Publications by authors named "Joanna Dulińska-Litewka"

33 Publications

Recent Progress in Discovering the Role of Carotenoids and Their Metabolites in Prostatic Physiology and Pathology with a Focus on Prostate Cancer-A Review-Part I: Molecular Mechanisms of Carotenoid Action.

Antioxidants (Basel) 2021 Apr 10;10(4). Epub 2021 Apr 10.

Nutrition and Health Research Group, Department of Population Health, Luxembourg Institute of Health, 1 A-B, rue Thomas Edison, L-23 1445 Strassen, Luxembourg.

Among the vast variety of plant-derived phytochemicals, the group of carotenoids has continuously been investigated in order to optimize their potential application in the area of dietary intervention and medicine. One organ which has been especially targeted in many of these studies and clinical trials is the human prostate. Without doubt, carotenoids (and their endogenous derivatives-retinoids and other apo-carotenoids) are involved in intra- and intercellular signaling, cell growth and differentiation of prostate tissue. Due to the accumulation of new data on the role of different carotenoids such as lycopene (LC) and β-carotene (BC) in prostatic physiology and pathology, the present review aims to cover the past ten years of research in this area. Data from experimental studies are presented in the first part of the review, while epidemiological studies are disclosed and discussed in the second part. The objective of this compilation is to emphasize the present state of knowledge regarding the most potent molecular targets of carotenoids and their main metabolites, as well as to propose promising carotenoid agents for the prevention and treatment of prostatic diseases.
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http://dx.doi.org/10.3390/antiox10040585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069951PMC
April 2021

Inhibition Effect of Chloroquine and Integrin-Linked Kinase Knockdown on Translation in Melanoma Cells.

Int J Mol Sci 2021 Apr 1;22(7). Epub 2021 Apr 1.

Chair of Medical Biochemistry, Jagiellonian University Medical College, ul. Kopernika 7, 31-034 Kraków, Poland.

The twofold role of autophagy in cancer is often the therapeutic target. Numerous regulatory pathways are shared between autophagy and other molecular processes needed in tumorigenesis, such as translation or survival signaling. Thus, we have assumed that ILK knockdown should promote autophagy, and used together with chloroquine, an autophagy inhibitor, it could generate a better anticancer effect by dysregulation of common signaling pathways. Expression at the protein level was analyzed using Western Blot; siRNA transfection was done for ILK. Analysis of cell signaling pathways was monitored with phospho-specific antibodies. Melanoma cell proliferation was assessed with the crystal violet test, and migration was evaluated by scratch wound healing assays. Autophagy was monitored by the accumulation of its marker, LC3-II. Our data show that ILK knockdown by siRNA suppresses melanoma cell growth by inducing autophagy through AMPK activation, and simultaneously initiates apoptosis. We demonstrated that combinatorial treatment of melanoma cells with CQ and siILK has a stronger antitumor effect than monotherapy with either of these. It generates the synergistic antitumor effects by the decrease of translation of both global and oncogenic proteins synthesis. In our work, we point to the crosstalk between translation and autophagy regulation.
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http://dx.doi.org/10.3390/ijms22073682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037356PMC
April 2021

Recent Progress in Discovering the Role of Carotenoids and Metabolites in Prostatic Physiology and Pathology-A Review-Part II: Carotenoids in the Human Studies.

Antioxidants (Basel) 2021 Feb 20;10(2). Epub 2021 Feb 20.

Nutrition and Health Research Group 1 A-B, Population Health Department, Luxembourg Institute of Health, rue Thomas Edison, L-23 1445 Strassen, Luxembourg.

Among the vast variety of plant-derived phytochemicals, the group of carotenoids has continuously been investigated in order to optimize their potential application in the area of dietary intervention related to chronic diseases. One organ that has been especially targeted in many of these studies and clinical trials is the human prostate. Without doubt, carotenoids (and their endogenous derivatives-retinoids and apo-carotenoids) are involved in a plethora of intra- and intercellular signaling, cell growth, and differentiation of prostate tissue. Due to the accumulation of new data on the role of different carotenoids, such as lycopene (LYC) and β-carotene (BC), in prostatic physiology and pathology, the present review aimed to cover the past ten years of research in this regard. Data from experimental studies are presented in the first part of the review, while epidemiological studies are disclosed in this second part. The objective of this compilation was to emphasize the present state of knowledge about the most potent molecular targets of carotenoids, as well as to propose promising carotenoid agents for the prevention and possible treatment of prostatic diseases.
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http://dx.doi.org/10.3390/antiox10020319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924028PMC
February 2021

Dysregulation of Transcription Factor Activity During Formation of Cancer-Associated Fibroblasts.

Int J Mol Sci 2020 Nov 19;21(22). Epub 2020 Nov 19.

Center for Medical Genomics OMICRON, Jagiellonian University Medical College, 31-034 Kraków, Poland.

