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Papers by Nikola Tanic

Research paper thumbnail of Frontiers in Anti-Cancer Drug Discovery

Research paper thumbnail of Dihydroflavonoid glycosides from Viscum album and their inhibitory effects on hepatic lipid accumulation and target identification

Phytochemistry, Dec 1, 2022

Five undescribed dihydroflavonoid glycoside derivatives, namely albvisosides A-E, together with t... more Five undescribed dihydroflavonoid glycoside derivatives, namely albvisosides A-E, together with two known compounds were isolated from the roots and stem leaves of Viscum album L. var. album. (European mistletoe). Their structures were determined by HRESIMS, 1D and 2D NMR, and ECD analysis. Albvisoside B exhibits significant inhibitory effect on hepatic lipid accumulation in HepG2 cells at very low concentrations (EC 50 : 0.7 nM). Using proteome integral solubility alteration assay, the direct targets or downstream effectors of albvisoside B were elucidated. As a result, 97 proteins were identified based on ligand-induced alterations in the protein thermal stability. Bioinformatics analysis indicated that albvisoside B primarily ameliorated oleic acid-induced lipid accumulation by regulating the selenoamino acids metabolism signaling pathway. RPL3, ADAM17, and RPL14 were likely to be involved in mediating the lipid-lowering effect of albvisoside B.

Research paper thumbnail of Identification of Suitable Reference Genes for Gene Expression Studies in Tissues from Fructose-Fed Rats

Advanced Science Letters, Feb 1, 2012

Research paper thumbnail of 1008 Characterization of Newly Established P-glycoprotein Over-expressing Multi-drug Resistant Human Cancer Cell Lines

European Journal of Cancer, Jul 1, 2012

Research paper thumbnail of Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD

BMC Molecular Biology, 2010

Background: Selection of appropriate endogenous control is a critical step in gene expression ana... more Background: Selection of appropriate endogenous control is a critical step in gene expression analysis. The aim of this study was to evaluate expression stability of four frequently used endogenous controls: β-actin, glyceraldehyde-3-phosphate dehydrogenase, β 2-microglobulin and RNA polymerase II polypeptide A in peripheral blood mononuclear cells from war veterans with and without posttraumatic stress disorder (PTSD). The study was designed as to identify suitable reference gene(s) for normalization of gene expression in peripheral blood mononuclear cells in response to war trauma and/or PTSD. Results: The variability in expression of the four endogenous controls was assessed by TaqMan Real-time RT-PCR in peripheral blood mononuclear cells from: war veterans with current PTSD, those with lifetime PTSD, trauma controls and healthy subjects. Expression stability was analyzed by GeNorm and NormFinder software packages, and by direct comparison of Ct values. Both, GeNorm and NormFinder identified β-actin and glyceraldehyde-3-phosphate dehydrogenase as a pair of genes with the lowest stability value. Conclusions: The combination of β-actin and glyceraldehyde-3-phosphate dehydrogenase appeared to be the most suitable reference for studying alterations in gene expression in peripheral blood mononuclear cells related to vulnerability and resilience to PTSD, as well as to trauma-provoked developing of this disorder and recovery from it. Using glyceraldehyde-3-phosphate dehydrogenase, β-actin and β 2-microglobulin as individual endogenous controls would provide satisfactory data, while RNA polymerase II polypeptide A could not be recommended.

Research paper thumbnail of Potential of the dual mTOR kinase inhibitor AZD2014 to overcome paclitaxel resistance in anaplastic thyroid carcinoma

Cellular oncology, May 22, 2018

Purpose Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-re... more Purpose Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-resistance is often associated with changes in activity of the RAS/MAPK/ERK and PI3K/AKT/mTOR pathways and/or a high expression of ATP binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). To assess the therapeutic efficacy in ATC of a combination of the dual mTOR kinase inhibitor vistusertib (AZD2014) and paclitaxel (PTX), we generated a new cell line (Rho-) via the selection of human thyroid carcinoma 8505C cells that exhibit a low accumulation of rhodamine 123, which serves as a P-gp and BCRP substrate. Methods Immunohistochemistry was used for P-gp and BCRP expression analyses in primary ATC patient samples. Spheroid formation and immunodeficient NSG mice were used for performing in vitro and in vivo tumorigenicity assays, respectively. MTT, flow-cytometry, fluorescent microscopy, cell death and proliferation assays, as well as migration, invasion and gelatin degradation assays, were used to assess the potential of AZD2014 to enhance the effects of PTX. ATC xenografts in SCID mice were used for evaluating in vivo treatment efficacies. Results Rho-cells were found to be 10-fold more resistant to PTX than 8505C cells and, in addition, to be more tumorigenic. We also found that AZD2014 sensitized Rho-cells to PTX by inhibiting proliferation and by inducing autophagy. The combined use of AZD2014 and PTX efficiently inhibited in vitro ATC cell migration and invasion. Subsequent in vivo xenograft studies indicated that the AZD2014 and PTX combination effectively suppressed ATC tumor growth. Conclusions Our data support results from recent phase I clinical trials using combinations of AZD2014 and PTX for the treatment of solid tumors. Such combinations may also be employed for the design of novel targeted ATC treatment strategies.

