Publications by authors named "Jana Steigerova"

9 Publications

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Molecular mechanisms of plant steroids and study of their interaction with nuclear receptors in prostate cancer cells.

Food Chem Toxicol 2020 Mar 27;137:111164. Epub 2020 Jan 27.

Laboratory of Growth Regulators, Faculty of Science, Palacky University, Institute of Experimental Botany of the Czech Academy of Sciences, Slechtitelu 27, CZ-78371, Olomouc, Czech Republic.

Plant hormone brassinosteroids (BRs) have multiple important functions in plants. They have also been found to exhibit anti-tumor, anti-angiogenic and anti-proliferative activity. The experimental part of this article describes the effects of BR biosynthetic precursors on prostate cancer cells. The experiments were performed with LNCaP and DU-145 prostate cancer cell lines. These were cultivated and treated with tested BRs in different concentrations and time intervals. The tested compounds were found to affect cell viability, nuclear receptor expression, cell cycle and apoptosis in the tumor cells. IC concentrations were determined based on MTT test and the two most active compounds (cathasterone and 6-oxocampestanol) were used in the next experiments. Cathasterone was the most effective of all tested compounds and effectively inhibited integrity of cell spheres. It was found that both BRs had no significant effect on the cell cycle in LNCaP at IC concentration, while in DU-145 a significant block in G/G phase after the BR treatment was observed. The effect of BRs on the nuclear steroid receptors was manifested by changes in their expression and localization. BRs demonstrated their significant effect on prostate cancer cells and the compounds have potential used in anticancer drug research and cancer treatment.
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http://dx.doi.org/10.1016/j.fct.2020.111164DOI Listing
March 2020

Targeting genotoxic and proteotoxic stress-response pathways in human prostate cancer by clinically available PARP inhibitors, vorinostat and disulfiram.

Prostate 2019 03 29;79(4):352-362. Epub 2018 Nov 29.

Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.

Background: Castration-resistant prostate cancer (PCa) represents a serious health challenge. Based on mechanistically-supported rationale we explored new therapeutic options based on clinically available drugs with anticancer effects, including inhibitors of PARP1 enzyme (PARPi), and histone deacetylases (vorinostat), respectively, and disulfiram (DSF, known as alcohol-abuse drug Antabuse) and its copper-chelating metabolite CuET that inhibit protein turnover.

Methods: Drugs and their combination with ionizing radiation (IR) were tested in various cytotoxicity assays in three human PCa cell lines including radio-resistant stem-cell like derived cells. Mechanistically, DNA damage repair, heat shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and immunoblotting.

Results: We observed enhanced sensitivity to PARPi/IR in PC3 cells consistent with lower homologous recombination (HR) repair. Vorinostat sensitized DU145 cells to PARPi/IR and decreased mutant p53. Vorinostat also impaired HR-mediated DNA repair, as determined by Rad51 foci formation and downregulation of TOPBP1 protein, and overcame radio-resistance of stem-cell like DU145-derived cells. All PCa models responded well to CuET or DSF combined with copper. We demonstrated that DSF interacts with copper in the culture media and forms adequate levels of CuET indicating that DSF/copper and CuET may be considered as comparable treatments. Both DSF/copper and CuET evoked hallmarks of UPR in PCa cells, documented by upregulation of ATF4, CHOP and phospho-eIF2α, with ensuing heat shock response encompassing activation of HSF1 and HSP70. Further enhancing the cytotoxicity of CuET, combination with an inhibitor of the anti-apoptotic protein survivin (YM155, currently undergoing clinical trials) promoted the UPR-induced toxicity, yielding synergistic effects of CuET and YM155.

Conclusions: We propose that targeting genotoxic and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration-resistant PCa.
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http://dx.doi.org/10.1002/pros.23741DOI Listing
March 2019

The novel brassinosteroid analog BR4848 inhibits angiogenesis in human endothelial cells and induces apoptosis in human cancer cells in vitro.

J Steroid Biochem Mol Biol 2018 04 4;178:263-271. Epub 2018 Jan 4.

Laboratory of Growth Regulators, Institute of Experimental Botany ASCR & Palacký University, Šlechtitelů 27, 78371, Olomouc, Czech Republic.

We report the synthesis and detailed biological study of the synthetic brassinosteroid analog 2α,3α-dihydroxy-6-oxo-5α-androstan-17β-yl N-(tert-butoxycarbonyl)-D,L-valinate (BR4848). The panel of cancer cell lines was used for characterization of its antiproliferative activity, yet had no adverse effects in normal human fibroblasts. In HeLa cells, BR4848-induced apoptosis was accompanied by increase of apoptotic subG cells, PARP-1 and caspase-7 fragmentation, downregulation of Bcl-2 and Mcl-1, an increase in caspase activity and G/M phase cell cycle arrest. Antiproliferative properties of BR4848 were exhibited by inhibition of phosphorylation of Akt, Erk1/2 and FAK. Furthermore, the developed analog exhibited in vitro antiangiogenic activity in human umbilical vein endothelial cells (HUVECs). BR4848-induced apoptosis accompanied with G/M arrest was detected in endothelial cells. BR4848 also inhibited adhesion, tube formation and migration of endothelial cells by inhibition of FAK, Erk 1/2, CDK5, VEGFR2, TNFα-stimulated production of IL-6, angiopoietin-2 and Jagged1. Finally, BR4848 did not modulate the activity nor nuclear translocation of any of the steroid receptors (ERα, ERβ, AR, MR and PR) included in reporter cell-based assays, which excludes the genomic activity of steroid receptors as a contributing factor to the observed biological activities of BR4848.
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http://dx.doi.org/10.1016/j.jsbmb.2018.01.005DOI Listing
April 2018