The reciprocal interactions between cancer cells and the quiescent fibroblasts leading to the activation of cancer-associated fibroblasts (CAFs) serve an important role in cancer progression. Here, we investigated the activation of transcription factors (TFs) in prostate fibroblasts (WPMY cell line) co-cultured with normal prostate or tumorous cells (RWPE1 and RWPE2 cell lines, respectively). After indirect co-cultures, we performed mRNA-seq and predicted TF activity using mRNA expression profiles with the Systems EPigenomics Inference of Regulatory Activity (SEPIRA) package and the GTEx and mRNA-seq data of 483 cultured fibroblasts. The initial differential expression analysis between time points and experimental conditions showed that co-culture with normal epithelial cells mainly promotes an inflammatory response in fibroblasts, whereas with the cancerous epithelial, it stimulates transformation by changing the expression of the genes associated with microfilaments. TF activity analysis revealed only one positively regulated TF in the RWPE1 co-culture alone, while we observed dysregulation of 45 TFs (7 decreased activity and 38 increased activity) uniquely in co-culture with RWPE2. Pathway analysis showed that these 45 dysregulated TFs in fibroblasts co-cultured with RWPE2 cells may be associated with the RUNX1 and PTEN pathways. Moreover, we showed that observed dysregulation could be associated with expression. We conclude that phenotypic changes in fibroblast responses to co-culturing with cancer epithelium result from orchestrated dysregulation of signaling pathways that favor their transformation and motility rather than proinflammatory status. This dysregulation can be observed both at the TF and transcriptome levels.
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http://dx.doi.org/10.3390/ijms21228749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699520PMC
November 2020

From carotenoid intake to carotenoid blood and tissue concentrations - implications for dietary intake recommendations.

Nutr Rev 2021 04;79(5):544-573

Population Health Department, Luxembourg Institute of Health, Strassen, Luxembourg.

There is uncertainty regarding carotenoid intake recommendations, because positive and negative health effects have been found or are correlated with carotenoid intake and tissue levels (including blood, adipose tissue, and the macula), depending on the type of study (epidemiological vs intervention), the dose (physiological vs supraphysiological) and the matrix (foods vs supplements, isolated or used in combination). All these factors, combined with interindividual response variations (eg, depending on age, sex, disease state, genetic makeup), make the relationship between carotenoid intake and their blood/tissue concentrations often unclear and highly variable. Although blood total carotenoid concentrations <1000 nmol/L have been related to increased chronic disease risk, no dietary reference intakes (DRIs) exist. Although high total plasma/serum carotenoid concentrations of up to 7500 nmol/L are achievable after supplementation, a plateauing effect for higher doses and prolonged intake is apparent. In this review and position paper, the current knowledge on carotenoids in serum/plasma and tissues and their relationship to dietary intake and health status is summarized with the aim of proposing suggestions for a "normal," safe, and desirable range of concentrations that presumably are beneficial for health. Existing recommendations are likewise evaluated and practical dietary suggestions are included.
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http://dx.doi.org/10.1093/nutrit/nuaa008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025354PMC
April 2021

Probing the recognition specificity of αβ integrin and syndecan-4 using force spectroscopy.

Micron 2020 10 1;137:102888. Epub 2020 Jun 1.

Chair of Medical Biochemistry Jagiellonian University Medical College, Kopernika 7, 31-034 Kraków, Poland.

The knowledge on how cells interact with microenvironment is particularly important in understanding the interaction of cancer cells with surrounding stroma, which affects cell migration, adhesion, and metastasis. The main cell surface receptors responsible for the interaction with extracellular matrix (ECM) are integrins, however, they are not the only ones. Integrins are accompanied to other molecules such as syndecans. The role of the latter has not yet been fully established. In our study, we would like to answer the question of whether integrins and syndecans, possessing similar functions, share also similar unbinding properties. By using single molecule force spectroscopy (SMFS), we conducted measurements of the unbinding properties of αβ and syndecan-4 in the interaction with vitronectin (VN), which, as each ECM protein, possesses two binding sites specific to integrins and syndecans. The unbinding force and the kinetic off rate constant derived from SMFS describe the stability of single molecular complex. Obtained data show one barrier transition for each complex. The proposed model shows that the unbinding of αβ from VN proceeds before the unbinding of SDC-4. However, despite different unbinding kinetics, the access to both receptors is needed for cell growth and proliferation.
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http://dx.doi.org/10.1016/j.micron.2020.102888DOI Listing
October 2020

Could the kinetin riboside be used to inhibit human prostate cell epithelial-mesenchymal transition?

Med Oncol 2020 Feb 6;37(3):17. Epub 2020 Feb 6.

Chair of Pathomorphology, Jagiellonian University Medical College, Kraków, Poland.

The epithelial-mesenchymal transition (EMT) is a molecular process connected to higher expression of vimentin and increased activity of transcription factors (Snail, Twist) which restrains E-cadherin. EMT has been linked to prostate cancer metastatic potential, therapy resistance, and poor outcomes. Kinetin riboside (9-(b-dribofuranosyl)-6-furfurylaminopurine, KR) is a naturally occurring cytokinin, which induces apoptosis and shows strong antiproliferative activity against various human cancer cell lines. To establish the effect of KR on human prostate cell lines, expression of, e.g. AR, E-, N-cadherins, Vimentin, Snail, Twist, and MMPs, was analysed at mRNA and protein levels using Western Blot and RT-PCR and/or RQ-PCR techniques. KR inhibited the growth of human prostate cancer cells, but also, to a small extent, of normal cells. This effect depended on the type of the cells and their androgen sensitivity. KR also decreased the level of p-Akt, which takes part in androgen signalling modulation. The antiapoptotic Bcl-2 protein was down-regulated in cancer cell lines, while that of Bax is up-regulated upon KR exposure. KR contributed to re-expression of the E-cadherin as well as to significant changes in cell migration. Taken together, our results indicate for the first time that KR can be proposed as a factor for signalling pathways regulation that participates in the inhibition of development of aggressive forms of prostate cancer, and may alter the approach to therapeutic interventions. We propose KR as a potent inhibitor of EMT in human prostate cells.
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http://dx.doi.org/10.1007/s12032-020-1338-1DOI Listing
February 2020

Aberrant promoter methylation may be responsible for the control of CD146 (MCAM) gene expression during breast cancer progression.

