Search

Human thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing enzyme which plays a crucial role in regulating numerous redox signalling pathways within the cell. While its functioning is important in all cells, levels of TrxR1 expression are higher in cancer cells, possibly as an adaptation to much higher levels of reactive oxygen species and the need for more extensive DNA synthesis. This makes TrxR1 an attractive target for cancer therapy development. Inspired by the structure of disulphide compounds which have advanced through various stages of clinical development, we designed a series of dithiodiglycolic acid derivatives. These were prepared from respective thiol synthons using an iodine- or benzotriazolyl chloride-promoted oxidative disulphide bond formation. Inhibition of TrxR present in cell lysates from human neuroblastoma cells (SH-SY5Y) and rat liver cells indicated several compounds with a potential for TrxR inhibition. Some of these compounds were also tested for growth inhibition against two human cancer cell lines and normal human keratinocytes., Graphical Abstract

Background: Glioblastoma (GBM) highly expresses Src tyrosine kinase involved in survival, proliferation, angiogenesis and invasiveness of tumor cells. Src activation also reduces reactive oxygen species (ROS) generation, whereas Src inhibitors are able to increase cellular ROS levels. Methods: Pro-oxidative effects of two pyrazolo[3,4-d]pyrimidine derivatives—Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306—were investigated in human GBM cells U87 and patient-derived GBM-6. ROS production and changes in mitochondrial membrane potential were assessed by flow cytometry. The expression levels of superoxide dismutase 1 (SOD1) and 2 (SOD2) were studied by Western blot. DNA damage, cell death induction and senescence were also examined in GBM-6 cells. Results: Si306 and pro-Si306 more prominently triggered ROS production and expression of antioxidant enzymes in primary GBM cells. These effects were followed by mitochondrial membrane potential disruption, double-strand DNA breaks and senescence that eventually led to necrosis. Conclusion: Src kinase inhibitors, Si306 and pro-Si306, showed significant pro-oxidative potential in patient-derived GBM cells. This feature contributes to the already demonstrated anti-glioblastoma properties of these compounds in vitro and in vivo and encourages clinical investigations.

Drug resistance remains the major cause of cancer treatment failure especially at the late stage of the disease. However, based on their versatile chemistry, metal and metalloid compounds offer the possibility to design fine-tuned drugs to circumvent and even specifically target drug-resistant cancer cells. Based on the paramount importance of platinum drugs in the clinics, two main areas of drug resistance reversal strategies exist: overcoming resistance to platinum drugs as well as multidrug resistance based on ABC efflux pumps. The current review provides an overview of both aspects of drug design and discusses the open questions in the field. The areas of drug resistance covered in this article involve: 1) Altered expression of proteins involved in metal uptake, efflux or intracellular distribution, 2) Enhanced drug efflux via ABC transporters, 3) Altered metabolism in drug-resistant cancer cells, 4) Altered thiol or redox homeostasis, 5) Altered DNA damage recognition and enhanced DNA damage repair, 6) Impaired induction of apoptosis and 7) Altered interaction with the immune system. This review represents the first collection of metal (including platinum, ruthenium, iridium, gold, and copper) and metalloid drugs (e.g. arsenic and selenium) which demonstrated drug resistance reversal activity. A special focus is on compounds characterized by collateral sensitivity of ABC transporter-overexpressing cancer cells. Through this approach, we wish to draw the attention to open research questions in the field. Future investigations are warranted to obtain more insights into the mechanisms of action of the most potent compounds which target specific modalities of drug resistance. ortuguese Foundation for Science and Technology UIDB/00100/2020, PTDC/QUI-QIN/28662/2017 Ministry of Education, Science and Techno-logical Development of Republic of Serbia 451-03-9/ 2021-14/200116 Ministerio de Ciencia e Innovación CTQ2016-78703-P

Currently, available glioblastoma (GBM) treatment remains ineffective, with relapse after initial response and low survival rate of GBM patients. The reasons behind limited capacities for GBM treatment are high tumor heterogeneity, invasiveness, and occurrence of drug resistance. Therefore, developing novel therapeutic strategies is of utmost importance. Thioredoxin reductase (TrxR) is a novel, promising target due to its overexpression in many cancer types and important role in cancer progression. Previous research on Ugi-type Michael acceptors-inhibitors of TrxR showed desirable anticancer properties, with significant selectivity toward cancer cells. Herein, two TrxR inhibitors, 5 and 6, underwent in-depth study on multidrug-resistant (MDR) glioma cell lines. Besides the antioxidative effects, 5 and 6 induced cell death, decreased cell proliferation, and suppressed invasion and migration of glioma cells. Both compounds showed a synergistic effect in combination with temozolomide (TMZ), a first-line chemotherapeutic for GBM treatment. Moreover, 5 and 6 affected activity of P-glycoprotein extrusion pump that could be found in cancer cells and in the blood-brain barrier (BBB), thus showing potential for suppressing MDR phenotype in cancer cells and evading BBB. In conclusion, investigated TrxR inhibitors are effective anticancer compounds, acting through inhibition of the thioredoxin system and perturbation of antioxidative defense systems of glioma cells. They are suitable for combining with other chemotherapeutics, able to surpass the BBB and overcome MDR. Thus, our findings suggest further exploration of Ugi-type Michael acceptors-TrxR inhibitors' potential as an adjuvant therapy for GBM treatment.