Clonality testing of lymphoproliferative disorders in a large cohort of primary and consultant biopsies.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2017 Jun 14;161(2):197-205. Epub 2017 Mar 14.

Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Czech Republic.

Background: Lymphoproliferative disease often presents the clinician and pathologist with a diagnostic dilemma, particularly in the early course of the disease.

Methods: We used modified BIOMED-2 protocols to detect monoclonal expansions of immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) genes in 957 formalin-fixed paraffin-embedded samples from 717 patients. To eliminate false-positive results, heteroduplex analysis was used after PCR reactions. The impact of different fixatives on DNA quality and performance of PCR was assessed.

Results: In the class of B lymphomas we detected clonal IgH rearrangement in nearly 80% of cases and in the class of T lymphomas in 64% of cases. Performance of the assays was 94.7% and 92.5% for IgH and TCR clonality, respectively. Clonality rates in various B and T lymphomas were in concordance with previous studies. We also present 10 difficult cases where PCR analysis of IgH and TCR gene rearrangements significantly contributed to a decision on the correct diagnosis.

Conclusion: These results confirm that the PCR-based analysis is suitable as a routine method and is helpful in establishing a diagnosis in morphologically unclear cases.
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http://dx.doi.org/10.5507/bp.2017.006DOI Listing
June 2017

Stimulating effect of normal-dosing of fibrates on cell proliferation: word of warning.

Lipids Health Dis 2016 Sep 22;15(1):164. Epub 2016 Sep 22.

Department of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15, Olomouc, Czech Republic.

Background: Fibrates are widely used hypolipidemic drugs, which serve as ligand of peroxisome proliferator-activated receptor α (PPARα). Recently, they have also been considered as potential anticancer agents. We studied effect of fibrates treatment on cell proliferation, expression of CYP2J2 and concomitant changes in expression of cell cycle regulatory proteins in three different human cell lines: HEK293, HepG2, and HT-29.

Methods: We used WST-1 viability test, western blot and immunocytochemistry for detection of proteins of interests and analysis of cell cycle.

Results: Our results showed that at lower concentrations of all tested fibrates, viability of all tested cell lines is increased, whereas at higher concentrations, repression is apparent. Unfortunately, the viability of tested cells is predominantly increased in a range of concentration which is reached in patient plasma. This phenomenon is accompanyed by elevation of CYP2J2, increased number of cyclin E-positive cells and decreased number of Cdc25A-positive cells in all tested cell lines, and elevated cyclin A expression in HepG2 and HT-29. These changes are concentration-dependent. We suppose that increased level of CYP2J2 could explain enhanced cell proliferation in lower concentration of fibrates.

Conclusion: Based on our results, we suggested there is no anti-cancer effect of fibrates in tested carcinoma cell lines.
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http://dx.doi.org/10.1186/s12944-016-0335-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034623PMC
September 2016

Structure activity relationship studies on cytotoxicity and the effects on steroid receptors of AB-functionalized cholestanes.

J Steroid Biochem Mol Biol 2016 May 11;159:154-69. Epub 2016 Mar 11.

Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR & Palacký University, Šlechtitelů 27, 78371 Olomouc, Czech Republic. Electronic address:

Structure-activity relationship analysis and profiling of a library of AB-functionalized cholestane derivatives closely related to brassinosteroids (BRs) were performed to examine their antiproliferative activities and activities on steroid hormone receptors. Some of the compounds were found to have strong cytotoxic activity in several human normal and cancer cell lines. The presence of a 3-hydroxy or 3-oxo group and 2,3-vicinal diol or 3,4-vicinal diol moiety were found to be necessary for optimum biological activity, as well as a six-membered B ring. According to the profiling of all steroid receptors in both agonist and antagonist mode, the majority of the cholestanes were weakly active or inactive compared to the natural ligands. Estrogenic activity was detected for two compounds, two compounds possessed antagonistic properties on estrogen receptors and seven compounds showed agonistic activity. Two active cholestane derivatives were shown to strongly influence cell viability, proliferation, cell cycle distribution, apoptosis and molecular pathways responsible for these processes in hormone-sensitive/insensitive (MCF7/MDA-MB-468) breast cancer cell lines.
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http://dx.doi.org/10.1016/j.jsbmb.2016.03.017DOI Listing
May 2016

Mechanisms of natural brassinosteroid-induced apoptosis of prostate cancer cells.

Food Chem Toxicol 2012 Nov 23;50(11):4068-76. Epub 2012 Aug 23.