Acta Biochim Pol 2019 Dec;66(4):619-625

Jagiellonian University - Medical College, Faculty of Medicine, Chair of Medical Biochemistry, Kraków, Poland.

The CD146 (also known as MCAM, MUC-18, Mel-CAM) was initially reported in 1987, as a protein crucial for the invasiveness of malignant melanoma. Recently, it has been confirmed that CD146 has been involved in progression and poor overall survival of many cancers including breast cancer. Importantly, in independent studies, CD146 was reported to be a trigger of epithelial to mesenchymal transition in breast cancer cells. The goal of our current study was to verify the potential involvement of epigenetic mechanism behind the regulation of CD146 expression in breast cancer cells, as it has been previously reported in prostate cancer. First, we analysed the response of breast cancer cell lines, differing in the initial CD146 mRNA and protein content, to epigenetic modifier, 5-aza-2-deoxycytidine, and subsequently the methylation status of CD146 gene promoter was investigated, using direct bisulfite sequencing. We observed that treatment with demethylating agent led to induction of CD146 expression in all analysed breast cancer cell lines, both at mRNA and protein level, what was accompanied by increased expression of selected mesenchymal markers. Importantly, CD146 gene promoter analysis showed aberrant CpG island methylation in 2 out of 3 studied breast cancer cells lines, indicating epigenetic regulation of CD146 gene expression. In conclusion, our study revealed, for the first time, that aberrant methylation maybe involved in expression control of CD146, a very potent EMT inducer in breast cancer cells. Altogether, the data obtained may provide the basis for novel therapies as well as diagnostic approaches enabling sensitive and very accurate detection of breast cancer cells.
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http://dx.doi.org/10.18388/abp.2019_2907DOI Listing
December 2019

Dihydrotestosterone increases the risk of bladder cancer in men.

Hum Cell 2019 Jul 22;32(3):379-389. Epub 2019 May 22.

Chair of Medical Biochemistry, Jagiellonian University Medical College, Kraków, Poland, ul.Kopernika 7, 31-034, Kraków, Poland.

Men are at a higher risk of developing bladder cancer than women. Although the urinary bladder is not regarded as an sex organ, it has the potential to respond to androgen signals. The mechanisms responsible for the gender differences remain unexplained. Androgen receptor (AR) after binding with 5α-dihydrotestosteron (DHT) undergoes a conformational change and translocates to nucleus to induce transcriptional regulation of target genes. However androgen/AR signaling can also be activated by interacting with several signaling molecules and exert its non-genomic function. The aim of present study was to explain whether the progression of bladder cancer in men is dependent on androgen/AR signaling. Studies were carried out on human bladder cancer cell lines: HCV29, T24, HT1376 and HTB9. Bladder cancer cells were treated for 48 h with 10 nM DHT or not, with replacement after 24 h. Expression of cell signaling proteins, was analyzed using Western Blot and RT-PCR. Subcellular localization of protein was studied using the ProteoExtract Subcellular Proteome Extraction Kit and Western blot analysis. We showed that DHT treatment significantly increased AR expression in bladder cell line HCV29. We also observed DHT-mediated activation of Akt/GSK-3β signaling pathway which plays a central role in cancer progression. Presented results also show that androgen/AR signaling is implicated in phosphorylation of eIF4E which can promote epithelial-mesenchymal transition (EMT). We indicate that AR plays an essential role in bladder cancer progression in male patients. Therefore, androgen-activated AR signaling is an attractive regulatory target for the inhibition or prevention of bladder cancer incidence in men.
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http://dx.doi.org/10.1007/s13577-019-00255-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570698PMC
July 2019

The Epigenetic Modifier 5-Aza-2-deoxycytidine Triggers the Expression of Gene in Prostate Cancer Cells.

Anticancer Res 2019 May;39(5):2395-2403

Chair of Medical Biochemistry, Faculty of Medicine, Jagiellonian University - Medical College, Cracow, Poland

Background/aim: During cancer progression cells undergo epithelial-to-mesenchymal transition (EMT). Although EMT is a complex process, recently, it has been reported that CD146 overexpression in prostate cancer cells is sufficient to induce mesenchymal phenotype. The following study aimed to investigate whether the expression of CD146 is altered by an epigenetic modifier in prostate cancer cells, in vitro.

Materials And Methods: Three human prostate cancer cell lines were treated with 5-aza-2-deoxycytidine; the expression of CD146 and EMT-related factors was analyzed by RT-PCR and western Blot. The methylation status of the CD146 promoter area was assessed using bisulfite sequencing.

Results: Our data showed that, the expression of CD146 was evidently increased in all three studied cell lines in response to a demethylating agent, both at the mRNA and protein level, suggesting epigenetic regulation of the analyzed gene. However, there was no methylation in the studied CpG island in CD146 gene promoter. Moreover, the demethylating agent induced the expression of EMT-related transcription factors (SNAI1, SNAI2, TWIST1 and ZEB1), the pattern of which differed among the cell lines, as well as alterations in cell morphology; altogether accounting for the mesenchymal phenotype.

