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Norepinephrine (NE), the stress hormone, stimulates many bacterial species’ growth and virulence, including Escherichia coli. However, the hormone’s impact on the adherent-invasive E. coli (AIEC) implicated in Crohn’s disease is poorly understood. In the study, we have investigated the effect of NE on the interaction of six AIEC strains isolated from an intestinal biopsy from 6 children with Crohn’s disease with Caco-2 cells. Our study focused on type 1 fimbria and CEACAM6 molecules serving as docking sites for these adhesins. The study results demonstrated that the hormone significantly increased the adherence and invasion of AIEC to Caco-2 cells in vitro. However, the effect was not associated with the impact of NE on the increased proliferation rate of AIEC or the fimA gene expression vital for their interaction with intestinal epithelial cells. Instead, the carcinoembryonic antigen-related cell-adhesion-molecule-6 (CEACAM6) level was increased significantly in NE-treated Caco-2 cells infected with AIEC in contrast to control uninfected NE-treated cells. These results indicated that NE influenced the interaction of AIEC with intestinal epithelium by increasing the level of CEACAM6 in epithelial cells, strengthening their adherence and invasion.

Breast cancer (BC) is the leading cause of death among cancer patients worldwide. In 2020, almost 12% of all cancers were diagnosed with BC. Therefore, it is important to search for new potential markers of cancer progression that could be helpful in cancer diagnostics and successful anti-cancer therapies. In this study, we investigated the potential role of the lysine acetyltransferases KAT6A and KAT6B in the outcome of patients with invasive breast carcinoma. The expression profiles of KAT6A/B in 495 cases of IDC and 38 cases of mastopathy (FBD) were examined by immunohistochemistry. KAT6A/B expression was also determined in the breast cancer cell lines MCF-7, BT-474, SK-BR-3, T47D, MDA-MB-231, and MDA-MB-231/BO2, as well as in the human epithelial mammary gland cell line hTERT-HME1 - ME16C, both at the mRNA and protein level. Statistical analysis of the results showed that the nuclear expression of KAT6A/B correlates with the estrogen receptor status: KAT6A NUC vs. ER r = 0.2373 and KAT6B NUC vs. ER r = 0.1496. Statistical analysis clearly showed that KAT6A cytoplasmic and nuclear expression levels were significantly higher in IDC samples than in FBD samples (IRS 5.297 ± 2.884 vs. 2.004 ± 1.072, p < 0.0001; IRS 5.133 ± 4.221 vs. 0.1665 ± 0.4024, p < 0.0001, respectively). Moreover, we noticed strong correlations between ER and PR status and the nuclear expression of KAT6A and KAT6B (nucKAT6A vs. ER, p = 0.0048; nucKAT6A vs. PR p = 0.0416; nucKAT6B vs. ER p = 0.0306; nucKAT6B vs. PR p = 0.0213). Significantly higher KAT6A and KAT6B expression was found in the ER-positive cell lines T-47D and BT-474, whereas significantly lower expression was observed in the triple-negative cell lines MDA-MB-231 and MDA-MB-231/BO2. The outcomes of small interfering RNA (siRNA)-mediated suppression of KAT6A/B genes revealed that within estrogen receptor (ER) positive and negative cell lines, MCF-7 and MDA-MB-231, attenuation of KAT6A led to concurrent attenuation of KAT6A, whereas suppression of KAT6B resulted in simultaneous attenuation of KAT6A. Furthermore, inhibition of KAT6A/B genes resulted in a reduction in estrogen receptor (ER) mRNA and protein expression levels in MCF-7 and MDA-MMB-231 cell lines. Based on our findings, the lysine acetyltransferases KAT6A and KAT6B may be involved in the progression of invasive ductal breast cancer. Further research on other types of cancer may show that KAT6A and KAT6B could serve as diagnostic and prognostic markers for these types of malignancies., (© 2024. The Author(s).)

Metabolic endotoxemia (ME) is associated with bacterial lipopolysaccharide (LPS, endotoxin) and increased levels of saturated fatty acids (SFAs) in the bloodstream, causing systemic inflammation. ME usually accompanies obesity and a diet rich in fats, especially SFAs. Numerous studies confirm the effect of ME-related endotoxin on microglial activation. Our study aimed to assess lipid metabolism and immune response in microglia pre-stimulated with TNFα (Tumor Necrosis Factor α) and then with endotoxin and palmitic acid (PA). Using ELISA, we determined cytokines IL-1β, IL-10, IL-13 (interleukin-1β, -10, -13, and TGFβ (Transforming Growth Factor β) in the culture medium from microglial cells stimulated for 24 h with TNFα and then treated with LPS (10 ng/mL) and PA (200 µM) for 24 h. HMC3 (Human Microglial Cells clone 3) cells produced negligible amounts of IL-1β, IL-10, and IL-13 after stimulation but secreted moderate levels of TGFβ. Changes in lipid metabolism accompanied changes in TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) expression. HMC3 stimulation with endotoxin increased TREM2 expression, while PA treatment decreased it. Endotoxin increased ceramide levels, while PA increased triglyceride levels. These results indicated that pre-stimulation of microglia with TNFα significantly affects its interactions with LPS and PA and modulates lipid metabolism, which may lead to microglial activation silencing and neurodegeneration.

Background: The tetraspanin (TSPAN) family comprises 33 membrane receptors involved in various physiological processes in humans. Tetrasapanins are surface proteins expressed in cells of various organisms. They are localised to the cell membrane by four transmembrane domains (TM4SF). These domains bind several cell surface receptors and signalling proteins to tetraspanin-enriched lipid microdomains (TERM or TEM). Tetraspanins play a critical role in anchoring many proteins. They also act as a scaffold for cell signalling proteins., Aim: To summarise how tetraspanins 6, 7 and 8 contribute to the carcinogenesis process in different types of cancer., Methods: To provide a comprehensive review of the role of tetraspanins 6, 7 and 8 in cancer biology, we conducted a thorough search in PubMed, Embase and performed manual search of reference list to collect and extract data., Discussion: The assembly of tetraspanins covers an area of approximately 100-400 nm. Tetraspanins are involved in various biological processes such as membrane fusion, aggregation, proliferation, adhesion, cell migration and differentiation. They can also regulate integrins, cell surface receptors and signalling molecules. Tetraspanins form direct bonds with proteins and other members of the tetraspanin family, forming a hierarchical network of interactions and are thought to be involved in cell and membrane compartmentalisation. Tetraspanins have been implicated in cancer progression and have been shown to have multiple binding partners and to promote cancer progression and metastasis. Clinical studies have documented a correlation between the level of tetraspanin expression and the prediction of cancer progression, including breast and lung cancer., Conclusions: Tetraspanins are understudied in almost all cell types and their functions are not clearly defined. Fortunately, it has been possible to identify the basic mechanisms underlying the biological role of these proteins. Therefore, the purpose of this review is to describe the roles of tetraspanins 6, 7 and 8., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Cancer is the leading cause of death worldwide. The World Health Organization (WHO) estimates that 10 million fatalities occurred in 2023. Breast cancer (BC) ranked first among malignancies with 2.26 million cases, lung cancer (LC) second with 2.21 million cases, and colon and rectum cancers (CC, CRC) third with 1.93 million cases. These results highlight the importance of investigating novel cancer prognoses and anti-cancer markers. In this study, we investigated the potential effects of alpha-2 macroglobulin and its receptor, LRP1, on the outcomes of breast, lung, and colorectal malignancies. Immunohistochemical staining was used to analyze the expression patterns of A2M and LRP1 in 545 cases of invasive ductal breast carcinoma (IDC) and 51 cases of mastopathies/fibrocystic breast disease (FBD); 256 cases of non-small cell lung carcinomas (NSCLCs) and 45 cases of non-malignant lung tissue (NMLT); and 108 cases of CRC and 25 cases of non-malignant colorectal tissue (NMCT). A2M and LRP1 expression levels were also investigated in breast (MCF-7, BT-474, SK-BR-3, T47D, MDA-MB-231, and MDA-MB-231/BO2), lung (NCI-H1703, NCI-H522, and A549), and colon (LS 180, Caco-2, HT-29, and LoVo) cancer cell lines. Based on our findings, A2M and LRP1 exhibited various expression patterns in the examined malignancies, which were related to one another. Additionally, the stroma of lung and colorectal cancer has increased levels of A2M/LRP1 areas, which explains the significance of the stroma in the development and maintenance of tumor homeostasis. A2M expression was shown to be downregulated in all types of malignancies under study and was positively linked with an increase in cell line aggressiveness. Although more invasive cells had higher levels of A2M expression, an IHC analysis showed the opposite results. This might be because exogenous alpha-2-macroglobulin is present, which has an inhibitory effect on several cancerous enzymes and receptor-dependent signaling pathways. Additionally, siRNA-induced suppression of the transcripts for A2M and LRPP1 revealed their connection, which provides fresh information on the function of the LRP1 receptor in A2M recurrence in cancer. Further studies on different forms of cancer may corroborate the fact that both A2M and LRP1 have high potential as innovative therapeutic agents., Competing Interests: None., (AJCR Copyright © 2024.)