Research paper thumbnail of Importance of Copy Number Alterations of FGFR1 and C-MYC Genes in Triple Negative Breast Cancer

Journal of Medical Biochemistry, Mar 1, 2019

Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical course a... more Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical course and is unresponsive to anti-HER2 and endocrine therapy. TNBC is difficult to treat and is often lethal. Given the need to find new targets for therapy we explored clinicopathological significance of copy number gain of FGFR1 and c-MYC. Our aim was to determine the impact of FGFR1 and c-MYC copy number gain on clinical course and outcome of TNBC. Methods: FGFR1 and c-MYC gene copy number alterations were evaluated in 78 archive TNBC samples using TaqMan based quantitative real time PCR assays. Results: 50% of samples had increased c-MYC copy number. c-MYC copy number gain was associated with TNBC in contrast to ER positive cancers. Our results showed significant correlation between c-MYC copy number gain and high grade of TNBCs. This suggests that c-MYC copy number could be an useful prognostic marker for TNBC patients. c-MYC copy number gain was associated with high pTNM stage as well as lobular and medullary tumor subtypes. 43% of samples had increased FGFR1 copy number. No correlations between FGFR1 copy number gain and clinicopathological variables were observed. Conclusions: We identified c-MYC copy number gain as a prognostic marker for TNBC. Our results indicate that c-Kratak sadr`aj Uvod: Trostruko negativne karcinome dojke karakteri{e agresivan klini~ki tok i neosetljivost na endokrinu i anti-HER2 tera piju. Ovi tumori se te{ko le~e i ~esto su letalni. Zbog potrebe za novim tipovima terapije, ispitali smo klini~ko-pa-tolo{ki zna~aj pove}anja broja kopija FGFR1 i c-MYC onkoge na. Cilj rada je bio da se utvrdi uticaj pove }anja broja kopija FGFR1 i c-MYC na klini~ki tok i ishod trostruko negativnog karcinome dojke. Metode: Promene u broju kopija FGFR1 i c-MYC gena odre-|ene su kvantitivnim PCR-om u realnom vremenu kod 78 arhivskih uzoraka trostruko negativnog karcinome dojke. Rezultati: 50% ispitanih uzoraka je imalo pove}an broj kopija c-MYC. Pove}anje broja kopija c-MYC gena je asocirano sa trostruko negativnim karcinomima dojke u pore-|enju sa ER pozitivnim karcinomima. Amplifikacija c-MYC je asocirana sa visokim gradusom trostruko negativnih karcinoma. Iz ovog rezultata proizilazi da bi se broj kopija c-MYC mogao smatrati korisnim prognosti~kim markerom za TNBC pacijente. Pove}anje broja kopija c-MYC gena je asocirano i sa visokim stadijumom tumora kao i sa lobularnim i medularnim podtipom. 43% ispitanih uzoraka je imalo pove}an broj kopija FGFR1. Nisu utvr|ene nikakve korelacije izme|u pove}anja broja kopija FGFR1 i klini~kih i histopatolo{kih parametara tumora.

Research paper thumbnail of The impact of TP53 and PTEN tumor suppressor genes on response to different breast cancer treatment modalities

Биомедицинска истраживања

Introduction. Breast cancer (BC) is the most frequent type of malignancy and the leading cause of... more Introduction. Breast cancer (BC) is the most frequent type of malignancy and the leading cause of cancer related death among women worldwide. BC is exceptionally heterogeneous disease and therefore distinct treatment modalities are necessary to address these differences. The aim of our study was to investigate the impact of TP53 and PTEN tumor suppressor genes (TSGs) inactivation on BC response to different treatment modalities and their possible cooperation, on post-operative BC samples. Methods. Patients were classified, based on applied adjuvant therapy, into four distinct groups: those that received hormonal therapy (HT) only, hormonal therapy combined with chemotherapy (HT/CHT), hormonal therapy combined with chemo and biological therapy (HT/CHT/H), and other systemic therapies that exclude HT. Functional inactivation of TP53 and PTEN TSG’s were studied by mutation, loss of heterozygosity (LOH) and hypermethylation analysis. Results. Our results revealed that TP53 gene was al...

Research paper thumbnail of Beyond Traditional Use of Alchemilla vulgaris: Genoprotective and Antitumor Activity In Vitro

Molecules, Nov 22, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Friend or foe: ABCG2, ABCC1 and ABCB1 expression in triple-negative breast cancer

Breast Cancer, 2021

ATP-binding cassette (ABC) transporters are responsible for the efflux of a wide variety of anti-... more ATP-binding cassette (ABC) transporters are responsible for the efflux of a wide variety of anti-cancer agents and have been implicated in the chemoresistance of various solid tumors. Chemoresistance is a major cause of therapeutic failure, especially in the highly aggressive triple-negative breast cancer (TNBC) in which, unlike estrogen receptor-expressing (ER+) BC, both endocrine and targeted treatments are ineffectual. We aimed to investigate the level and frequency of expression of the three most important ABC transporter, ABCG2, ABCC1, and ABCB1, according to breast cancer subtype. We evaluated ABCG2, ABCC1, and ABCB1 protein expressions in 124 primary breast tumors (78 samples were classified as TNBC, while 46 were classified as ER+) by immunohistochemistry and correlated it to clinicopathological characteristics and outcome. All three transporters had significantly higher expression and were more frequently expressed in TNBC compared to ER+ tumors (p < 0.0001). ABCG2 and ABCC1 had a very high level of expression in TNBC that was significantly greater compared to ABCB1 (p < 0.0001). ABCB1 expression was associated with TNBC metastatic spread (p = 0.03). In contrast, TNBC patients with high ABCG2 expression level had significantly longer disease-free interval (p = 0.03) and overall survival (p = 0.007). ABCG2, ABCC1, and ABCB1 expression in breast cancer is subtype-specific and associated with triple-negative tumors. The expression of ABCB1 may be useful as a marker of metastatic spread. Moreover, unexpectedly, our results showed a beneficial effect of ABCG2 expression on TNBC clinical behavior. These findings could have implications for the implementation of future TNBC treatment strategies.