Conclusion: The demethylating agent 5-aza-2-deoxycytidine triggers the expression of CD146 in prostate cancer cells independently on the methylation status of the analyzed CpG island fragment in CD146 gene promoter. Moreover, demethylation treatment induces a mesenchymal profile in prostate cancer cells.
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http://dx.doi.org/10.21873/anticanres.13357DOI Listing
May 2019

Superparamagnetic Iron Oxide Nanoparticles-Current and Prospective Medical Applications.

Materials (Basel) 2019 Feb 19;12(4). Epub 2019 Feb 19.

Department of Chemistry, Jagiellonian University, 2 Gronostajowa St., 30-387 Kraków, Poland.

The recent, fast development of nanotechnology is reflected in the medical sciences. Superparamagnetic Iron Oxide Nanoparticles (SPIONs) are an excellent example. Thanks to their superparamagnetic properties, SPIONs have found application in Magnetic Resonance Imaging (MRI) and magnetic hyperthermia. Unlike bulk iron, SPIONs do not have remnant magnetization in the absence of the external magnetic field; therefore, a precise remote control over their action is possible. This makes them also useful as a component of the advanced drug delivery systems. Due to their easy synthesis, biocompatibility, multifunctionality, and possibility of further surface modification with various chemical agents, SPIONs could support many fields of medicine. SPIONs have also some disadvantages, such as their high uptake by macrophages. Nevertheless, based on the ongoing studies, they seem to be very promising in oncological therapy (especially in the brain, breast, prostate, and pancreatic tumors). The main goal of our paper is, therefore, to present the basic properties of SPIONs, to discuss their current role in medicine, and to review their applications in order to inspire future developments of new, improved SPION systems.
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http://dx.doi.org/10.3390/ma12040617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416629PMC
February 2019

Targeting the hypoxia pathway in malignant plasma cells by using 17-allylamino-17-demethoxygeldanamycin.

Acta Biochim Pol 2018 15;65(1):101-109. Epub 2018 Mar 15.

Chair of Medical Biochemistry, Jagiellonian University Medical College, Kraków, Poland.

Multiple myeloma (MM) is characterized as a clonal expansion of malignant plasma cells in the bone marrow, which is often associated with pancytopenia and osteolytic bone disease. Interestingly, myeloma-infiltrated bone marrow is considered to be hypoxic, providing selection pressure for a developing tumour. Since HSP90 was shown to participate in stabilization of the subunit of the key transcription factor HIF-1, which controls the hypoxic response, the aim of this study was to investigate the influence of a HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), on MM cells cultured under low oxygenation conditions. We confirmed that 17-AAG inhibits hypoxic induction of the HIF-1 target genes in malignant plasma cells and demonstrate the concentration range of severe hypoxia-specific cytotoxicity. Next, we selected the malignant plasma cells under severe hypoxia/re-oxygenation culture conditions in the presence or absence of 17-AAG and subsequently, the cells which survived were further expanded and analyzed. Interestingly, we have noticed significant changes in the survival and the response to anti-MM drugs between the parental cell lines and those selected in cyclic severe hypoxia in the presence and absence of 17-AAG. Importantly, we also observed that the lack of oxygen itself, irrespectively of HIF-1 inhibition, is the main/pivotal factor driving the selection process in the experiments presented here.
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http://dx.doi.org/10.18388/abp.2017_1630DOI Listing
August 2018

The effect of "NutramilTM Complex," food for special medical purpose, on breast and prostate carcinoma cells.

PLoS One 2018 14;13(2):e0192860. Epub 2018 Feb 14.

Department of Human Nutrition, Faculty of Food Technology, University of Agriculture, Krakow, Poland.

NutramilTM Complex is a multicomponent food product that meets the requirements of a food for special medical purpose. As a complete, high-energy diet it consists of properly balanced nutrients, vitamins and minerals. The aim of this study was to assess the effect of NutramilTM Complex on breast and prostate carcinoma cells. Our results showed that NutramilTM Complex reduced the viability and proliferation of breast and prostate cancer cells and that this process was associated with the induction of apoptosis via activation of caspase signalling. Data showed elevated levels of p53 tumour suppressor, up-regulation of p38 MAPK and SAPK / JNK proteins and downregulation of anti-apoptotic ERK1/2, AKT1 and HSP27. Treatment with NutramilTM Complex also affected the expression of the BCL2 family genes. Results also showed down-regulation of anti-apoptotic BCL-2 and up-regulation of pro-apoptotic members such as BAX, BAD, BID. In addition, we also observed regulation of many other genes, including Iκβα, Chk1 and Chk2, associated with apoptotic events. Taken together, our results suggest activation of the mitochondrial apoptotic pathway as most likely mechanism of anti-carcinogenic activity of NutramilTM Complex.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192860PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812662PMC
April 2018

Effects of resveratrol, curcumin, berberine and other nutraceuticals on aging, cancer development, cancer stem cells and microRNAs.

Aging (Albany NY) 2017 06;9(6):1477-1536

Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare "Alberto Monroy", Palermo, Italy.

