Bartlomiej E. Krazinski

Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine luteal cells

The objective of the study was to examine the expression of the genes coding for proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) in porcine luteal cells isolated from corpora lutea (CL) collected on days 3-6, 8-10 and 13-16 of the oestrous cycle. Total RNA was purified from non-incubated cells and from cells incubated for 48 h in the absence or presence of luteinising hormone (LH). The semi-quantitative RT-PCR technique, involving coamplification of the target and control cDNA (beta-actin or 18S rRNA), was used to examine gene expression. It was found that the genes coding for opioid precursors are expressed in both non-incubated and incubated porcine luteal cells representing the early, mid- and late luteal phase. In non-incubated cells, only POMC mRNA content changed during CL development, whereas the expression of PENK and PDYN genes remained relatively constant. Additionally, the treatment of cells with LH markedly affected the expression of POMC and PENK, but no influence on PDYN expression was observed. The present study indicates that porcine luteal cells may produce opioid peptides and that gene expression of their precursors (except for PDYN) may be modulated in these cells by LH. Moreover, the present results support the involvement of opioid peptides in local regulation within the CL of the pig.

Divergent expression patterns of SATB1 mRNA and SATB1 protein in colorectal cancer and normal tissues

Special AT-rich sequence-binding protein 1 (SATB1) is a ‘genome organizer,’ and it has been proposed as a factor that affects the development and progression of various human neoplasms, including colorectal cancer (CRC). This study aimed to compare SATB1 expression in a group of CRC patients and healthy subjects at the mRNA and protein levels. We collected paired tumor tissue and unchanged mucosa of the large intestine from 102 CRC patients as well as 53 biopsies of normal colon mucosa obtained from healthy patients during screening colonoscopy. Tissue samples were quantified for SATB1 mRNA by quantitative PCR, while SATB1 protein expression was determined by Western blotting and immunohistochemistry. SATB1 mRNA level in tumor tissues was over twofolds lower than in samples of corresponding unchanged tissues and fourfolds lower than in biopsies of healthy colon mucosa. Western blotting analysis revealed that SATB1 protein content in tumor and unchanged tissues of CRC patients was over sixfold and fivefolds higher than in biopsies of healthy colon mucosa, respectively. Immunohistochemical staining demonstrated higher nuclear and cytoplasmic SATB1 reactivity in the tumor tissue compared to unchanged mucosa of CRC patients. Despite these differences, SATB1 mRNA, protein, and immunoreactivity levels did not correlate with patients’ clinicopathological data and their overall survival, but the latter analysis was limited by a relatively short period of follow-up. In conclusion, we suggest that some as yet unidentified posttranscriptional mechanisms that regulate SATB1 expression may be altered in the CRC tissue.

Optical Biosensing System for the Detection of Survivin mRNA in Colorectal Cancer Cells Using a Graphene Oxide Carrier-Bound Oligonucleotide Molecular Beacon

The anti-apoptotic protein survivin is one of the most promising cancer biomarkers owing to its high expression in human cancers and rare occurrence in normal adult tissues. In this work, we have investigated the role of supramolecular interactions between a graphene oxide (GO) nanosheet nanocarrier and a survivin molecular beacon (SurMB), functionalized by attaching fluorophore Joe and quencher Dabcyl (SurMB-Joe). Molecular dynamics simulations revealed hydrogen bonding of Joe moiety and Dabcyl to GO carriers that considerably increase the SurMB-GO bonding strength. This was confirmed in experimental work by the reduced fluorescence background in the OFF state, thereby increasing the useful analytical signal range for mRNA detection. A new mechanism of hairpin–hairpin interaction of GO@SurMB with target oligonucleotides has been proposed. A low limit of detection, LOD = 16 nM (S/N = 3), has been achieved for complementary tDNA using GO@SurMB-Joe nanocarriers. We have demonstrated an efficient internalization of SurMB-Joe-loaded GO nanocarriers in malignant SW480 cells. The proposed tunability of the bonding strength in the attached motifs for MBs immobilized on nanocarriers, via structural modifications, should be useful in gene delivery systems to enhance the efficacy of gene retention, cell transfection and genomic material survivability in the cellular environment.