Research paper thumbnail of Importance of Copy Number Alterations of FGFR1 and C-MYC Genes in Triple Negative Breast Cancer

Journal of Medical Biochemistry, 2018

Summary Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical ... more Summary Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical course and is unresponsive to anti-HER2 and endocrine therapy. TNBC is difficult to treat and is often lethal. Given the need to find new targets for therapy we explored clinicopathological significance of copy number gain of FGFR1 and c-MYC. Our aim was to determine the impact of FGFR1 and c-MYC copy number gain on clinical course and outcome of TNBC. Methods: FGFR1 and c-MYC gene copy number alterations were evaluated in 78 archive TNBC samples using TaqMan based quantitative real time PCR assays. Results: 50% of samples had increased c-MYC copy number. c-MYC copy number gain was associated with TNBC in contrast to ER positive cancers. Our results showed significant correlation between c-MYC copy number gain and high grade of TNBCs. This suggests that c-MYC copy number could be an useful prognostic marker for TNBC patients. c-MYC copy number gain was associated with high pTNM stage as ...

Research paper thumbnail of Reverse Transcription of 18S rRNA with Poly(dT)18 and Other Homopolymers

Plant Molecular Biology Reporter, 2012

Ribosomal 18S RNA is widely used as a housekeeping gene in expression studies, including end-poin... more Ribosomal 18S RNA is widely used as a housekeeping gene in expression studies, including end-point PCR, Northern analysis, and real-time experiments. However, there are two disadvantages and two points of error introduction in using 18S rRNA as a reference gene. First, 18S has no poly(A) tail, so it is commonly reverse transcribed with specific primers or random hexamers, independently from poly(dT)-primed transcripts. Secondly, due to its abundance, the 18S cDNA must be extensively diluted to be comparable to the tested genes. In this study, 18S rRNA from five taxonomically diverse plant species, including Physcomitrella patens, Adiantum capillus-veneris, Centaurium erythraea, Arabidopsis thaliana, and Zea mays, was successfully reverse transcribed (RT) using poly(dT)18. As all other homopolymers, including poly(dA)18, poly(dC)18, and poly(dG)18, could serve as RT primers, it was concluded that homopolymers anneal by mispriming at the sites of complementary homopolymeric runs or segments rich in complementary base. Poly(dC)18 was the most efficient as RT primer, and the only one which interfered with subsequent PCR, giving species-specific pattern of products. Poly(dT)-primed RT reactions were less efficient in comparison to specific primer or random hexamer-primed reactions. Homopolymeric priming of 18S in RT reactions is general in terms of RNA origin and the method of RNA isolation and is possibly applicable to other tailless housekeeping genes.

Research paper thumbnail of Abstract B2: Alterations of p53, PTEN, and EGFR genes in glioblastoma multiforme

Clinical Cancer Research, 2010

Glioblastomas are the most frequent and the most aggressive human brain tumors. Glioblastoma mult... more Glioblastomas are the most frequent and the most aggressive human brain tumors. Glioblastoma multiforme is one of the most intensively investigated human malignancy but the molecular mechanisms associated with the evolution of this type of tumor are still poorly understood. The aim of this study was to investigate alterations in EGFR oncogene and PTEN and p53 tumor supressor genes in 35 glioma specimen and to evaluate their role in glioma pathogenesis. DNA from tumor and blood of 35 patients with glioma was isolated and used to perform analysis of p53 mutational status using single-strand conformational polymorphism analysis (PCR- SSCP) and sequencing, evaluate loss of heterozigosity (LOH) in p53 and PTEN by fragment analysis, and to asseess EGFR gene amplification by differential PCR. Obtained results were further correlated with clinicopathological parameters. Loss of heterozygosity of PTEN was the most frequent alteration present in 62.86% of samples, followed by EGFR amplificati...

Research paper thumbnail of Citrus flavanones mildly interfere with pituitary-thyroid axis in old-aged male rats

Acta Histochemica, 2017

Please cite this article in press as: Miler, M., et al., Citrus flavanones mildly interfere with ... more Please cite this article in press as: Miler, M., et al., Citrus flavanones mildly interfere with pituitary-thyroid axis in old-aged male rats.