Numerous studies support the hypothesis that grape skin extract may have the ability to inhibit health complications such as diabetes, kidney lipotoxicity, prostate cancer, inflammation in the lung, and oxidative damage after exposure to cigarette smoke, etc. The use of grape seed extract in treatments of mice and rats showed the inhibition of lipid peroxidation and DNA fragmentation in brain and liver tissue, and the reduction of myocardial infarct size. In addition, the protective effect of grape seed extract was found on nephrotoxicity, gastrointestinal injury, and regarding the serum chemistry changes. In vitro, the grape seed extract showed inhibition of various human cancer cell lines. Our research with Montenegrin grapevine variety Vranac showed that the quality parameters of grape could be managed by irrigation, during suitable climatic conditions. The results of biological tests with colorectal carcinoma cells lines represent novel information concerning anticancer properties of grape skin extracts. Ozturk M, Dilfuza E, Pešić M, editors. Biodiversity and Biomedicine: Our Future provides. Cambridge (Massachusetts, United States): Academic Press - Elsevier; 2020. p. 385-401.

Simple Summary P-glycoprotein (P-gp) is an ATP-binding cassette transporter whose overexpression in cancer cells is one of the main causes of multidrug resistance (MDR). Tyrosine kinase inhibitors (TKIs) have been reported to interact with ABC transporters and in some cases, increase the susceptibility of cancer cells to chemotherapy. We investigated the potential of novel TKI pyrazolo[3,4-d] pyrimidines and their prodrugs to inhibit P-gp in two MDR cancer cell lines with P-gp overexpression. The tested compounds were able to suppress P-gp by inhibiting its ATPase activity. Interestingly, prodrugs displayed a stronger potential to modulate P-gp and showed higher interaction energies in the docking simulations compared to their parent drugs. Furthermore, prodrugs showed significant potential to inhibit P-gp activity even in prolonged treatment and therefore to enhance the efficacy of doxorubicin and paclitaxel in MDR cancer cells. All of these characteristics imply that the new TKIs could be considered a valuable strategy for combating resistant cancers, especially in combination with other chemotherapeutics. Abstract Tyrosine kinase inhibitors (TKIs) often interact with the multidrug resistant (MDR) phenotype of cancer cells. In some cases, TKIs increase the susceptibility of MDR cancer cells to chemotherapy. As the overexpression of membrane transporter P-glycoprotein (P-gp) is the most common alteration in MDR cancer cells, we investigated the effects of TKI pyrazolo[3,4-d]pyrimidines on P-gp inhibition in two cellular models comprising sensitive and corresponding MDR cancer cells (human non-small cell lung carcinoma and colorectal adenocarcinoma). Tested TKIs showed collateral sensitivity by inducing stronger inhibition of MDR cancer cell line viability. Moreover, TKIs directly interacted with P-gp and inhibited its ATPase activity. Their potential P-gp binding site was proposed by molecular docking simulations. TKIs reversed resistance to doxorubicin and paclitaxel in a concentration-dependent manner. The expression studies excluded the indirect effect of TKIs on P-gp through regulation of its expression. A kinetics study showed that TKIs decreased P-gp activity and this effect was sustained for seven days in both MDR models. Therefore, pyrazolo[3,4-d]pyrimidines with potential for reversing P-gp-mediated MDR even in prolonged treatments can be considered a new therapeutic strategy for overcoming cancer MDR.