Natural products or nutraceuticals have been shown to elicit anti-aging, anti-cancer and other health-enhancing effects. A key target of the effects of natural products may be the regulation of microRNA (miR) expression which results in cell death or prevents aging, diabetes, cardiovascular and other diseases. This review will focus on a few natural products, especially on resveratrol (RES), curcumin (CUR) and berberine (BBR). RES is obtained from the skins of grapes and other fruits and berries. RES may extend human lifespan by activating the sirtuins and SIRT1 molecules. CUR is isolated from the root of turmeric (). CUR is currently used in the treatment of many disorders, especially in those involving an inflammatory process. CUR and modified derivatives have been shown to have potent anti-cancer effects, especially on cancer stem cells (CSC). BBR is also isolated from various plants () and has been used for centuries in traditional medicine to treat diseases such as adult- onset diabetes. Understanding the benefits of these and other nutraceuticals may result in approaches to improve human health.
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http://dx.doi.org/10.18632/aging.101250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509453PMC
June 2017

Anti-androgen 2-hydroxyflutamide modulates cadherin, catenin and androgen receptor phosphorylation in androgen-sensitive LNCaP and androgen-independent PC3 prostate cancer cell lines acting via PI3K/Akt and MAPK/ERK1/2 pathways.

Toxicol In Vitro 2017 Apr 2;40:324-335. Epub 2017 Feb 2.

Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.

This study aimed to investigate rapid effect of anti-androgen 2-hydroxyflutamide (HF) on cadherin/catenin complex and androgen receptor (AR) phosphorylation in prostate cancer cell lines. In addition, a role of PI3K/Akt and MAPK/ERK1/2 pathways in mediating these effects was explored. We have demonstrated that in androgen-sensitive LNCaP cells HF induced rapid increase of E-cadherin phosphorylation at Ser 838/840 (p<0.05) in MAPK/ERK1/2-dependent manner, whereas phosphorylation of β-catenin at Tyr 654 was unchanged. Concomitantly, the reduction of the level of AR phosphorylated at Ser210/213 was found (p<0.01). In androgen-independent PC3 cells HF decreased Tyr 860 N-cadherin and Tyr 645 β-catenin phosphorylation (p<0.01), acting via both MAPK/ERK1/2 and PI3K/Akt pathways. Further, we evidenced that MAPK/ERK1/2 and PI3K/Akt pathways were differentially influenced by HF in LNCaP and PC3 cells. In LNCaP cells, both Akt (p<0.01) and ERK1/2 (p<0.001) phosphorylation were negatively regulated and this effect was mediated by Raf-1 (p<0.05). In contrast, in PC3 cells HF stimulated Akt (p<0.001) and ERK1/2 (p<0.001) activation, but had no effect on the crosstalk between PI3K/Akt and MEK/ERK1/2 pathways at the Raf-1 kinase level. Our findings expand the role of anti-androgen into non-genomic signaling, creating a link between anti-androgen action and phosphorylation of adherens junction proteins in prostate cancer cells.
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http://dx.doi.org/10.1016/j.tiv.2017.01.019DOI Listing
April 2017

Roles of GSK-3 and microRNAs on epithelial mesenchymal transition and cancer stem cells.

Oncotarget 2017 Feb;8(8):14221-14250

Department of Biomedical and Biotechnological Sciences - Oncological, Clinical and General Pathology Section, University of Catania, Catania, Italy.

Various signaling pathways exert critical roles in the epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs). The Wnt/beta-catenin, PI3K/PTEN/Akt/mTORC, Ras/Raf/MEK/ERK, hedgehog (Hh), Notch and TP53 pathways elicit essential regulatory influences on cancer initiation, EMT and progression. A common kinase involved in all these pathways is moon-lighting kinase glycogen synthase kinase-3 (GSK-3). These pathways are also regulated by micro-RNAs (miRs). TP53 and components of these pathways can regulate the expression of miRs. Targeting members of these pathways may improve cancer therapy in those malignancies that display their abnormal regulation. This review will discuss the interactions of the multi-functional GSK-3 enzyme in the Wnt/beta-catenin, PI3K/PTEN/Akt/mTORC, Ras/Raf/MEK/ERK, Hh, Notch and TP53 pathways. The regulation of these pathways by miRs and their effects on CSC generation, EMT, invasion and metastasis will be discussed.
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http://dx.doi.org/10.18632/oncotarget.13991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355173PMC
February 2017

Integrin-linked kinase regulates cadherin switch in bladder cancer.

Tumour Biol 2016 Nov 28;37(11):15185-15191. Epub 2016 Sep 28.

Chair of Medical Biochemistry, Jagiellonian University Medical College, ul.Kopernika 7, 31-034, Kraków, Poland.

Cadherin switch is specific of epithelial-mesenchymal transition (EMT) and is closely related to tumor cell invasion. However, the molecular mechanism that promotes the phenotypic changes remains unclear and elusive. We found that integrin-linked kinase (ILK) is a key factor involved in cadherin switch. The expression and activity of ILK are elevated in a variety of cancers but its mechanisms are not exactly understood. In this report, we studied the role and mechanism of ILK in EMT of human bladder cancer. We showed that silencing of ILK expression by small interfering RNA (siRNA) significantly abolished the nuclear translocation or the presence of markers associated with EMT like Snail, Twist, Zeb, and beta-catenin. ILK knockdown by siRNA suppressed N-cadherin expression and increased re-expression of E-cadherin in bladder cancer cells. We suggest that ILK is a major signaling factor involved in EMT. It is essential to understand the molecular mechanism of EMT in aim to possibly use it in search for new therapeutic targets.
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http://dx.doi.org/10.1007/s13277-016-5354-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126188PMC
November 2016

The role of 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) in metabolic reprogramming of cancer cells.

Postepy Hig Med Dosw (Online) 2016 Sep 13;70(0):938-950. Epub 2016 Sep 13.