Laboratory of Molecular Pathology, Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc, Czech Republic.

Brassinosteroids (BRs) are a group of polyhydroxylated sterol derivatives with important regulatory roles in various plant physiological processes. The aim of this study was to examine the mechanism of the antiproliferative activity of natural BRs 28-homocastasterone (28-homoCS) and 24-epibrassinolide (24-epiBL) in hormone-sensitive and -insensitive (LNCaP and DU-145, respectively) human prostate cancer cell lines. The effects of BRs on prostate cancer cells were surveyed using flow cytometry, Western blotting, TUNEL, DNA ladder assays and immunofluorescence analyses. The studied BRs inhibited cell growth and induced G(1) blocks in LNCaP cells accompanied by reductions in cyclin D(1), CDK4/6 and pRb expression. Following BR treatment of DU-145 cells, increases in proportions of cells in the G(2)/M phase of cell cycle were observed, accompanied by down-regulation of cyclins A and B(1). Changes in AR localization patterns in LNCaP cells treated with BRs were shown by immunofluorescence analysis. Furthermore, apoptotic detection methods demonstrated induction of apoptosis mediated by BRs in both cell lines, although changes in the expression of apoptosis-related proteins were modulated differently by 28-homoCS and 24-piBL in each cell line. The studied BRs seem to exert potent growth inhibitory and pro-apoptotic effects and could be therefore highly valuable new candidates for prostate anticancer drugs.
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http://dx.doi.org/10.1016/j.fct.2012.08.031DOI Listing
November 2012

Brassinosteroids cause cell cycle arrest and apoptosis of human breast cancer cells.

Chem Biol Interact 2010 Dec 15;188(3):487-96. Epub 2010 Sep 15.

Department of Pathology, Palacký University, Hněvotínská 3, 775 15 Olomouc, Czech Republic.

Brassinosteroids (BRs) are plant hormones that appear to be ubiquitous in both lower and higher plants. Recently, we published the first evidence that some natural BRs induce cell growth inhibitory responses in several human cancer cell lines without affecting normal non-tumor cell growth (BJ fibroblasts). The aim of the study presented here was to examine the mechanism of the antiproliferative activity of the natural BRs 28-homocastasterone (28-homoCS) and 24-epibrassinolide (24-epiBL) in human hormone-sensitive and -insensitive (MCF-7 and MDA-MB-468, respectively) breast cancer cell lines. The effects of 6, 12 and 24h treatments with 28-homoCS and 24-epiBL on cancer cells were surveyed using flow cytometry, Western blotting, TUNEL assays and immunofluorescence analyses. The studied BRs inhibited cell growth and induced blocks in the G(1) cell cycle phase. ER-α immunoreactivity was uniformly present in the nuclei of control MCF-7 cells, while cytoplasmic speckles of ER-α immunofluorescence appeared in BR-treated cells (IC(50), 24h). ER-β was relocated to the nuclei following 28-homoCS treatment and found predominantly at the periphery of the nuclei in 24-epiBL-treated cells after 24h of treatment. These changes were also accompanied by down-regulation of the ERs following BR treatment. In addition, BR application to breast cancer cells resulted in G(1) phase arrest. Furthermore, TUNEL staining and double staining with propidium iodide and acridine orange demonstrated the BR-mediated induction of apoptosis in both cell lines, although changes in the expression of apoptosis-related proteins were modulated differently by the BRs in each cell line. The studied BRs seem to exert potent growth inhibitory effects via interactions with the cell cycle machinery, and they could be highly valuable leads for agents for managing breast cancer.
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http://dx.doi.org/10.1016/j.cbi.2010.09.006DOI Listing
December 2010

Immunohistochemical assessment of E-cadherin and beta-catenin in trichofolliculomas and trichoepitheliomas.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2007 Dec;151(2):251-5

Department of Pathology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.

Background: Trichofolliculomas and trichoepitheliomas are benign skin neoplasms originating from hair follicle cells. They result from defects in the signaling pathways that regulate hair follicle morphogenesis and regeneration. Thus they seem to be an excellent model of these processes. It is known that the E-cadherin/beta-catenin system of adhesion molecules plays a crucial role in the maintenance of tissue architecture.

Aim: The aim of the present study was to investigate their involvement in benign hair follicle tumor development.

Methods: Semiquantitative intensity of expression were examined in formalin-fixed and paraffin-embedded tissue sections of 53 trichoepitheliomas, 15 trichofolliculomas and 19 normal skin samples by indirect immunohistochemistry.

Results: The intensity of E-cadherin/beta-catenin expression in tumor cells did not differ from controls. However, normal hair follicles cells exhibited membranous E-cadherin/beta-catenin expression, whereas both types of tumors, particularly trichoepitheliomas, showed E-cadherin/beta-catenin expression with a predominantly cytoplasmic localization.

Conclusions: We suggest that this dystopic distribution of the E-cadherin/beta-catenin complex in hair follicle tumor cells may be a marker of cell-cell adhesion disruption which may contribute to the tumor formation.
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http://dx.doi.org/10.5507/bp.2007.042DOI Listing
December 2007
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