Research paper thumbnail of Changes in expression of GFAP, ApoE and APP mRNA and protein levels in the adult rat brain following cortical injury

Archives of Biological Sciences, 2013

The recovery period following cortical injury (CI) is characterized by a dynamic and highly compl... more The recovery period following cortical injury (CI) is characterized by a dynamic and highly complex interplay between beneficial and detrimental events. The aim of this study was to examine the expressions of Glial Fibrillary Acidic Protein (GFAP), Apolipoprotein E (ApoE) and Amyloid Precursor Protein (APP), all of which are involved in brain plasticity and neurodegeneration. Our results reveal that CI strongly influenced GFAP, ApoE and APP mRNA expression, as well as GFAP and ApoE protein expression. Considering the pivotal role of these proteins in the brain, the obtained results point to their potential contribution in neurodegeneration and consequent Alzheimer's disease development.

Research paper thumbnail of Targeted Anti-Cancer Therapy, Acquiring and Overcoming Multi-Drug Resistance

Frontiers in Anti-Cancer Drug Discovery, 2014

Research paper thumbnail of could P53 And Ras Alterations Be Responsible For The Progression Of Cerebellar Glioblastomas? : m237

Clinical Chemistry and Laboratory Medicine, 2015

Research paper thumbnail of Identification of genes associated with non-small-cell lung cancer promotion and progression

Lung Cancer, 2010

Lung cancer is the most common cause of neoplasia-related death worldwide. One of the crucial ear... more Lung cancer is the most common cause of neoplasia-related death worldwide. One of the crucial early events in carcinogenesis is the induction of genomic instability and mutator phenotype. We investigated genomic instability in 30 patients with non-small-cell lung cancer (NSCLC) by comparing DNA fingerprints of paired tumor and normal tissues using arbitrarily primed polymerase chain reaction (AP-PCR). Selected 21 DNA bands with altered mobility were isolated from polyacrylamide gels, cloned and sequenced. Obtained sequences were submitted to homology search in GenBank database which revealed the following genes: TSPAN14, CDH12, RDH10, CYP4Z1, KIR, E2F4, PHACTR3, PHF20, PRAME family member and SLC2A13. Following the identification of these genes we examined their relation to the clinicopathological parameters and survival of the patients. Our study revealed that genetic alterations of TSPAN14, SLC2A13 and PHF20 appeared prevalently in tumors of grade 1, stage I suggesting that structural changes of these genes could play a role in NSCLC promotion. Contrary to this CYP4Z1, KIR and RDH10 were prevalently mutated in tumors of grade 3, stage III suggesting that they could play a role in NSCLC progression. E2F4, PHACTR3, PRAME family member and CDH12 most probably play important role in NSCLC geneses. In conclusion, our study revealed altered genes previously not described in regard to this type of cancer.

Research paper thumbnail of Coalterations of p53 and PTEN tumor suppressor genes in non–small cell lung carcinoma patients

Translational Research, 2011

The inactivation of p53 and PTEN tumor suppressor genes is a common genetic event in lung cancer.... more The inactivation of p53 and PTEN tumor suppressor genes is a common genetic event in lung cancer. However, data on the effect of the joint inactivation of tumor-suppressor genes in non-small cell lung carcinoma (NSCLC) are lacking. The purpose of this study was to investigate the alterations in PTEN and p53 genes, as well as to evaluate their mutual role in NSCLC pathogenesis and their impact on survival rate. To that end, polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP), sequencing, methylation-specific PCR, and fragment analysis were used. The results obtained were correlated with clinicopathologic parameters, the level of genomic instability, and patient survival. Overall, 13% of specimens had aberrant p53 only, 13% had inactive PTEN only, and 50% of samples had both genes altered. Correlation analyses showed that the mutual inactivation of p53 and PTEN was a frequent event that was associated significantly with the increased level of genomic instability and lymph node invasion implying their synergistic effect in promoting metastatic phenotype of this kind of cancer. In addition, our results revealed a significant association of joint alterations of these genes with dramatically shortened survival indicating that aberrant p53 and PTEN could be used as an adverse prognostic factor for NSCLC patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; outcome. Our findings established the relevance of the combinatorial inactivation of p53 and PTEN in NSCLC progression and identified a subgroup of patients with a particularly aggressive disease.

Research paper thumbnail of TP53 and C-MYC Co-Alterations – A Hallmark of Oral Cancer Progression / SIMULTANA ALTERACIJA TP53 I C-MYC GENA – OBELEŽJE PROGRESIJE ORALNIH KARCINOMA

Journal of Medical Biochemistry, 2013

Summary Background: Head and neck squamous cell carcinoma, including oral cancer, is the sixth mo... more Summary Background: Head and neck squamous cell carcinoma, including oral cancer, is the sixth most common cancer worldwide. Despite advances in surgery and treatment, the 5-year survival rate has not improved significantly. There- fore, reliable molecular markers for oral cancer progression are badly needed. Methods: We conducted a copy number analysis to esti- mate amplification status of c-myc, cycD1 and EGFR onco- genes, mutational PCR-SSCP analysis to determine activa- tion of H-ras oncogene and inactivation of TP55 tumour suppressor gene and methylation specific PCR analysis to evaluate hypermethylation of p16 and MGMT genes. Results: c-myc oncogene was amplified in 56.7%, cycD1 in 20% and EGFR in 16.7% of Oral Squamous Cell Carci- noma (OSCC) cases while H-ras was activated in 33.3% of samples. Amplification of c-myc was significantly associat- ed with the tumour grade 2. Interestingly, EGFR and H-ras alterations were mutually exclusive. p16 and MGMT were inactivated by hyper...