Katedra Biochemii Lekarskiej, Uniwersytet Jagielloński-Collegium Medicum, Kraków.

The high rate of glucose breakdown is the fingerprint of cancer. Increased glycolysis allows tumour cells to fulfil their high energetic and biosynthetic demands. Interestingly, however, rather than metabolizing glucose in the oxidative phosphorylation pathway, cancer cells generally use glucose for aerobic glycolysis. This phenomenon is known as the Warburg effect and is considered as one of the most fundamental forms of metabolic reprogramming during cancerogenesis. Changes in the rate of glycolytic activity of cancer cells are caused mainly by the increased expression of glycolytic enzymes as a consequence of activation of oncogenes or loss of tumour suppressors. In addition, the hypoxic tumour environment also triggers upregulation of a series of genes involved in glucose metabolism. Among the metabolic enzymes that are modulated by these factors in cancer cells are the 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatases (PFKFBs), a family of bifunctional enzymes that control the levels of fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. Fru-2,6-P2 strongly activates glucose breakdown in glycolysis through allosteric modulation of the rate-limiting enzyme of glycolysis, phosphofructokinase-1 (PFK-1). Thus far, many studies have reported a correlation between aberrant PFKFB expression level and the grade of tumour aggressiveness, which directly indicates that these enzymes may play a crucial role in cancerogenesis. The objective of this review is to highlight the recent studies on aberrant expression of PFKFBs and its influence on cancer progression.
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http://dx.doi.org/10.5604/17322693.1218187DOI Listing
September 2016

Critical Roles of EGFR Family Members in Breast Cancer and Breast Cancer Stem Cells: Targets for Therapy.

Curr Pharm Des 2016 ;22(16):2358-88

Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Brody Building 5N98C, Greenville, NC 27858 USA.

The roles of the epidermal growth factor receptor (EGFR) signaling pathway in various cancers including breast, bladder, brain, colorectal, esophageal, gastric, head and neck, hepatocellular, lung, neuroblastoma, ovarian, pancreatic, prostate, renal and other cancers have been keenly investigated since the 1980's. While the receptors and many downstream signaling molecules have been identified and characterized, there is still much to learn about this pathway and how its deregulation can lead to cancer and how it may be differentially regulated in various cell types. Multiple inhibitors to EGFR family members have been developed and many are in clinical use. Current research often focuses on their roles and other associated pathways in cancer stem cells (CSCs), identifying sites where therapeutic resistance may develop and the mechanisms by which microRNAs (miRs) and other RNAs regulate this pathway. This review will focus on recent advances in these fields with a specific focus on breast cancer and breast CSCs. Relatively novel areas of investigation, such as treatments for other diseases (e.g., diabetes, metabolism, and intestinal parasites), have provided new information about therapeutic resistance and CSCs.
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http://dx.doi.org/10.2174/1381612822666160304151011DOI Listing
November 2017

Deregulation of the EGFR/PI3K/PTEN/Akt/mTORC1 pathway in breast cancer: possibilities for therapeutic intervention.

Oncotarget 2014 Jul;5(13):4603-50

Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University Greenville, NC 27858 USA.

The EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance and metastasis. The expression of this pathway is frequently altered in breast cancer due to mutations at or aberrant expression of: HER2, ERalpha, BRCA1, BRCA2, EGFR1, PIK3CA, PTEN, TP53, RB as well as other oncogenes and tumor suppressor genes. In some breast cancer cases, mutations at certain components of this pathway (e.g., PIK3CA) are associated with a better prognosis than breast cancers lacking these mutations. The expression of this pathway and upstream HER2 has been associated with breast cancer initiating cells (CICs) and in some cases resistance to treatment. The anti-diabetes drug metformin can suppress the growth of breast CICs and herceptin-resistant HER2+ cells. This review will discuss the importance of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway primarily in breast cancer but will also include relevant examples from other cancer types. The targeting of this pathway will be discussed as well as clinical trials with novel small molecule inhibitors. The targeting of the hormone receptor, HER2 and EGFR1 in breast cancer will be reviewed in association with suppression of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148087PMC
http://dx.doi.org/10.18632/oncotarget.2209DOI Listing
July 2014

A comparative study of glycoproteomes in androgen-sensitive and -independent prostate cancer cell lines.

Mol Cell Biochem 2014 Jan 9;386(1-2):189-98. Epub 2013 Oct 9.

Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Mickiewicza 30 Ave, 30-059, Krakow, Poland,

Prostate cancer is one of the most common malignancies in men and is predicted to be the second leading cause of cancer-related deaths. After 6-18 months, hormone ablation treatment results in androgen-independent growth of cancer cells, metastasis and progression. The mechanism of androgen-independent growth of prostatic carcinoma cells is still unknown. Identification of factors that facilitate the transition from androgen-dependent to independent states is crucial in designing future diagnostics and medication strategies. To understand the biochemical meaning of hormone dependency deprivation, glycoproteins enriched profiles were compared between DU145 (hormone non-responding) and LNCaP (hormone responding) prostate cancer cells. These results allow for anticipation on the important role of glycosylation in malignant transformation. Both Tn antigen and complex antennary N-oligosaccharides were recognized. Their occurrence might be involved in the development and progression of tumor, and failure of hormone ablation therapy. Among identified proteins in androgen-sensitive cells nucleolin (P19338) was found that is widely described as apoptosis inhibitor, and also transporter of molecules from the membrane to the cytoplasm or nucleus. In addition, 14-3-3 protein family (P27348, P31946, P61981, P63104, P62258, Q04917, and P31947) was investigated across available databases as it forms stable complexes with glycoproteins. Our studies indicate that isoforms: sigma and eta were found in androgen-dependent prostate cancer cells, while other isoforms were present in androgen non-responding cells. 14-3-3 binding partners are involved in cancer pathogenesis. These findings may contribute to a better understanding of prostate cancer tumorigenesis and to a more efficient prognosis and individual therapy in a future. However, it still remains to be revealed how important those changes are for androgen dependency loss in prostate cancer patients carried out on clinically relevant populations.
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http://dx.doi.org/10.1007/s11010-013-1857-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867656PMC
January 2014