A checkpoint protein called the V-domain Ig suppressor of T cell activation (VISTA) is important for controlling immune responses. Immune cells that interact with VISTA have molecules, or receptors, known as VISTA receptors. Immune system activity can be modified by the interaction between VISTA and its receptors. Since targeting VISTA or its receptors may be beneficial in certain conditions, VISTA has been studied in relation to immunotherapy for cancer and autoimmune illnesses. The purpose of this study was to examine the expression levels and interactions between VISTA and its receptors, VSIG3 and PSGL-1, in breast cancer tissues. IHC analysis revealed higher levels of proteins within the VISTA/VSIG3/PSGL-1 axis in cancer tissues than in the reference samples (mastopathies). VISTA was found in breast cancer cells and intratumoral immune cells, with membranous and cytoplasmic staining patterns. VISTA was also linked with pathological grade and VSIG3 and PSGL-1 levels. Furthermore, we discovered that the knockdown of one axis member boosted the expression of the other partners. This highlights the significance of VISTA/VSIG3/PSGL-1 in tumor stroma and microenvironment remodeling. Our findings indicate the importance of the VISTA/VSIG3/PSGL-1 axis in the molecular biology of cancer cells and the immune microenvironment., (© 2024. The Author(s).)

Background: Recent studies have indicated that the skin lymphatic system and interstitium may play a role in the pathophysiology of arterial hypertension (AH)., Objectives: We aimed to determine whether the set of pathway parameters described previously in rodents would allow for the distinction between hypertensive and normotensive patients., Material and Methods: Molecular and histopathological parameters from the skin and blood of patients with AH (AH group, n = 53), resistant AH (RAH group, n = 32) and control (C group, n = 45) were used, and a statistical multivariate bootstrap methodology combining partial least squares-discriminant analysis (PLS-DA) and selectivity ratio (SR) were applied., Results: The C vs RAH model presented the best prediction performance (AUC test = 0.90) and had a sensitivity and specificity of 73.68% and 83.33%, respectively. However, the parameters selected for the C vs AH group model were the most important for the pathway described in the rodent model, i.e., greater density of the skin lymphatic vessels (D2-40 expression) and greater number of macrophages (CD68 expression), higher expression of the messenger ribonucleic acid (mRNA) of nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGFC) and podoplanin (PDPN) in the skin, greater concentration of hyaluronic acid (HA) in the skin, and lower serum concentration of VEGF-C., Conclusions: Our study suggests that the NFAT5/VEGF-C/lymphangiogenesis pathway, previously described in rodent studies, may also be present in human HA. Further experiments are needed to confirm our findings.

Purpose: Recent studies, conducted mainly on the rodent model, have demonstrated that regulatory pathway in the skin provided by glycosaminoglycans, nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGF-C) and process of lymphangiogenesis may play an important role in extrarenal regulation of sodium (Na + ) balance, body water volume, and blood pressure. We aimed to investigate the concentrations and relations among the main factors of this pathway in human skin to confirm that this regulatory axis also exists in humans., Patients and Methods: Skin specimens from patients diagnosed with arterial hypertension and from control group were histologically and molecularly examined., Results: The primary hypertensive and control groups did not differ in Na +  ​concentrations in the skin. However, the patients with hypertension and higher skin Na + concentration had significantly greater density of skin lymphatic vessels. Higher skin Na + concentration was associated with higher skin water content. In turn, skin water content correlated with factors associated with lymphangiogenesis, i.e. NFAT5, VEGF-C, and podoplanin (PDPN) mRNA expression in the skin. The strong mutual pairwise correlations of the expressions of NFAT5, VEGF-C, vascular endothelial growth factor D (VEGF-D) and PDPN mRNA were noted in the skin in all of the studied groups., Conclusions: Our study confirms that skin interstitium and the lymphatic system may be important players in the pathophysiology of arterial hypertension in humans. Based on the results of our study and existing literature in this field, we propose the hypothetical model which might explain the phenomenon of salt-sensitivity., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The transcription factor SOX18 has been shown to play a crucial role in lung cancer progression and metastasis. In this study, we investigated the effect of Sm4, a SOX18 inhibitor, on cell cycle regulation in non-small cell lung cancer (NSCLC) cell lines LXF-289 and SK-MES-1, as well as normal human lung fibroblast cell line IMR-90. Our results demonstrated that Sm4 treatment induced cytotoxic effects on all three cell lines, with a greater effect observed in NSCLC adenocarcinoma cells. Sm4 treatment led to S-phase cell accumulation and upregulation of p21, a key regulator of the S-to-G2/M phase transition. While no significant changes in SOX7 or SOX17 protein expression were observed, Sm4 treatment resulted in a significant upregulation of SOX17 gene expression. Furthermore, our findings suggest a complex interplay between SOX18 and p21 in the context of lung cancer, with a positive correlation observed between SOX18 expression and p21 nuclear presence in clinical tissue samples obtained from lung cancer patients. These results suggest that Sm4 has the potential to disrupt the cell cycle and target cancer cell growth by modulating SOX18 activity and p21 expression. Further investigation is necessary to fully understand the relationship between SOX18 and p21 in lung cancer and to explore the therapeutic potential of SOX18 inhibition in lung cancer.