Research paper thumbnail of Frontiers in Anti-Cancer Drug Discovery

Research paper thumbnail of Dihydroflavonoid glycosides from Viscum album and their inhibitory effects on hepatic lipid accumulation and target identification

Phytochemistry, Dec 1, 2022

Five undescribed dihydroflavonoid glycoside derivatives, namely albvisosides A-E, together with t... more Five undescribed dihydroflavonoid glycoside derivatives, namely albvisosides A-E, together with two known compounds were isolated from the roots and stem leaves of Viscum album L. var. album. (European mistletoe). Their structures were determined by HRESIMS, 1D and 2D NMR, and ECD analysis. Albvisoside B exhibits significant inhibitory effect on hepatic lipid accumulation in HepG2 cells at very low concentrations (EC 50 : 0.7 nM). Using proteome integral solubility alteration assay, the direct targets or downstream effectors of albvisoside B were elucidated. As a result, 97 proteins were identified based on ligand-induced alterations in the protein thermal stability. Bioinformatics analysis indicated that albvisoside B primarily ameliorated oleic acid-induced lipid accumulation by regulating the selenoamino acids metabolism signaling pathway. RPL3, ADAM17, and RPL14 were likely to be involved in mediating the lipid-lowering effect of albvisoside B.

Research paper thumbnail of Identification of Suitable Reference Genes for Gene Expression Studies in Tissues from Fructose-Fed Rats

Advanced Science Letters, Feb 1, 2012

Research paper thumbnail of 1008 Characterization of Newly Established P-glycoprotein Over-expressing Multi-drug Resistant Human Cancer Cell Lines

European Journal of Cancer, Jul 1, 2012

Research paper thumbnail of Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD

BMC Molecular Biology, 2010

Background: Selection of appropriate endogenous control is a critical step in gene expression ana... more Background: Selection of appropriate endogenous control is a critical step in gene expression analysis. The aim of this study was to evaluate expression stability of four frequently used endogenous controls: β-actin, glyceraldehyde-3-phosphate dehydrogenase, β 2-microglobulin and RNA polymerase II polypeptide A in peripheral blood mononuclear cells from war veterans with and without posttraumatic stress disorder (PTSD). The study was designed as to identify suitable reference gene(s) for normalization of gene expression in peripheral blood mononuclear cells in response to war trauma and/or PTSD. Results: The variability in expression of the four endogenous controls was assessed by TaqMan Real-time RT-PCR in peripheral blood mononuclear cells from: war veterans with current PTSD, those with lifetime PTSD, trauma controls and healthy subjects. Expression stability was analyzed by GeNorm and NormFinder software packages, and by direct comparison of Ct values. Both, GeNorm and NormFinder identified β-actin and glyceraldehyde-3-phosphate dehydrogenase as a pair of genes with the lowest stability value. Conclusions: The combination of β-actin and glyceraldehyde-3-phosphate dehydrogenase appeared to be the most suitable reference for studying alterations in gene expression in peripheral blood mononuclear cells related to vulnerability and resilience to PTSD, as well as to trauma-provoked developing of this disorder and recovery from it. Using glyceraldehyde-3-phosphate dehydrogenase, β-actin and β 2-microglobulin as individual endogenous controls would provide satisfactory data, while RNA polymerase II polypeptide A could not be recommended.

Research paper thumbnail of Potential of the dual mTOR kinase inhibitor AZD2014 to overcome paclitaxel resistance in anaplastic thyroid carcinoma

Cellular oncology, May 22, 2018

Purpose Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-re... more Purpose Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-resistance is often associated with changes in activity of the RAS/MAPK/ERK and PI3K/AKT/mTOR pathways and/or a high expression of ATP binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). To assess the therapeutic efficacy in ATC of a combination of the dual mTOR kinase inhibitor vistusertib (AZD2014) and paclitaxel (PTX), we generated a new cell line (Rho-) via the selection of human thyroid carcinoma 8505C cells that exhibit a low accumulation of rhodamine 123, which serves as a P-gp and BCRP substrate. Methods Immunohistochemistry was used for P-gp and BCRP expression analyses in primary ATC patient samples. Spheroid formation and immunodeficient NSG mice were used for performing in vitro and in vivo tumorigenicity assays, respectively. MTT, flow-cytometry, fluorescent microscopy, cell death and proliferation assays, as well as migration, invasion and gelatin degradation assays, were used to assess the potential of AZD2014 to enhance the effects of PTX. ATC xenografts in SCID mice were used for evaluating in vivo treatment efficacies. Results Rho-cells were found to be 10-fold more resistant to PTX than 8505C cells and, in addition, to be more tumorigenic. We also found that AZD2014 sensitized Rho-cells to PTX by inhibiting proliferation and by inducing autophagy. The combined use of AZD2014 and PTX efficiently inhibited in vitro ATC cell migration and invasion. Subsequent in vivo xenograft studies indicated that the AZD2014 and PTX combination effectively suppressed ATC tumor growth. Conclusions Our data support results from recent phase I clinical trials using combinations of AZD2014 and PTX for the treatment of solid tumors. Such combinations may also be employed for the design of novel targeted ATC treatment strategies.