Advances in targeting signal transduction pathways.

Oncotarget 2012 Dec;3(12):1505-21

Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, USA.

Over the past few years, significant advances have occurred in both our understanding of the complexity of signal transduction pathways as well as the isolation of specific inhibitors which target key components in those pathways. Furthermore critical information is being accrued regarding how genetic mutations can affect the sensitivity of various types of patients to targeted therapy. Finally, genetic mechanisms responsible for the development of resistance after targeted therapy are being discovered which may allow the creation of alternative therapies to overcome resistance. This review will discuss some of the highlights over the past few years on the roles of key signaling pathways in various diseases, the targeting of signal transduction pathways and the genetic mechanisms governing sensitivity and resistance to targeted therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681490PMC
http://dx.doi.org/10.18632/oncotarget.802DOI Listing
December 2012

The role of mRNA E6/E7 HPV high oncogenic risk expression in colposcopy of cervical intraepithelial neoplasia (CIN).

Przegl Lek 2012 ;69(9):651-7

Jagiellonian University, Medical College, Department of Gynecology and Oncology, Krakow, Poland.

Unlabelled: The aim of this paper is the evaluation of colposcopy and mRNA E6/E7 HPV detection--as the marker of persistent human papilloma virus (HPV) infection in the triage of abnormal Pap smears and in the assessment of cervical intraepithelial neoplasia progression risk. The clinical material consisted of 85 women, participating the national cervical cancer screening in the period of April 2010, and October 2010, reffered to the Outpatient Clinic of Gynecologic Oncology and Female Genital Tract Neoplasms Prophylaxy of the Jagiellonian University Medical College in Krakow, Poland. All subjects were offered gynecological evaluation, Pap smear, colposcopy, DNA HPV (Hybrid Capture2, Qiagen) and mRNA E6/E7 testing (NulciSens, Biomerieux). In case of positive tests colposcopically directed cervical biopsy with histopathologic evaluation were performed.

Results: The presence of mRNA E6/E7 HPV transcripts correlated with high grade squamous intraepithelial lesions, statistically significantly. There was statistically difference between colposcopic, histologic concordance comparing to mRNA E6/E7 HPV colposcopic histologic concordance (p < 0.001).

Conclusions: The presence of mRNA E6/E7 HR HPV may be assumed as specific marker of high grade cervical lesions. The combination of mRNA E6/E7 HR HPV ewith colposcopic evaluation increases the colposcopy concordanece with final histologic findings.
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March 2013

Changes in cellular response to the damage induced in PC-3 prostate cancer cells by proton microbeam irradiation.

Gen Physiol Biophys 2012 Mar;31(1):11-8

The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland.

The aim of this research was to find out whether the passage number effect may influence on the PC-3 cells (the human prostate cancer line derived from bone metastases) response to proton radiation. 2 MeV horizontally focused proton microbeam was used as a radiation source. The cells were treated with a counted number of H(+) ions (50-8000) corresponding to doses of 1.3-209 Gy/cell. For comparison, cell death was also induced by UVC radiation. All cells were stained with Hoechst 33342 and propidium iodide and visualized under a fluorescence microscope. Necrosis was observed at: a) 8000 protons per cell (corresponding to ∼209 Gy/cell) after 2-4 passages, b) 3200 protons per cell (corresponding to ∼84 Gy/cell) for cells after 11-14 passages and c) only 800 protons per cell (corresponding to ∼2 Gy/cell ) after 47-50 passages. Apoptosis was efficiently induced, by protons, only in cells after 50 passages. The results showed that the laboratory conditions affected cellular response of PC-3 cell line to the proton irradiation. The cellular response to the radiation treatment strongly depends on number of passages.
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http://dx.doi.org/10.4149/gpb_2012_001DOI Listing
March 2012

Cancer cell detection in tissue sections using AFM.

Arch Biochem Biophys 2012 Feb 23;518(2):151-6. Epub 2011 Dec 23.

The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland.

Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Young's modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.
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http://dx.doi.org/10.1016/j.abb.2011.12.013DOI Listing
February 2012

[Beta-carotene regulates the expression of proapoptotic BAX and CAPN2 in HL-60, U-937 and TF-1 - human acute myeloid leukemia cell lines; microarray, RQ-PCR and Western Blot analysis].

Przegl Lek 2011 ;68(5):258-62

Katedra i Klinika Hematologii Collegium Medicum UJ w Krakowie.