Colorectal cancer patients exhibit increased levels of galanin in serum and colon tissues

Galanin (GAL) is a 30-amino acid neuropeptide that is expressed in both the central and peripheral nervous system, including the enteric nervous system (ENS). Increased GAL concentrations have been identified in the blood of colorectal cancer (CRC) patients. The aim of the present study was to assess whether sections of the colon wall containing ENS plexuses or CRC tumor are associated with increased GAL concentrations. Blood samples were collected from 68 CRC patients and 39 healthy volunteers. In addition, samples of CRC tumors and colon wall tissue in close proximity to and distant from the neoplastic tissue were obtained from 22 CRC patients. The GAL concentration of sera and tissue homogenates obtained from three sections of the colon wall (mucosa with submucosa, muscularis externa and CRC tumor) was analyzed by ELISA. The localization of GAL was evaluated using immunohistochemistry and morphometry was used to measure the distribution of GAL-immunoreactive (GAL-Ir) myenteric plexuses in the vicinity of cancer invasion and in sections of the colon wall distant from the tumor. The GAL serum concentration of CRC patients was 2.4 times higher than that of the control group. The GAL concentration was highest in the homogenates of neoplastic tissue and mucosa obtained from the control (distant) section of the intestinal wall, followed by that in the mucosa and muscularis externa proximal to the tumor. The lowest GAL concentrations were identified within the muscular layer of the colon wall located distant from the tumor. Strong GAL immunoreactivity was identified in the cancer cells, intestinal epithelium and the submucosal and myenteric plexuses. Morphometric analysis revealed that the GAL-Ir myenteric plexuses in the vicinity of tumor infiltration were significantly smaller in size than those in the intact section of the large intestine. Furthermore, no associations were identified between the clinicopathological characteristics of CRC patients and GAL serum and tissue concentration. The increased GAL serum concentrations observed in CRC patients in comparison to healthy controls may result from GAL secretion by CRC tumors, however, other sources of GAL cannot be excluded. The atrophy of myenteric plexuses within close proximity to the tumor may affect the colon function of CRC patients. In conclusion, investigation into the presence of GAL in the colon wall and serum of CRC patients revealed that serum and tissue GAL levels may present useful potential biomarkers in CRC patients.

PLAGL1 protein is differentially expressed in the nephron segments and collecting ducts in human kidney

INTRODUCTION PLAGL1 (pleiomorphic adenoma gene-like 1) is a C2H2-type zinc finger transcription factor associated with the regulation of cell growth and development. Although PLAGL1 expression in kidney was assessed by biochemical methods, the exact localization of the PLAGL1 protein in human kidney has not yet been described. MATERIAL AND METHODS Macroscopically unchanged specimens of kidney tissue were collected from 39 patients undergoing nephrectomy due to renal cell carcinoma. H & E staining of paraffin sections was used to assess histology of the kidney whereas immunohistochemistry was used to localize PLAGL1 protein in kidney compartments. In addition, database sequences search for putative PLAGL1 binding sites among the kidney-related genes was performed. RESULTS PLAGL1 staining intensity differed depending on the kidney compartment. Strong PLAGL1 immunoreactivity was found in thick ascending limbs of Henles loop, distal tubules and collecting ducts, whereas PLAGL1 expression in proximal tubules and renal corpuscles (including podocytes) was moderate and weak, respectively. By the in sillico screening of promoter sequences for PLAGL1 specific DNA-binding sites GGG-GCCCC we designated 43 candidate genes for PLAGL1-regulated genes. Analysis of their functional annotations identified three significantly over-represented gene sets: inositol phosphate metabolic processes (GO), endocrine and other factor-regulated calcium reabsorption (KEGG) and calcium signaling pathways (KEGG). CONCLUSION Differences in the renal expression of PLAGL1 suggest that this protein may be involved in the regulation of several cellular pathways both as transcriptional factor and coactivator/corepressor of other tran-scription factors reflecting its role in the cell type-specific control of gene expression.