Glioblastoma is one of the most aggressive tumours with a poor response to treatment and a poor prognosis for patients. One of the proteins expressed in glioblastoma tissue is CHI3L1 (YKL-40), which is upregulated and known for its angiogenesis-supporting and pro-tumour immunomodulatory effects in a variety of cancers. In this paper we present the anti-angiogenic, anti-migratory and immunomodulatory effects of the compound G721-0282, an inhibitor of CHI3L1. The inhibitor-induced changes were investigated using conventional techniques as well as the novel label-free digital holographic tomography (DHT), a quantitative phase imaging technique that allows the reconstruction of the refractive index (RI), which is used as an image contrast for 3D visualisation of living cells. DHT allowed digital staining of individual cells and intercellular structures based only on their specific RI. Quantitative spatially resolved analysis of the RI data shows that the concentration of G721-0282 leads to significant changes in the density of cells and their intracellular structures (in particular the cytoplasm and nucleus), in the volume of lipid droplets and in protein concentrations. Studies in the U-87 MG glioblastoma cell line, THP-1 monocytes differentiated into macrophages, human microvascular endothelial cells (HMEC-1) and in the spheroid model of glioblastoma composed of U-87 MG, HMEC-1 and macrophages suggest that inhibition of CHI3L1 may have potential in the antitumour treatment of glioblastoma. In this paper, we also propose a spheroid model for in vitro studies that mimics this type of tumour., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: MicroRNAs are endogenous, small noncoding RNA molecules that play a pivotal role in the regulation of gene expression. MicroRNAs are involved in many biological processes such as proliferation, cell differentiation, neovascularization, and apoptosis. Studies on microRNA expression may contribute to a better understanding of the pathomechanism of chronic inflammatory demyelinating polyneuropathy (CIDP) and consequently enable the development of new therapeutic measures using antisense miRNAs (antagomirs). In this study, we evaluated the level of miR-31-5p in the serum of patients with CIDP and its correlation with the miR-31-5p level and clinical presentation and electrophysiological and biochemical parameters., Methods: The study group consisted of 48 patients, mean age 61.60 ± 11.76, who fulfilled the diagnostic criteria of a typical variant of CIDP. The expression of miR-31-5p in patient serum probes was investigated by droplet digital PCR. The results were correlated with neurophysiological findings and the patient's clinical and biochemical parameters., Results: The mean copy number of miRNA-31 in 100  μ l serum was 1288.64 ± 2001.02 in the CIDP group of patients, while in the control group, it was 3743.09 ± 4026.90. There was a significant positive correlation (0.426) between IgIV treatment duration and miR-31-5p expression. Patients without IgIV treatment showed significantly lower levels of miR-31 compared to the treated group (259.44 ± 304.02 vs. 1559.48 ± 2168.45; p = 0.002). The group of patients with body weight > 80 kg showed statistically significantly lower levels of miRNA-31-5p than the patients with lower body weight (934.37 ± 1739.66 vs. 1784.62 ± 2271.62, respectively; p = 0.014). Similarly, the patients with elevated cerebrospinal fluid (CSF) protein levels had significantly higher miRNA-31-5p expression than those with normal protein levels (1393.93 ± 1932.27 vs. 987.38 ± 2364.10, respectively; p = 0.044)., Conclusion: The results may support the hypothesis that miR-31-5p is strongly involved in the autoimmune process in CIDP. The positive correlation between higher miR-31-5p levels and duration of IVIg treatment may be an additional factor explaining the efficacy of prolonged IVIg therapy in CIDP., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Edyta Dziadkowiak et al.)

Testin is a protein expressed in normal human tissues, being responsible, with other cytoskeleton proteins, for the proper functioning of cell−cell junction areas and focal adhesion plaques. It takes part in the regulation of actin filament changes during cell spreading and motility. Loss of heterozygosity in the testin-encoding gene results in altered protein expression in many malignancies, as partly described for cervical cancer. The aim of our study was the assessment of the immunohistochemical (IHC) expression of testin in cervical cancer and its analysis in regard to clinical data as well the expression of the Ki-67 antigen and p16 protein. Moreover, testin expression was assessed by Western blot (WB) in commercially available cell lines. The IHC analysis disclosed that the expression of testin inversely correlated with p16 (r = −0.2104, p < 0.0465) and Ki-67 expression (r = −0.2359, p < 0.0278). Moreover, weaker testin expression was observed in cancer cases vs. control ones (p < 0.0113). The WB analysis of testin expression in the cervical cancer cell lines corresponded to the IHC results and showed a weaker expression compared to that in the control cell line. When we compared the expression of testin in cervical cancer cell lines, we found a weaker expression in HPV-negative cell lines. In summary, we found that the intensity of testin expression and the number of positive cells inversely correlated with the expression of Ki-67 (a marker of proliferation) and p16 (a marker of cell cycle dysregulation). This study shows that the combined assessment of testin, Ki-67 and p16 expression may improve cervical cancer diagnostics.

Aim: To assess the effect of physical training on the selected parameters of the immune system regarding CD3, CD4, CD8, CD11, CD161, CD45A cell counts in rats treated with N-methyl-N-nitrosourea (MNU). Material and Methods: Thirty-eight female Sprague-Dawley rats were injected intraperitoneally with MNU and were divided into three groups, i.e., sedentary control (SC), the group of moderate-intensity training (MIT) and the group of high-intensity training (HIT). Physical training was supervised immediately after MNU administration and was conducted 5 days per week for 12 weeks on a three-position treadmill. Results: A significant difference was found between SC and training groups in terms of the number of induced tumors per rat (1.57 vs. 0.4, p = 0.05) and in the following lymphocyte subpopulations: CD4+/CD8+ (p = 0.01), CD3−/CD11b+ (p = 0.02), CD3−/CD161+ (p = 0.002), CD3−/CD161− (p = 0.002), CD3+/CD45RA+ (p = 0.003) and CD3−/CD45RA+ (p = 0.005). In terms of the intensity of physical training, the highest efficacy was found for MIT and the following lymphocyte subpopulations: CD3−/CD11b+ (SC vs. MIT, p < 0.001), CD3−/CD161+ (SC vs. MIT, p = 0.002), CD3−/CD161− (SC vs. MIT, p = 0.002), CD3+/CD45RA+ (SC vs. MIT, p = 0.02) and CD3−/CD45RA+ (SC vs. MIT, p < 0.001, MIT vs. HIT, p = 0.02). Furthermore, negative correlations were found between the number of apoptotic cells and CD3−/CD11b (r = −0.76, p = 0.01) in SC and between the number of induced tumors and CD3+/CD8+ (r = −0.61, p = 0.02) and between their volume and CD+/CD8+ (r = −0.56, p = 0.03) in the group of rats undergoing training. Conclusions: Physical training, particularly MIT, affected immune cell function and an altered immune response can be considered a mechanism underlying the effect of exercise on breast cancer development.

Inhaled nitric oxide (iNO) remains one of the treatment modalities in shock, and in addition to its vasoactive properties, iNO exerts immunomodulatory effects. We used a porcine model of endotoxemia with shock resuscitation (control) and additional treatment with iNO and a steroid (treatment group). After 20 h, bone marrow (BM), peripheral blood (PB), and bronchoalveolar lavage fluid (BALF) were collected to analyze the immunophenotype and mitochondrial membrane potential (Δφ) in three subsets of monocytes. In both groups, SLA-DR expression decreased twofold on the circulating CD14+CD163+ and CD14−CD163+ monocytes, while it did not change on the CD14+CD163+. Δφ increased only in the CD14−CD163+ subpopulation (0.8 vs. 2.0, p < 0.001). The analysis of compartment-specific alterations showed that nearly 100% of BALF CD14+CD163+ and CD14−CD163+ monocytes expressed SLA-DR, and it was higher compared to PB (32% and 20%, p < 0.0001) and BM (93% and 67%, p < 0.001, respectively) counterparts. BALF CD14+CD163+ had a threefold higher Δφ than PB and BM monocytes, while the Δφ of the other subsets was highest in PB monocytes. We confirmed the compartmentalization of the monocyte response during endotoxemic shock, which highlights the importance of studying tissue-resident cells in addition to their circulating counterparts. The iNO/steroid treatment did not further impair monocyte fitness.