Beta carotene (BC) is a nutritional compound widespread in foods which can influence vital cellular functions--differentiation, proliferation and apoptosis of normal and cancer cells. However its role in the carcinogenesis remains controversial. We performed a microarray expression analysis in three human acute leukemia cell lines (HL-60, U937 and TF-1) exposed to 10mM BC and found that BC stimulated the apoptosis in all studied cell lines. This effect was most evident in the HL-60 cell line and correlated with increased expression of proapoptotic BAX and CAPN2 genes. The micro-array findings were replicated by the quantitative BAX and CAPN2 expression analysis using real-time PCR and by Western Blot on protein level. The biological tests (TUNEL method) for apoptosis showed consistent proapoptotic effects in all studied cell lines. In this paper the stimulatory effect of BC on apoptosis (enhanced expression of proapoptotic genes and proteins) in human acute myeloid leukemia cells was confirmed. The most potent activation of apoptosis in the HL-60 cells is in line with other investigators observations suggesting distinct molecular mechanism of apoptosis stimulation by BC in different human acute myeloid leukemia cells.
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February 2012

Expression of vascular endothelial growth factors VEGF- C and D, VEGFR-3, and comparison of lymphatic vessels density labeled with D2-40 antibodies as a prognostic factors in vulvar intraepithelial neoplasia (VIN) and invasive vulvar cancer.

Neuro Endocrinol Lett 2011 ;32(4):530-9

Department of Obstetrics and Perinatology, Medical College of Jagiellonian University, Cracow University Hospital, Cracow, Poland.

Objective: The aim of this study was to compare the immunohistochemical expression of vascular endothelial growth factors VEGF-C and D, as well as the expression of VEGFR-3 in VIN and vulvar invasive cancer and to compare the density of lymphatic marker D2-40 antibody in both groups, and to compare them with different clinicopathologic features.

Materials & Methods: The study was performed using tissue material and clinical data from 100 women diagnosed with VIN and 100 women diagnosed with invasive vulvar cancer.

Results: No significant differences were found in the expression of VEGF-C and -D or VEGFR-3 between those patients with VIN and those with invasive vulvar cancers. Weak expression of VEGF-C was confirmed only in two cases of the analyzed series; in all cases, expression of VEGF-D and VEGFR-3 was observed. The strongest expression of VEGF-D and VEGFR-3 was observed in the group of invasive cancers. The highest density of lymphatic vessels per 2 mm was observed in VIN. In the cancer group, small lymphatic vessels with a narrow oval lumen were observed. Moreover, in two cases of vulvar cancer, the presence of intratumoral lymphatic vessels was observed.

Conclusions: These results suggest that lymphangiogenesis begins at the preinvasive stage of vulvar carcinogenesis and suggests the important role of VEGF-C, VEGF-D, VEGFR-3 and LV (D2-40) as prognostic factors in the process of carcinogenesis in the vulvar area.
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October 2011

Effects of kinetin riboside on proliferation and proapoptotic activities in human normal and cancer cell lines.

J Cell Biochem 2011 Aug;112(8):2115-24

Jagiellonian University Medical College, Kraków, Poland.

Kinetin riboside (KR) is a N6-substituted derivative of adenosine. It is a natural compound which occurs in the milk of coconuts on the nanomole level. KR was initially shown to selectively inhibit proliferation of cancer cells and induce their apoptosis. We observed that KR inhibited growth (20-80%) of not only human cancer, but also normal cells and that this effect strongly depended on the type of cells. The anti-apoptotic Bcl-2 protein was downregulated, while proapoptotic Bax was upregulated in normal as well as in cancer cell lines, upon exposure to KR. Cytochrome c level increased in the cytosol upon treatment of cells with KR. The activity of caspases (ApoFluor®Green Caspase Activity Assay), as well as caspase-3 (caspase-3 activation assay) were increased mainly in cancer cells. The expression of procaspase 9 and its active form in the nucleus as well as in cytosol of KR-treated cells was elevated. In contrast, no effect of KR on caspase 8 expression was noted. The results indicated that non-malignant cells were less sensitive to KR then their cancer analogs and that KR most likely stimulated apoptosis mechanism of cancer cells through the intrinsic pathway.
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http://dx.doi.org/10.1002/jcb.23132DOI Listing
August 2011

The mechanism of contribution of integrin linked kinase (ILK) to epithelial-mesenchymal transition (EMT).

Adv Enzyme Regul 2011 28;51(1):195-207. Epub 2010 Oct 28.

Chair of Medical Biochemistry Jagiellonian University Medical College, Kraków, Poland.

Integrin linked kinase (ILK) is ubiquitously expressed serine/threonine protein kinase, a binding partner of β1 and β3 integrin subunit as a cytoplasmic effector of integrin receptors that functionally links them to the actin cytoskeleton.We postulate that ILK is important enzyme involved in epithelial-mesenchymal transition (EMT) a critical event in the process of cancer progression. Commonly used EMT molecular markers include among others increased expression of N-cadherin and vimentin, nuclear localization of β-catenin, and the decrease of E-cadherin synthesis. In this study we were able to show that N-cadherin expression in melanoma cells is dependent on ILK signaling and the translocation of β-catenin to the nucleus. Silencing of ILK expression by siRNA significantly inhibited the stabilization and subsequent nuclear translocation of β-catenin and the expression of N-cadherin, a crucial molecule in the EMT, which facilitates association with fibroblast and endothelial cells during invasion of various cancers. The results allow to cautiously speculate on the important role of ILK in the cross-talk between integrins and cadherins accompanying EMT in melanoma.
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http://dx.doi.org/10.1016/j.advenzreg.2010.09.005DOI Listing
August 2011