Research paper thumbnail of Importance of Copy Number Alterations of FGFR1 and C-MYC Genes in Triple Negative Breast Cancer

Journal of Medical Biochemistry, Mar 1, 2019

Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical course a... more Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical course and is unresponsive to anti-HER2 and endocrine therapy. TNBC is difficult to treat and is often lethal. Given the need to find new targets for therapy we explored clinicopathological significance of copy number gain of FGFR1 and c-MYC. Our aim was to determine the impact of FGFR1 and c-MYC copy number gain on clinical course and outcome of TNBC. Methods: FGFR1 and c-MYC gene copy number alterations were evaluated in 78 archive TNBC samples using TaqMan based quantitative real time PCR assays. Results: 50% of samples had increased c-MYC copy number. c-MYC copy number gain was associated with TNBC in contrast to ER positive cancers. Our results showed significant correlation between c-MYC copy number gain and high grade of TNBCs. This suggests that c-MYC copy number could be an useful prognostic marker for TNBC patients. c-MYC copy number gain was associated with high pTNM stage as well as lobular and medullary tumor subtypes. 43% of samples had increased FGFR1 copy number. No correlations between FGFR1 copy number gain and clinicopathological variables were observed. Conclusions: We identified c-MYC copy number gain as a prognostic marker for TNBC. Our results indicate that c-Kratak sadr`aj Uvod: Trostruko negativne karcinome dojke karakteri{e agresivan klini~ki tok i neosetljivost na endokrinu i anti-HER2 tera piju. Ovi tumori se te{ko le~e i ~esto su letalni. Zbog potrebe za novim tipovima terapije, ispitali smo klini~ko-pa-tolo{ki zna~aj pove}anja broja kopija FGFR1 i c-MYC onkoge na. Cilj rada je bio da se utvrdi uticaj pove }anja broja kopija FGFR1 i c-MYC na klini~ki tok i ishod trostruko negativnog karcinome dojke. Metode: Promene u broju kopija FGFR1 i c-MYC gena odre-|ene su kvantitivnim PCR-om u realnom vremenu kod 78 arhivskih uzoraka trostruko negativnog karcinome dojke. Rezultati: 50% ispitanih uzoraka je imalo pove}an broj kopija c-MYC. Pove}anje broja kopija c-MYC gena je asocirano sa trostruko negativnim karcinomima dojke u pore-|enju sa ER pozitivnim karcinomima. Amplifikacija c-MYC je asocirana sa visokim gradusom trostruko negativnih karcinoma. Iz ovog rezultata proizilazi da bi se broj kopija c-MYC mogao smatrati korisnim prognosti~kim markerom za TNBC pacijente. Pove}anje broja kopija c-MYC gena je asocirano i sa visokim stadijumom tumora kao i sa lobularnim i medularnim podtipom. 43% ispitanih uzoraka je imalo pove}an broj kopija FGFR1. Nisu utvr|ene nikakve korelacije izme|u pove}anja broja kopija FGFR1 i klini~kih i histopatolo{kih parametara tumora.

Research paper thumbnail of The impact of TP53 and PTEN tumor suppressor genes on response to different breast cancer treatment modalities

Биомедицинска истраживања

Introduction. Breast cancer (BC) is the most frequent type of malignancy and the leading cause of... more Introduction. Breast cancer (BC) is the most frequent type of malignancy and the leading cause of cancer related death among women worldwide. BC is exceptionally heterogeneous disease and therefore distinct treatment modalities are necessary to address these differences. The aim of our study was to investigate the impact of TP53 and PTEN tumor suppressor genes (TSGs) inactivation on BC response to different treatment modalities and their possible cooperation, on post-operative BC samples. Methods. Patients were classified, based on applied adjuvant therapy, into four distinct groups: those that received hormonal therapy (HT) only, hormonal therapy combined with chemotherapy (HT/CHT), hormonal therapy combined with chemo and biological therapy (HT/CHT/H), and other systemic therapies that exclude HT. Functional inactivation of TP53 and PTEN TSG’s were studied by mutation, loss of heterozygosity (LOH) and hypermethylation analysis. Results. Our results revealed that TP53 gene was al...

Research paper thumbnail of Beyond Traditional Use of Alchemilla vulgaris: Genoprotective and Antitumor Activity In Vitro

Molecules, Nov 22, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Research paper thumbnail of Friend or foe: ABCG2, ABCC1 and ABCB1 expression in triple-negative breast cancer

Breast Cancer, 2021

ATP-binding cassette (ABC) transporters are responsible for the efflux of a wide variety of anti-... more ATP-binding cassette (ABC) transporters are responsible for the efflux of a wide variety of anti-cancer agents and have been implicated in the chemoresistance of various solid tumors. Chemoresistance is a major cause of therapeutic failure, especially in the highly aggressive triple-negative breast cancer (TNBC) in which, unlike estrogen receptor-expressing (ER+) BC, both endocrine and targeted treatments are ineffectual. We aimed to investigate the level and frequency of expression of the three most important ABC transporter, ABCG2, ABCC1, and ABCB1, according to breast cancer subtype. We evaluated ABCG2, ABCC1, and ABCB1 protein expressions in 124 primary breast tumors (78 samples were classified as TNBC, while 46 were classified as ER+) by immunohistochemistry and correlated it to clinicopathological characteristics and outcome. All three transporters had significantly higher expression and were more frequently expressed in TNBC compared to ER+ tumors (p < 0.0001). ABCG2 and ABCC1 had a very high level of expression in TNBC that was significantly greater compared to ABCB1 (p < 0.0001). ABCB1 expression was associated with TNBC metastatic spread (p = 0.03). In contrast, TNBC patients with high ABCG2 expression level had significantly longer disease-free interval (p = 0.03) and overall survival (p = 0.007). ABCG2, ABCC1, and ABCB1 expression in breast cancer is subtype-specific and associated with triple-negative tumors. The expression of ABCB1 may be useful as a marker of metastatic spread. Moreover, unexpectedly, our results showed a beneficial effect of ABCG2 expression on TNBC clinical behavior. These findings could have implications for the implementation of future TNBC treatment strategies.