The term "nanosilica" refers to materials containing ultrafine particles. They have gained a rapid increase in popularity in a variety of applications and in numerous aspects of human life. Due to their unique physicochemical properties, SiO 2 nanoparticles have attracted significant attention in the field of biomedicine. This study aimed to elucidate the mechanism underlying the cellular response to stress which is induced by the exposure of cells to both biogenic and pyrogenic silica nanoparticles and which may lead to their death. Both TEM and fluorescence microscopy investigations confirmed molecular changes in cells after treatment with silica nanoparticles. The cytotoxic activity of the compounds and intracellular RNS were determined in relation to HMEC-1 cells using the fluorimetric method. Apoptosis was quantified by microscopic assessment and by flow cytometry. Furthermore, the impact of nanosilica on cell migration and cell cycle arrest were determined. The obtained results compared the biological effects of mesoporous silica nanoparticles extracted from Urtica dioica L. and pyrogenic material and indicated that both types of NPs have an impact on RNS production causing apoptosis, necrosis, and autophagy. Although mesoporous silica nanoparticles did not cause cell cycle arrest, at the concentration of 50 μg/mL and higher they could disturb redox balance and stimulate cell migration.

Non-small cell lung cancer (NSCLC) is a subtype of the most frequently diagnosed cancer in the world. Its epidemiology depends not only on tobacco exposition but also air quality. While the global trends in NSCLC incidence have started to decline, we can observe region-dependent differences related to the education and the economic level of the patients. Due to an increasing understanding of NSCLC biology, new diagnostic and therapeutic strategies have been developed, such as the reorganization of histopathological classification or tumor genotyping. Precision medicine is focused on the recognition of a genetic mutation in lung cancer cells called "driver mutation" to provide a variety of specific inhibitors of improperly functioning proteins. A rapidly growing group of approved drugs for targeted therapy in NSCLC currently allows the following mutated proteins to be treated: EGFR family (ERBB-1, ERBB-2), ALK, ROS1, MET, RET, NTRK, and RAF. Nevertheless, one of the most frequent NSCLC molecular sub-types remains without successful treatment: the K-Ras protein. In this review, we discuss the current NSCLC landscape treatment focusing on targeted therapy and immunotherapy, including first- and second-line monotherapies, immune checkpoint inhibitors with chemotherapy treatment, and approved predictive biomarkers.

Non-small cell lung cancer (NSCLC) is the most commonly diagnosed cancer and the most frequent cause of cancer-associated mortality worldwide. Tesmin (MTL5) is a 60 kDa protein which has cysteine rich motifs, characteristic of metallothioneins. Tesmin expression was first observed in germ cells during spermatogenesis. Increased tesmin expression in NSCLC has been described previously. Minichromosome maintenance proteins (MCMs) serve a critical role in replication and cell cycle progression, i.e. in NSCLC. The aim of the present study was to evaluate the localization and intensity of tesmin, MCM5 and MCM7 protein expression in NSCLC and their association with the clinicopathological data of patients. Archival paraffin blocks of 243 cases of NSCLC and 104 non-cancerous tissue samples from the surgical margin (control) were obtained from patients treated at the Clinic of Thoracic Surgery of Wroclaw Medical University (Wroclaw, Poland) between 2010 and 2016, and were used for tissue microarrays and immunohistochemical (IHC) experiments. Laser capture microdissection was used for the isolation of cancer cells from 36 frozen samples of NSCLC and 8 control samples, and subsequently, MTL5, MCM5 and MCM7 mRNA expression was detected separately by reverse transcription-quantitative PCR. Positive cytoplasmic and nuclear tesmin, as well as nuclear MCM5 and MCM7 IHC expression were observed in 95.1, 83.67, 95.51 and 100% of the NSCLC cases, respectively. MTL5, MCM5 and MCM7 mRNA expression was observed in 91.66% of the cancer cases for all genes. The statistical analysis revealed increased tesmin IHC expression in cancer cells compared with the control. A positive correlation was observed between the IHC expression of nuclear tesmin and MCM5 proteins (r=0.33; P<0.0001) and nuclear tesmin and MCM7 proteins (r=0.315; P<0.0001). In addition, a positive correlation between the mRNA expression levels of MTL5 and MCM5 (r=0.421; P<0.05), MTL5 and MCM7 (r=0.557; P<0.01) was demonstrated. The survival analysis revealed that the presence of IHC cytoplasmic tesmin expression was a positive prognostic marker in NSCLC (P=0.0524). Furthermore, in vitro experiments performed on the NCI-H1703 cell line revealed that silencing of MTL5 mRNA and tesmin caused the downregulation of the expression levels of MCM5 and MCM7 and decreased the number of cells in the G2 phase. A positive association among tesmin, MCM5 and MCM7 could indicate a possible role of tesmin in the proliferation of NSCLC cancer cells., (Copyright: © Grzegrzolka et al.)

The SOX family proteins are proved to play a crucial role in the development of the lymphatic ducts and the cardiovascular system. Moreover, an increased expression level of the SOX18 protein has been found in many malignances, such as melanoma, stomach, pancreatic breast and lung cancers. Another SOX family protein, the SOX30 transcription factor, is responsible for the development of male germ cells. Additionally, recent studies have shown its proapoptotic character in non-small cell lung cancer cells. Our preliminary studies showed a disparity in the amount of mRNA of the SOX18 gene relative to the amount of protein. This is why our attention has been focused on microRNA (miRNA) molecules, which could regulate the SOX18 gene transcript level. Recent data point to the fact that, in practically all types of cancer, hundreds of genes exhibit an abnormal methylation, covering around 5-10% of the thousands of CpG islands present in the promoter sequences, which in normal cells should not be methylated from the moment the embryo finishes its development. It has been demonstrated that in non-small-cell lung cancer (NSCLC) cases there is a large heterogeneity of the methylation process. The role of the SOX18 and SOX30 expression in non-small-cell lung cancers (NSCLCs) is not yet fully understood. However, if we take into account previous reports, these proteins may be important factors in the development and progression of these malignancies.

A neoplastic tumor consists of cancer cells that interact with each other and non-cancerous cells that support the development of the cancer. One such cell are tumor-associated macrophages (TAMs). These cells secrete many chemokines into the tumor microenvironment, including especially a large amount of CCL18. This chemokine is a marker of the M2 macrophage subset; this is the reason why an increase in the production of CCL18 is associated with the immunosuppressive nature of the tumor microenvironment and an important element of cancer immune evasion. Consequently, elevated levels of CCL18 in the serum and the tumor are connected with a worse prognosis for the patient. This paper shows the importance of CCL18 in neoplastic processes. It includes a description of the signal transduction from PITPNM3 in CCL18-dependent migration, invasion, and epithelial-to-mesenchymal transition (EMT) cancer cells. The importance of CCL18 in angiogenesis has also been described. The paper also describes the effect of CCL18 on the recruitment to the cancer niche and the functioning of cells such as TAMs, regulatory T cells (T reg ), cancer-associated fibroblasts (CAFs) and tumor-associated dendritic cells (TADCs). The last part of the paper describes the possibility of using CCL18 as a therapeutic target during anti-cancer therapy.