Hairpin–Hairpin Molecular Beacon Interactions for Detection of Survivin mRNA in Malignant SW480 Cells

Cancer biomarkers offer unique prospects for the development of cancer diagnostics and therapy. One of such biomarkers, protein survivin (Sur), exhibits strong antiapoptotic and proliferation-enhancing properties and is heavily expressed in multiple cancers. Thus, it can be utilized to provide new modalities for modulating the cell-growth rate, essential for effective cancer treatment. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe, encoded to recognize Sur messenger RNA (mRNA). Contrary to the expectations, we have found that both the complementary target oligonucleotide strands as well as the single- and double-mismatch targets, instead of exhibiting the anticipated simple random conformations, preferentially formed secondary structure motifs by folding into small-loop hairpin structures. Such a conformation may interfere with, or even undermine, the biorecognition process. To gain better understanding of the interactions involved, we have replaced the classical Tyagi-Kramer model of interactions between a straight target oligonucleotide strand and a hairpin MB with a new model to account for the hairpin-hairpin interactions as the biorecognition principle. A detailed mechanism of these interactions has been proposed. Furthermore, in experimental work, we have demonstrated an efficient transfection of malignant SW480 cells with SurMB probes containing a fluorophore Joe (SurMB-Joe) using liposomal nanocarriers. The green emission from SurMB-Joe in transfected cancer cells, due to the hybridization of the SurMB-Joe loop with Sur mRNA hairpin target, corroborates Sur overexpression. On the other hand, healthy human-colon epithelial cells CCD 841 CoN show only negligible expression of survivin mRNA. These experiments provide the proof-of-concept for distinguishing between the cancer and normal cells by the proposed hairpin-hairpin interaction method. The single nucleotide polymorphism sensitivity and a low detection limit of 26 nM (S/N = 3σ) for complementary targets have been achieved.

The Immunoexpression of YAP1 and LATS1 Proteins in Clear Cell Renal Cell Carcinoma: Impact on Patients’ Survival

The aim of the study was to determine by immunohistochemistry cellular localization and immunoreactivity levels of YAP1 and LATS1 proteins in paired sections of tumor and unchanged renal tissues of 54 clear cell renal cell carcinoma (ccRCC) patients. Associations between clinical-pathological and overall survival (OS; median follow-up was 40.6 months) data of patients and YAP1 and LATS1 immunoreactivity were analyzed by uni- and multivariate Cox regression model and log-rank test. YAP1 immunoreactivity was found in the nuclei of tumor cells in 64.8% of ccRCC patients, whereas only 24.1% of tumors revealed cytoplasmic YAP1 expression. LATS1 immunoexpression was observed only in the cytoplasm of tumor cells in 59.3% of patients. LATS1 immunoreactivity in cancer cells negatively correlated with the size of primary tumor. The overall YAP1 immunoreactivity did not correlate with clinical-pathological data of patients. However, the subgroup of ccRCC patients who presented with cytoplasmic YAP1 immunoexpression had significantly shorter OS (median = 26.8 months) than patients without cytoplasmic YAP1 expression (median undefined). Multivariate Cox analysis revealed that increased cytoplasmic YAP1 (HR = 4.53) and decreased LATS1 immunoreactivity levels (HR = 0.90) were associated with worse prognosis, being independent prognostic factors. These results suggest that YAP1 and LATS1 can be considered as new prognostic factors in ccRCC.

A novel approach for preventing esophageal stricture formation: olmesartan prevented apoptosis

Accidentally ingested corrosive substances can cause functional and structural damage to the esophageal tissue resulting in stricture formation. It has been reported that the administration of olmesartan (OLM) can have anti-inflammatory, antifibrotic and antiapoptotic effects on injured tissue. The aim of our study was to check if OLM could prevent formation of scars in the corrosive esophageal burn model. Fifty-one Wistar Albino rats were divided into six groups: Control, Sham, OLM, Sham + OLM, Burn, and Burn + OLM. Olmesartan (5 mg/kg) was given by gavage once per day for 21 consecutive days after injury. The morphology of the esophagus was assessed after Masson trichrome staining, and apoptosis was evaluated using the terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) method. The serum nucleosomes (as an indicator of apoptosis), serum p53 protein, and esophageal tissue p53 protein levels of each group were measured by immunoassays. Muscularis mucosa damage, submucosal collagen deposition, and tunica muscularis injury in the Burn + OLM group decreased significantly compared with the Burn group (p < 0.05). Similarly, the number of apoptotic cells in the Burn + OLM group decreased compared with the Burn group (p < 0.05). Serum levels of nucleosomes and p53 and tissue of p53 protein did not differ between the groups. Exogenously administered OLM can effectively prevent the occurrence of esophageal strictures caused by corrosive esophageal burns.