Research paper thumbnail of Importance of Copy Number Alterations of FGFR1 and C-MYC Genes in Triple Negative Breast Cancer

Journal of Medical Biochemistry, 2018

Summary Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical ... more Summary Background: Triple negative breast cancer (TNBC) is characterized by aggressive clinical course and is unresponsive to anti-HER2 and endocrine therapy. TNBC is difficult to treat and is often lethal. Given the need to find new targets for therapy we explored clinicopathological significance of copy number gain of FGFR1 and c-MYC. Our aim was to determine the impact of FGFR1 and c-MYC copy number gain on clinical course and outcome of TNBC. Methods: FGFR1 and c-MYC gene copy number alterations were evaluated in 78 archive TNBC samples using TaqMan based quantitative real time PCR assays. Results: 50% of samples had increased c-MYC copy number. c-MYC copy number gain was associated with TNBC in contrast to ER positive cancers. Our results showed significant correlation between c-MYC copy number gain and high grade of TNBCs. This suggests that c-MYC copy number could be an useful prognostic marker for TNBC patients. c-MYC copy number gain was associated with high pTNM stage as ...

Research paper thumbnail of Reverse Transcription of 18S rRNA with Poly(dT)18 and Other Homopolymers

Plant Molecular Biology Reporter, 2012

Ribosomal 18S RNA is widely used as a housekeeping gene in expression studies, including end-poin... more Ribosomal 18S RNA is widely used as a housekeeping gene in expression studies, including end-point PCR, Northern analysis, and real-time experiments. However, there are two disadvantages and two points of error introduction in using 18S rRNA as a reference gene. First, 18S has no poly(A) tail, so it is commonly reverse transcribed with specific primers or random hexamers, independently from poly(dT)-primed transcripts. Secondly, due to its abundance, the 18S cDNA must be extensively diluted to be comparable to the tested genes. In this study, 18S rRNA from five taxonomically diverse plant species, including Physcomitrella patens, Adiantum capillus-veneris, Centaurium erythraea, Arabidopsis thaliana, and Zea mays, was successfully reverse transcribed (RT) using poly(dT)18. As all other homopolymers, including poly(dA)18, poly(dC)18, and poly(dG)18, could serve as RT primers, it was concluded that homopolymers anneal by mispriming at the sites of complementary homopolymeric runs or segments rich in complementary base. Poly(dC)18 was the most efficient as RT primer, and the only one which interfered with subsequent PCR, giving species-specific pattern of products. Poly(dT)-primed RT reactions were less efficient in comparison to specific primer or random hexamer-primed reactions. Homopolymeric priming of 18S in RT reactions is general in terms of RNA origin and the method of RNA isolation and is possibly applicable to other tailless housekeeping genes.

Research paper thumbnail of Abstract B2: Alterations of p53, PTEN, and EGFR genes in glioblastoma multiforme

Clinical Cancer Research, 2010

Glioblastomas are the most frequent and the most aggressive human brain tumors. Glioblastoma mult... more Glioblastomas are the most frequent and the most aggressive human brain tumors. Glioblastoma multiforme is one of the most intensively investigated human malignancy but the molecular mechanisms associated with the evolution of this type of tumor are still poorly understood. The aim of this study was to investigate alterations in EGFR oncogene and PTEN and p53 tumor supressor genes in 35 glioma specimen and to evaluate their role in glioma pathogenesis. DNA from tumor and blood of 35 patients with glioma was isolated and used to perform analysis of p53 mutational status using single-strand conformational polymorphism analysis (PCR- SSCP) and sequencing, evaluate loss of heterozigosity (LOH) in p53 and PTEN by fragment analysis, and to asseess EGFR gene amplification by differential PCR. Obtained results were further correlated with clinicopathological parameters. Loss of heterozygosity of PTEN was the most frequent alteration present in 62.86% of samples, followed by EGFR amplificati...

Research paper thumbnail of Citrus flavanones mildly interfere with pituitary-thyroid axis in old-aged male rats

Acta Histochemica, 2017

Please cite this article in press as: Miler, M., et al., Citrus flavanones mildly interfere with ... more Please cite this article in press as: Miler, M., et al., Citrus flavanones mildly interfere with pituitary-thyroid axis in old-aged male rats.