Lung cancer is the most commonly diagnosed cancer and the most frequent cause of death worldwide. Tesmin (testis‑specific metallothionein‑like protein; MTL‑5) is a 60‑kDa protein which has cysteine‑rich motifs (CXC domain), characteristic of metallothioneins (MTs). Tesmin expression has been observed in germ cells during spermatogenesis, oogenesis and also in various cell nuclei after exposure to heavy metal ions. Yet, the role of tesmin in carcinogenesis is unknown. The aim of the present study was to evaluate the localization and intensity of tesmin expression in non‑small cell lung cancer (NSCLC) and its association with the clinicopathological data of patients. A total of 121 cases of NSCLC and 20 cases of non‑cancerous tissue samples from the surgical margin (control) were used for immunohistochemistry (IHC). In addition, 20 cases of frozen NSCLC tissues and 20 cases of control were used for the in vivo study. Normal lung fibroblasts (IMR‑90) and lung cancer cell lines NCI‑H1703 (lung squamous cell carcinoma), NCI‑H522 and A549 (both adenocarcinomas of the lung) were used for western blot analysis (WB) and RT‑PCR studies. Positive cytoplasmic tesmin expression was observed in 88.42% of the examined cases of NSCLC. Statistical analysis showed increased IHC tesmin expression in cancer cells compared to that noted in the controls. In addition, MTL5 mRNA and WB tesmin protein expression were also higher in cancer cases compared to the controls. A positive correlation between tesmin and Ki‑67 IHC expression was demonstrated (r=0.32; P<0.001). Higher WB tesmin expression was also associated with shorter overall survival (P<0.05, Mantel‑Cox test). The in vitro study revealed higher tesmin protein (WB) and MTL5 (qPCR) in lung cancer cell lines compared to the lung fibroblast control cell line. Higher tesmin expression in cancer cells compared to control cells may suggest a role of tesmin in NSCLC carcinogenesis. A positive correlation between tesmin and Ki‑67 could indicate a possible role of tesmin in the proliferation of NSCLC.

Background/aim: The expression of metallothionein I/II (MT-I/II) was examined in thyroids of Graves' disease (GD) and nodular goiter (NG) patients to determine its role as a potential marker of proliferation and autoimmune inflammation in the thyroid., Patients and Methods: MT-I/II and Ki-67 antigen expression was studied using immunohistochemistry in 72 GD and 24 NG patients., Results: MT-I/II expression was noted in the cytoplasm and nuclei of thyrocytes of GD and NG patients. Cytoplasmic and nuclear MT-I/II expression correlated strongly with GD (r=0.51; p<0.0001) and NG (r=0.50; p=0.0137). Cytoplasmic MT-I/II expression was significantly higher in GD (mean IRS 9.24±2.36) than in NG (mean IRS 7.13±2.51; p=0.0006) and correlated positively with Ki-67 antigen expression (r=0.28; p=0.0165). Nuclear MT-I/II expression was elevated in GD (mean 3.53±0.65) in comparison to NG (mean 2.96±0.86; p=0.028)., Conclusion: MT-I/II may be a potential marker of GD in the thyroid and may be potentially involved in thyrocytes' proliferation., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

There is a high demand for anticancer drugs due to the fact that the chemotherapeutics currently used have numerous side effects, which lowers the patient's quality of life. However, the latest antibody therapies are extremely expensive, hence the requirement to identify novel, equally effective but low-toxic treatments that have limited side effects. As a result of this, a number of research centres around the world are attempting to identify novel molecular markers that could be effective targets for anticancer therapy in the future. The SOX18 protein has been suggested to be a significant diagnostic and prognostic marker in various types of cancer. SRY-related HMG-box 18 (SOX18) is an important transcription factor involved in the development of cardiovascular and lymphatic vessels during embryonic development. In addition, it is involved in the progression of atherosclerosis and wound-healing processes. It has been observed that its level is higher in a number of cancer types, including melanoma, pancreas, stomach, liver, breast, lung, ovarian and cervical cancer. Furthermore, an association between a high expression of SOX18 in gastric cancer stromal cells and a poor prognosis has been demonstrated. The literature indicates how complex the pathogenesis of cancer is. Knowing the molecular basis of the pathogenesis of the tumor will allow for the effective use of targeted therapy, which may result in a higher success in treating patients. It is therefore important to identify novel and effective therapies as well as new proteins that could be potential markers. The SOX18 family, represented by the SOX18 protein, seems to be in this respect a promising element in modern anticancer therapy.

Background: Non-small cell lung carcinomas (NSCLCs), mainly adenocarcinoma (AC) and squamous cell carcinoma (LSCC), account for about 80% of all lung cancer cases. One of the proteins involved in NSCLC progression may be special AT-rich binding protein 1 (SATB1), a potent transcriptional regulator, able to control the expression of whole sets of genes simultaneously. SATB1 has been found to be associated with aggressive phenotype and poor prognosis in numerous malignancies, including breast, colon, ovary and prostate cancer. However, its role in NSCLC is still not fully understood. The aim of this study was to investigate the expression of SATB1 protein and mRNA in NSCLC and non-malignant lung tissue (NMLT) samples, as well as to determine possible relationships of SATB1 expression with both the expression of Ki-67 and the clinicopathological data of the patients., Materials and Methods: The study was performed on 277 NSCLC (158 AC, 119 LSCC) and 20 NMLT samples., Results: We observed increased SATB1 immunoreactivity in NSCLC when compared to NMLT, and in LSCC when compared to AC cases. We also noted that an elevated SATB1 immunoreactivity was associated with a poor degree of AC differentiation, whereas in LSCC, an inverse relationship was observed. Our analyses revealed that the expression of SATB1 positively correlated with Ki-67 index in NSCLC and LSCC, but not in AC cases. Finally, we found that high SATB1 expression was associated with a better overall survival of patients with NSCLC., Conclusion: SATB1 plays diverse roles in different NSCLC subtypes, and its expression may have a prognostic significance for patients with these tumours., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

This study evaluated the effects of inhaled nitric oxide (iNO) therapy combined with intravenous (IV) corticosteroids on hemodynamics, selected cytokines, and kidney messenger RNA toll-like receptor 4 (mRNA TLR4) expression in ischemia-reperfusion injury animal model. The primary endpoint was the evaluation of circulatory, respiratory, and renal function over time. We also investigated the profile of selected cytokines and high-mobility group box 1 (HMGB1) protein, as well as renal mRNA TLR4 activation determined by quantitative real-time polymerase chain reaction analysis. Pigs (n = 19) under sevoflurane AnaConDa anesthesia/sedation were randomized and subjected to abdominal laparotomy and alternatively suprarenal aortic cross-clamping (SRACC) for 90 min or sham surgery: Group 1 (n = 8) iNO (80 ppm) + IV corticosteroids (25 mg ×3) started 30 min before SRACC and continued 2 h after SRACC release, followed with decreased iNO (30 ppm) until the end of observation, Group 2 (n = 8) 90 min SRACC, Group 3 (n = 3)-sham surgery. Renal biopsies were sampled 1 hr before SRACC and at 3 and 20 h after SRACC release. Aortic clamping increased TLR4 mRNA expression in ischemic kidneys, but significant changes were recorded only in the control group ( P = 0.016). Treatment with iNO and hydrocortisone reduced TLR4 mRNA expression to pre-ischemic conditions, and the difference observed in mRNA expression was significant between control and treatment group after 3 h ( P = 0.042). Moreover, animals subjected to treatment with iNO and hydrocortisone displayed an attenuated systemic inflammatory response and lowered pulmonary vascular resistance plus increased oxygen delivery. The results indicated that iNO therapy combined with IV corticosteroids improved central and systemic hemodynamics, oxygen delivery, and diminished the systemic inflammatory response and renal mRNA TLR4 expression.

