VGSCs was studied in the model utilizing molecular biological and immunochemical techniques. Results: Reverse transcription PCR and immunohistochemistry showed that the expression of VGSC is conserved in the primary prostate tumors of Mat-LyLu and not eliminated in the corresponding lung metastases at both the mRNA and protein level. Conclusion: The results are consistent with the predominant role of VGSC in the metastatic progression of PCa and consistently support the VGSC hypothesis of cancer progression. J Clin Exp Invest 2013; 4 (4): 422-428