Research paper thumbnail of Changes in expression of GFAP, ApoE and APP mRNA and protein levels in the adult rat brain following cortical injury

Archives of Biological Sciences, 2013

The recovery period following cortical injury (CI) is characterized by a dynamic and highly compl... more The recovery period following cortical injury (CI) is characterized by a dynamic and highly complex interplay between beneficial and detrimental events. The aim of this study was to examine the expressions of Glial Fibrillary Acidic Protein (GFAP), Apolipoprotein E (ApoE) and Amyloid Precursor Protein (APP), all of which are involved in brain plasticity and neurodegeneration. Our results reveal that CI strongly influenced GFAP, ApoE and APP mRNA expression, as well as GFAP and ApoE protein expression. Considering the pivotal role of these proteins in the brain, the obtained results point to their potential contribution in neurodegeneration and consequent Alzheimer's disease development.

Research paper thumbnail of Targeted Anti-Cancer Therapy, Acquiring and Overcoming Multi-Drug Resistance

Frontiers in Anti-Cancer Drug Discovery, 2014

Research paper thumbnail of could P53 And Ras Alterations Be Responsible For The Progression Of Cerebellar Glioblastomas? : m237

Clinical Chemistry and Laboratory Medicine, 2015

Research paper thumbnail of Identification of genes associated with non-small-cell lung cancer promotion and progression

Lung Cancer, 2010

Lung cancer is the most common cause of neoplasia-related death worldwide. One of the crucial ear... more Lung cancer is the most common cause of neoplasia-related death worldwide. One of the crucial early events in carcinogenesis is the induction of genomic instability and mutator phenotype. We investigated genomic instability in 30 patients with non-small-cell lung cancer (NSCLC) by comparing DNA fingerprints of paired tumor and normal tissues using arbitrarily primed polymerase chain reaction (AP-PCR). Selected 21 DNA bands with altered mobility were isolated from polyacrylamide gels, cloned and sequenced. Obtained sequences were submitted to homology search in GenBank database which revealed the following genes: TSPAN14, CDH12, RDH10, CYP4Z1, KIR, E2F4, PHACTR3, PHF20, PRAME family member and SLC2A13. Following the identification of these genes we examined their relation to the clinicopathological parameters and survival of the patients. Our study revealed that genetic alterations of TSPAN14, SLC2A13 and PHF20 appeared prevalently in tumors of grade 1, stage I suggesting that structural changes of these genes could play a role in NSCLC promotion. Contrary to this CYP4Z1, KIR and RDH10 were prevalently mutated in tumors of grade 3, stage III suggesting that they could play a role in NSCLC progression. E2F4, PHACTR3, PRAME family member and CDH12 most probably play important role in NSCLC geneses. In conclusion, our study revealed altered genes previously not described in regard to this type of cancer.

Research paper thumbnail of Coalterations of p53 and PTEN tumor suppressor genes in non–small cell lung carcinoma patients

Translational Research, 2011

The inactivation of p53 and PTEN tumor suppressor genes is a common genetic event in lung cancer.... more The inactivation of p53 and PTEN tumor suppressor genes is a common genetic event in lung cancer. However, data on the effect of the joint inactivation of tumor-suppressor genes in non-small cell lung carcinoma (NSCLC) are lacking. The purpose of this study was to investigate the alterations in PTEN and p53 genes, as well as to evaluate their mutual role in NSCLC pathogenesis and their impact on survival rate. To that end, polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP), sequencing, methylation-specific PCR, and fragment analysis were used. The results obtained were correlated with clinicopathologic parameters, the level of genomic instability, and patient survival. Overall, 13% of specimens had aberrant p53 only, 13% had inactive PTEN only, and 50% of samples had both genes altered. Correlation analyses showed that the mutual inactivation of p53 and PTEN was a frequent event that was associated significantly with the increased level of genomic instability and lymph node invasion implying their synergistic effect in promoting metastatic phenotype of this kind of cancer. In addition, our results revealed a significant association of joint alterations of these genes with dramatically shortened survival indicating that aberrant p53 and PTEN could be used as an adverse prognostic factor for NSCLC patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; outcome. Our findings established the relevance of the combinatorial inactivation of p53 and PTEN in NSCLC progression and identified a subgroup of patients with a particularly aggressive disease.

Research paper thumbnail of TP53 and C-MYC Co-Alterations – A Hallmark of Oral Cancer Progression / SIMULTANA ALTERACIJA TP53 I C-MYC GENA – OBELEŽJE PROGRESIJE ORALNIH KARCINOMA

Journal of Medical Biochemistry, 2013

Summary Background: Head and neck squamous cell carcinoma, including oral cancer, is the sixth mo... more Summary Background: Head and neck squamous cell carcinoma, including oral cancer, is the sixth most common cancer worldwide. Despite advances in surgery and treatment, the 5-year survival rate has not improved significantly. There- fore, reliable molecular markers for oral cancer progression are badly needed. Methods: We conducted a copy number analysis to esti- mate amplification status of c-myc, cycD1 and EGFR onco- genes, mutational PCR-SSCP analysis to determine activa- tion of H-ras oncogene and inactivation of TP55 tumour suppressor gene and methylation specific PCR analysis to evaluate hypermethylation of p16 and MGMT genes. Results: c-myc oncogene was amplified in 56.7%, cycD1 in 20% and EGFR in 16.7% of Oral Squamous Cell Carci- noma (OSCC) cases while H-ras was activated in 33.3% of samples. Amplification of c-myc was significantly associat- ed with the tumour grade 2. Interestingly, EGFR and H-ras alterations were mutually exclusive. p16 and MGMT were inactivated by hyper...