Bone marrow adipocytes (BMAs) derived from mesenchymal stem cells (MSC) are an active and significant element of the bone marrow microenvironment. They are involved in metabolic functions, complex interactions with other stromal cells, and in the development and progression of tumours. Currently, there is little data regarding the role of BMAs in haematological malignancies. Due to this, we have attempted to characterise the BMAs in these malignancies in terms of quantity and morphology. Our study included 30 patients aged 22-76 with myelo- (n=17) and lymphoproliferative malignancies (n=13), both with and without bone marrow infiltration. Trepanobioptate was the evaluated material. The number and diameter of BMAs were measured, and the percentage of adipocytes (adipocyte fraction - AF), hematopoietic cells (hematopoietic fraction - HF) and trabecular bone (trabecular bone fraction - BF) was calculated. The obtained results were considered against the clinical parameters of age, sex, body weight, body surface area (BSA) and body mass index (BMI). We observed that as age increases, the number of BMA/mm 2 , the diameter of adipocytes and AF increase while BF and HF decrease. However, this relationship was not statistically significant. A significant correlation of BMA parameters was also not found in relation to weight, BMI and BSA, and the number and diameter of BMAs were comparable in both sexes. The trepanobioptate of infiltrated bone marrow showed a decreased number of BMA/mm 2 compared to the trepanobioptate from bone marrow without infiltration (97.44±69.16 vs. 164.14±54.16; p=0.010) with a marked difference in men (69.75±65.26 vs. 180.33±60.40; p=0.007). These trepanobioptate also showed an increase in the number of BMA/mm 2 with age (r=0.472; p=0.041), and with an increase of BMI, an increase in diameter of BMAs (r=0.625; p=0.007) and AF (r=0.546; p=0.023). The number and size of BMAs, as well as AF, BF and HF in patients with myeloproliferative malignancies did not differ significantly compared to patients with lymphoproliferative malignancies., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The molecular pathogenesis of the development of non-small cell lung carcinomas (NSCLCs) is extremely complex. Understanding the molecular basis of the development of this malignant tumor may enable the use of targeted therapy, which may result in a better treatment outome for these patients. Adenocarcinoma (AC) is the most common NSCLC subtype, equally common among smokers and non-smokers, and its pathogenesis remains unknown. The SOX18 protein is an important protein that plays a role in the development of blood and lymphatic vessels during the process of embryogenesis. Recent studies have also shown that the SOX18 protein may play a significant role in tumors, including lung cancers. In the present study, we analyzed the expression of the SOX18 protein and the mRNA level in postoperative samples of AC and non-malignant lung tissues (NMLTs), and a disparity in both levels was observed. Based on our previous observations that miR-7a and miR-24-3p are able to modulate SOX18 expression in NSCLC, the main aim of this study was to verify the miRNA modulation of the SOX18 transcript with the use of the MirTrap System in established lung cancer cell lines NCI-H1703, NCI-H522 and A549. The SOX18 mRNA expression level was significantly lower in AC than that noted in the NMLTs (P<0.0001). However, the protein levels were higher in AC cases compared to levels noted in the NMLTs (P<0.0001). Additionally, correlations between the RQ values of SOX18 in NMLT and AC cases (r=0.8195, P=0.0001), and between miR-7a and miR24-3p in AC cases (r=0.4344, P=0.0016), were noted. In conclusion, we confirmed that miR-7a and miR-24-3p are more highly expressed in NMLTs than in the AC samples, and that they modulate the SOX18 transcript in NSCLC cells.

Background: Metallothioneins (MTs) are involved in numerous cell processes such as binding and transport of zinc and copper ions, differentiation, proliferation and apoptosis, therefore contributing to carcinogenesis. Scarce data exist on their expression in benign and malignant lesions of the thyroid., Materials and Methods: mRNA expression of functional isoforms of MT genes (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT4) was studied in 17 nodular goiters (NG), 12 follicular adenomas (FA) and 26 papillary thyroid carcinomas (PTC)., Results: One-way ANOVA revealed significant differences in mRNA expression levels of MT1A (p<0.05), MT1E (p<0.005), MT1F (p<0.0001), MT1G (p<0.005), MT1X (p<0.0005) and MT2A (p<0.005) in the analyzed samples. Post hoc analysis confirmed a significantly lower expression of MT1A mRNA in PTC compared to NG (p<0.05). Significant down-regulation was also noted for other MT isoforms in PTC in comparison to NG: MT1E (p<0.05), MT1F (p<0.0001), MT1G (p<0.005), MT1X (p<0.0005) and MT2A (p<0.05). In addition, significant down-regulation of MT1F and MT1G in FA compared to NG was observed (p<0.005 and p<0.05, respectively)., Conclusion: Expression of functional MT isoforms may contribute to thyroid carcinogenesis and potentially serve as a diagnostic marker in distinguishing benign and malignant lesions., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The deregulation of the cell cycle is crucial for the processes of carcinogenesis and tumor progression. The cell cycle is under the control of cyclin-dependent kinases (CDKs) and their inhibitors (CDKIs). Proteins p16 and p27 are CDKIs, and their altered expression has been noticed in many types of cancers. The aim of this study has been to compare the expression levels of these proteins in laryngeal papillomas and carcinomas in order to confirm their predictive value in cancer development., Materials and Methods: Eighty-three cases of laryngeal lesions were analyzed: cancers (51) and papillomas (32). Immunohistochemical (IHC) reactions were performed in order to detect the expression levels of p16, p27 and Ki-67. Immunofluorescence (IF) and western blot were performed on HEp-2 cell line., Results: The expression of p16 and p27 was decreased in cancer cells in comparison to papillomas. Additionally, we observed cytoplasmic expression of p16 in cancers and lack of such expression in papillomas. The same pattern of expression of p16 was also detected by IF and western blot analysis in HEp-2 cell line. The expression of Ki-67 protein revealed no significant differences in the case of papilloma compared with cancer, however a significant correlation between tumor grade and the nuclear expression of Ki-67 was observed., Conclusion: There is a necessity in finding biomarkers, which would predict the risk level of malignant transformation. Our study has confirmed that altered expressions of the p16 and p27 proteins might be useful biomarkers in the progression of laryngeal squamous cell carcinomas (LSCC)., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Recent statistics show that lung cancer is the second most common malignant tumor in the world (14% of all cancers in the USA), both in terms of morbidity and mortality. The mortality of this type of tumor shows an increasing trend (28% for men and 26% for women). Lung squamous cell carcinoma (LSCC) is the second‑largest histological subtype of non‑small cell lung cancers (NSCLCs) after adenocarcinoma. SRY‑related HMG‑box 18 (SOX18) protein is an important transcription factor involved in the development of the cardiovascular system and the lymphatic ducts. In addition, it was observed that SOX18 functions in wound healing processes and the development of atherosclerosis. Likewise, an increased level of this protein was found in melanomas and malignant pancreatic, stomach and breast tumors. Furthermore, high expression of SOX18 in gastric cancer stromal cells was found to be associated with a poor patient prognosis. In the present study, we analyzed the expression of the SOX18 protein and the mRNA level in postoperative samples of LSCC and non‑malignant lung tissues (NMLTs), and a disparity in both levels was observed. Because of the fact that microRNAs (miRNAs) play important roles in the initiation and progression of lung cancer, the main aim of this study was to identify the miRNAs that interact with the SOX18 transcript in NSCLC cases. SOX18 mRNA expression level was significantly lower in the LSCC tissues than that noted in the NMLTs (p<0.01). However, protein levels were higher in the LSCC cases compared to these levels in the NMLTs (p<0.0001). We showed that miR‑7a and miR‑24‑3p were expressed more highly in the NMLTs than levels in the LSCC samples, and that they could be switched off in lung cancer tissue. Additionally, correlations between RQ‑values of SOX18 in NMLTs and LSCC samples (r=0.43, p=0.019), and between miR‑7a and miR24‑3p in NMLT cases (r=0.4, p=0.057) as well as in the LSCC samples (r=0.51, p=0.012) were noted. In conclusion, miRNAs interact with the mRNA of the SOX18 gene, but the mechanism by which they could be inhibited in cancer cells requires further examination.

The phenomenon of cancer cell resistance to chemotherapeutics is the main cause of insensitivity to anticancer therapy. Thus, the current challenge remains searching for substances sensitising the activity of cytostatic drugs. In this respect, resveratrol is a very promising therapeutic agent. It has pleiotropic effect on cancer cells, which can play a key role in numerous resistance mechanisms, both classical and atypical. The purpose of the present study was to assess the effect of resveratrol on the inhibition of human pancreatic cancer cell proliferation and on the level of cytostatic resistance-associated proteins. The study was performed on human pancreatic cancer cell lines EPP85-181P (control), EPP85-181RDB (daunorubicin resistance) and EPP85-181PRNOV (mitoxantrone resistance). The effect of resveratrol on the viability and proliferation of the studied cell lines was evaluated by SRB assay, whereas cell cycle arrest and cytostatic accumulation by FACS. Western blot analysis was used to determine the level of P-glycoprotein, topoisomerase II α and β and immunofluorescence technique to visualise the proteins in the cells. Resveratrol inhibited proliferation of all studied cell lines. Phase-specific cell cycle arrest depended on the type of cancer cells. Resveratrol decreased the level and activity of P-gp in EPP85-181RDB cells. In EPP85-181PRNOV cells, expression of both TopoII isoforms increased in a statistically significant manner. The results of in vitro studies support the possibility of potential use of resveratrol in breaking cancer cell resistance to chemotherapeutic drugs.

Background/aim: Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma (CTCL) characterized by malignant proliferation of mature T lymphocytes and primary skin involvement. Recent reports suggest that angiogenesis may play a role in the growth and spread of this malignancy. Cluster of differentiation 31 (CD31) is a protein classified into the Ig-superfamily of cell adhesion molecules, expressed on endothelial cells and majority of hematopoietic non-erythroid cells. The aim of our study was to explore the role of angiogenesis in MF., Materials and Methods: We evaluated the expression of CD31 in relation to clinicopathological data and potential impact on patients' outcome in MF utilizing immunohistochemistry (IHC) and western blot (WB) techniques in 73 and 19 MF and 21 and 4 control samples, respectively., Results: In the IHC study, statistical analysis revealed significantly higher CD31 expression in MF compared to the controls (p<0.0001) with highest expression in advanced stages vs. early ones and controls (p<0.0001 for both). In regard to skin involvement, expression was also elevated in more infiltrative (T3, tumors) and in more extensive (T4, erythroderma) cutaneous lesions compared to less infiltrative and limited skin lesions (T1, T2, patches and/or plaques) (p<0.01 for both). Regarding the extracutaneous spread, higher CD31 expression correlated with nodal involvement (N1-3 vs. N0; p<0.0001). In the WB study, statistical analysis revealed significantly higher CD31 expression only in advanced vs. early stage of MF (p<0.05). In regard to skin involvement, expression was also elevated in T3 and T4 as compared T1+T2 (p=0.08, p<0.05; respectively). Higher CD31 expression correlated with nodal involvement (N1-3 vs. N0; p<0.01). A strong significant correlation between CD31 expression at the protein level analyzed by IHC and WB was noticed (r=0.802, p<0.0001). Moreover, strong positive correlations between IHC expression of CD31 and podoplanin (PDPN; r=0.582, p<0.0001), vascular endothelial growth factor C (VEGFC; r=0.332, p<0.01) and Ki-67 (r=0.330, p<0.01) were disclosed., Conclusion: Expression of CD31 in MF skin biopsies provides new evidence for the role of angiogenesis in the progression of MF. Additionally, the new data revealed prompts for further research on potential use of CD31 as a new marker of the disease advancement, as well as the target of new therapeutic strategies., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Many studies are dedicated to exploring the molecular mechanisms of chemotherapy-resistance in breast cancer (BC). Some of them are focused on searching for candidate genes responsible for this process. The aim of this study was typing the candidate genes associated with the response to standard chemotherapy in the case of invasive ductal carcinoma. Frozen material from 28 biopsies obtained from IDC patients with different responses to chemotherapy were examined using gene expression microarray, Real-Time PCR (RT-PCR) and Western blot (WB). Based on the microarray results, further analysis of candidate gene expression was evaluated in 120 IDC cases by RT-PCR and in 224 IDC cases by immunohistochemistry (IHC). The results were correlated with clinical outcome and molecular subtype of the BC. Gene expression microarray revealed Prolactin-Induced Peptide (PIP) as a single gene differentially expressed in BC therapy responder or non-responder patients (p <0.05). The level of PIP expression was significantly higher in the BC therapy responder group than in the non-responder group at mRNA (p=0.0092) and protein level (p=0.0256). Expression of PIP mRNA was the highest in estrogen receptor positive (ER+) BC cases (p=0.0254) and it was the lowest in triple negative breast cancer (TNBC) (p=0.0336). Higher PIP mRNA expression was characterized by significantly longer disease free survival (DFS, p=0.0093), as well as metastasis free survival (MFS, p=0.0144). Additionally, PIP mRNA and PIP protein expression levels were significantly higher in luminal A than in other molecular subtypes and TNBC. Moreover significantly higher PIP expression was observed in G1, G2 vs. G3 cases (p=0.0027 and p=0.0013, respectively). Microarray analysis characterized PIP gene as a candidate for BC standard chemotherapy response marker. Analysis of clinical data suggests that PIP may be a good prognostic and predictive marker in IDC patients. Higher levels of PIP were related to longer DFS and MFS but not with OS.

Background: Metallothioneins (MTs) are low-molecular-weight and cysteine-rich proteins that bind heavy metal ions and oxygen-free radicals. MTs are commonly expressed in various tissues of mammals and are involved in regulation of cell proliferation and differentiation, and may be engaged in angiogenesis. Expression of MTs has been studied in many cancer types, especially breast cancer. The research results indicate that MTs may play important, although not yet fully known, roles in cancer angiogenesis. The aim of this study was to analyze the level of gene expression of selected MT isoforms induced with zinc ions in correlation with vascular endothelial growth factor (VEGF) isoforms in in vitro models of breast cancer., Materials and Methods: The studies were carried out in three breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231). An epithelial cell line derived from normal breast tissue (Me16c) was used as a control. The levels of expression of selected MT isoforms and selected genes involved in angiogenesis were studied with real-time PCR., Results: Expression of different MT isoforms was induced by zinc ions to differing degrees in individual breast cancer cell lines. An increase in the expression of some MT isoforms was associated with a slight increase in the level of expression of VEGFA., Conclusion: The research results may indicate certain correlation between an increased expression of selected MT isoforms and a pro-angiogenic factor VEGF in specific types of breast cancer cells., (Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: The epithelial-mesenchymal transition (EMT) has been observed in progression of in situ breast cancer to the invasive form and might be initiated by snail family zinc finger 2 (SLUG) and twist family bHLH transcription factor 1 (TWIST) protein overexpression. During this phenomenon, cells lose their epithelial phenotype and acquire mesenchymal features. The aim of the study was to examine the association of EMT markers SLUG and TWIST with clinicopathological data and the possibility of using these proteins as prognostic markers of breast cancer., Materials and Methods: Immunohistochemical analysis (IHC) of SLUG and TWIST expression was performed on archival paraffin samples of 19 cases with fibrocystic breast changes (control group), 148 cases of invasive ductal breast cancer (IDC) and 26 of invasive lobular breast cancer (ILC). Laser capture microdissection for isolation of cells from 17 frozen samples of IDC was employed and subsequently SLUG and TWIST mRNA expression in cancer and stromal cells was detected separately by real-time polymerase chain reaction., Results: SLUG and TWIST expression in IDC was significant higher in stromal cells regardless of the method of quantification used (p<0.001 for SLUG mRNA, and p<0.0001 for SLUG IHC, TWIST IHC and TWIST mRNA expression). Positive correlation of SLUG and TWIST protein and mRNA expression was observed in stromal cells of IDC (r=0.347; p<0.0001 and r=0.704; p<0.01, respectively). Expression of TWIST protein in IDC was higher in cancer cells of cases with shorter event-free survival period, as well as in stromal cells of cases with shorter overall survival period (p<0.05 for both)., Conclusion: Stromal cells could play a role in the regulation of EMT in breast cancer., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients' shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases.

Recent studies have demonstrated the involvement of SOX18 transcription factor in blood and lymphatic vessel development, as well as in wound healing processes. SOX18 expression has been noted in cancer cells of various tumours, including lung cancer. However, the exact role of SOX18 expression in non-small cell lung cancer (NSCLC) remains to be determined. The present study, therefore, assessed its expression in 198 cases of NSCLC, consisting of 94 adenocarcinomas (AC), 89 squamous cell carcinomas (SQC) and 15 large cell carcinomas (LCC). The analysis utilized immunohistochemistry (IHC) and, in 42 cases, molecular methods. SOX18 expression was also determined in NSCLC cell lines (NCI-H1703, NCI-H522 and A549) and in normal lung fibroblasts (IMR-90). SOX18 was found to be expressed in nuclei, as well as in the cytoplasm of cancer cells, in the majority of studied cases. SOX18 mRNA expression was significantly lower in NSCLC than in non-malignant lung tissue (p<0.0001). However, SOX18 protein expression levels were higher in NSCLC tissues (p<0.005) and in the examined lung cancer cell lines. No SOX18 expression was noted in the IMR-90 cell line. In paraffin sections, a positive correlation between the Ki-67 antigen and nuclear SOX18 expression (r=0.17, p<0.05) was noted. In univariate survival analysis, cytoplasmic SOX18 expression correlated with poor patient outcome in the whole study and in AC cohorts (both p<0.05). Based on these results, SOX18 may be involved in the progression of NSCLC.

Background: Nogo-B receptor (NgBR) has been shown to be involved in endothelial cell chemotaxis and morphogenesis. However, few studies analyzing its expression in cancer cells have been performed., Materials and Methods: We examined NgBR expression in 233 patients with invasive ductal breast carcinoma (IDC) and corresponding non-malignant breast tissues (NMBT) on mRNA (real-time polymerase chain reaction) and protein levels (immunohistochemistry; IHC and western-blot analysis). NgBR expression was found also analyzed in breast cancer cell lines of varying invasiveness., Results: NgBR expression was increased in IDC compared to NMBT on the mRNA (p=0.0007) and protein level (p=0.018). NgBR expression decreased significantly with IDC malignancy grade and correlated negatively with the Ki-67 antigen expression (r=-0.18; p=0.0005). High NgBR mRNA expression was associated with estrogen receptor negativity (p=0.0023) and the triple-negative phenotype of the tumors (p=0.0129)., Conclusion: NgBR may be involved in IDC development, however, its role in its progression requires further research., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Nogo-B was recently shown to be involved in proliferation, apoptosis and invasiveness of cancer cells, whereas its specific receptor (NgBR) was found to be up-regulated in estrogen receptor-α positive breast cancer. No data are currently available concerning their expression in non-small cell lung carcinomas (NSCLC)., Materials and Methods: Expression of Nogo isoforms and NgBR was studied in 191 NSCLC., Results: Higher Nogo-A/B immunoreactivity was noted in cancer cells of squamous cell carcinomas (SQC) compared to adenocarcinomas (p<0.001). Stage II-IV tumors had the lowest Nogo-A/B expression (p<0.0001) compared to stage I cases. Nogo-A/B expression decreased with increasing SQC malignancy grade (p=0.026). Significant NgBR mRNA down-regulation was associated with larger primary tumor size (p=0.039), lymph node involvement (p=0.039) and advancement stage (p=0.0054). Low NgBR mRNA expression predicted poor patients outcome (p=0.029)., Conclusion: The current data may point to the involvement of Nogo isoforms and NgBR in the pathogenesis of NSCLC., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: SOX18 is a transcription factor known to be involved in blood and lymphatic vessel, hair follicle development, and wound healing processes. In addition, it has been reported that SOX18 may influence cancer growth. The role of SOX18 expression in ovarian cancer (OC) has not been determined., Materials and Methods: SOX18 expression was assessed in 85 OC cases using immunohistochemical methods and in ovarian cancer cell lines on the mRNA and protein level., Results: SOX18 was expressed in cancer cell nuclei as well as the cytoplasm. Higher nuclear SOX18 expression was associated with presence of residual disease following surgical treatment (p=0.0158) and advanced disease stage (p=0.0056). Univariate survival analysis revealed that high SOX18 (p=0.0125) expression, presence of residual disease (p<0.0001) and advanced disease stage (p<0.0324) predicted poor patient outcome., Conclusion: SOX18 may be a new predictive marker for OC., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: SOX18 is a transcription factor known to be involved in hair follicle, blood and lymphatic vessel development, as well as wound healing processes (together with SOX7 and SOX17). In addition, it has been reported that SOX18 may affect the growth of cancer cells in vitro. Until now, the exact role of SOX18 expression in invasive ductal breast carcinoma (IDC) has remained unknown., Methods: In this study, we have investigated SOX18 expression in cancer cells and endothelial cells in 122 IDC samples using immunohistochemistry (IHC). SOX18 expression was also determined using real-time PCR and Western blotting in a series of breast cancer-derived cell lines (i.e., MCF-7, BT-474, SK-BR-3, MDA-MB-231, BO2)., Results: Using IHC, we observed SOX18 nuclear expression in cancer cells, as well as in blood and lymphatic vessels of the IDC samples tested. SOX18 expression in the IDC samples correlated with a higher malignancy grade (Grade 2 and Grade 3 versus Grade 1; p = 0.02 and p = 0.009, respectively) and VEGF-D expression (r = 0.27, p = 0.007). SOX18 expression was also associated with HER2 positivity (p = 0.02). A significantly higher SOX18 expression was found in the HER2-positive cell line BT-474, and a significantly lower expression in the triple negative cell lines MDA-MB-231 and BO2. Laser capture microdissection of IDC samples revealed significantly higher mRNA SOX7, SOX17 and SOX18 expression levels in the vessels as compared to the cancer cells (p = 0.02 and p = 0.0002, p < 0.0001, respectively). SOX18 positive intratumoral and peritumoral microvessel counts (MVC) were associated with higher malignancy grades (p = 0.04 and p = 0.02, respectively). Moreover, peritumoral SOX18 positive MVC were found to act as an independent marker for a poor prognosis (p = 0.04)., Conclusion: SOX18 expression may serve as a marker for a poor prognosis in IDC.

The renin-angiotensin system (RAS) is known mainly as a regulator of cardiovascular homeostasis. However, it has also been shown to mediate processes such as proliferation, apoptosis, angiogenesis, and carcinogenesis. Nonmelanoma skin cancers (NMSC) - including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) - are among the most common cancers. The aim of the present study was to determine the immunohistochemical expression of angiotensin-converting enzyme (ACE), angiotensin-converting enzyme 2 (ACE2), and Ki-67 antigen in archival samples of normal skin, actinic keratosis, and malignant skin lesions. Cytoplasmic-nuclear ACE immunoreactivity was observed in 99% of examined cases of both normal skin and cancers. Significantly higher ACE immunoreactivity occurred in normal skin, as compared with BCC and SCC (p < 0.01, p < 0.0001, respectively). Additionally, ACE immunoreactivity was also significantly higher in BCC, compared with SCC (p < 0.05). ACE2 immunoreactivity was noted in basal epidermal layers and in sebaceous gland cells in normal skin, though not in NMSC. These novel observations suggest that ACE and skin RAS may be involved in the pathogenesis of malignant skin lesions.