The efficacy of microtubule targeting agents in cancer treatment has been compromised by the development of drug resistance that may involve both, P-glycoprotein overexpression and the changes in β-tubulin isoforms' expression. The anti-Topoisomerase II activity of methyl 4-((E)-2-(methoxycarbonyl)vinyloxy)oct-2-ynoate (DTA0100) was recently reported. Herein, we further evaluated this propargylic enol ether derivative and found that it exerts inhibitory effect on tubulin polymerization by binding to colchicine binding site. DTA0100 mitotic arrest properties were investigated in two multi-drug resistant cancer cell lines with P-glycoprotein overexpression (colorectal carcinoma and glioblastoma). The sensitivity of multi-drug resistant cancer cell lines to DTA0100 was not significantly changed in contrast to microtubule targeting agents such as paclitaxel, vinblastine and colchicine. DTA0100 clearly induced microtubule depolymerization, leading to disturbance of cell cycle kinetics and subsequent apoptosis. The fine-tuning in β-tubulin isoforms expression observed in multi-drug resistant cancer cells may influence the efficacy of DTA0100. Importantly, DTA0100 blocked the P-glycoprotein function in both multi-drug resistant cancer cell lines without inducing the increase in P-glycoprotein expression. Therefore, DTA0100 acting as dual inhibitor of Topoisomerase II and microtubule formation could be considered as multi-potent anticancer agent. Besides, it is able to overcome the problem of drug resistance that emerges in the therapeutic approaches with either Topoisomerase II or microtubule targeting agents. This is the peer reviewed version of the following article: Podolski-Renić A, Banković J, Dinić J, Ríos-Luci C, Fernandes MX, Ortega N, Kovačević-Grujičić N, Martín VS, Padrón JM, Pešić M. DTA0100, dual topoisomerase II and microtubule inhibitor, evades paclitaxel resistance in P-glycoprotein overexpressing cancer cells. Eur. J. Pharm. Sci. 2017;105:159-168. [http://dx.doi.org/10.1016/j.ejps.2017.05.011] Related to: [https://ibiss-r.rcub.bg.ac.rs/handle/123456789/2762]

Heat Shock Protein 90 (Hsp90) chaperone interacts with a broad range of client proteins involved in cancerogenesis and cancer progression. However, Hsp90 inhibitors were unsuccessful as anticancer agents due to their high toxicity, lack of selectivity against cancer cells and extrusion by membrane transporters responsible for multidrug resistance (MDR) such as P-glycoprotein (P-gp). Recognizing the potential of new compounds to inhibit P-gp function and/or expression is essential in the search for effective anticancer drugs. Eleven Hsp90 inhibitors containing an isoxazolonaphtoquinone core were synthesized and evaluated in two MDR models comprised of sensitive and corresponding resistant cancer cells with P-gp overexpression (human non-small cell lung carcinoma and colorectal adenocarcinoma). We investigated the effect of Hsp90 inhibitors on cell growth inhibition, P-gp activity and P-gp expression. Structure&ndash, activity relationship analysis was performed in respect to cell growth and P-gp inhibition. Compounds 5, 7, and 9 directly interacted with P-gp and inhibited its ATPase activity. Their potential P-gp binding site was identified by molecular docking studies. In addition, these compounds downregulated P-gp expression in MDR colorectal carcinoma cells, showed good relative selectivity towards cancer cells, while compound 5 reversed resistance to doxorubicin and paclitaxel in concentration-dependent manner. Therefore, compounds 5, 7 and 9 could be promising candidates for treating cancers with P-gp overexpression.

Development of drug resistance in cancer has major implications for patients’ outcome. It is related to processes involved in the decrease of drug efficacy, which are strongly influenced by intratumor heterogeneity and changes in the microenvironment. Heterogeneity arises, to a large extent, from genetic mutations analogously to Darwinian evolution, when selection of tumor cells results from the adaptation to the microenvironment, but could also emerge as a consequence of epigenetic mutations driven by stochastic events. An important exogenous source of alterations is the action of chemotherapeutic agents, which not only affects the signalling pathways but also the interactions among cells. In this work we provide experimental evidence from in vitro assays and put forward a mathematical kinetic transport model to describe the dynamics displayed by a system of non-small-cell lung carcinoma cells (NCI-H460) which, depending on the effect of a chemotherapeutic agent (doxorubicin), exhibits a complex interplay between Darwinian selection, Lamarckian induction and the nonlocal transfer of extracellular microvesicles. The role played by all of these processes to multidrug resistance in cancer is elucidated and quantified.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity.

Summary Background C-Myc is one of the major cellular oncogenes overexpressed in non-small cell lung carcinoma (NSCLC). Its deregulated expression is necessary but not sufficient for malignant transformation. We evaluated expression of MYC gene in NSCLC patients and its association with alterations in the genes previously identified to be related to NSCLC pathogenesis, PHACTR3 and E2F4. Methods We analyzed MYC gene expression by qRT-PCR in 30 NSCLC patients’ samples and paired normal lung tissue. MYC expression was further statistically evaluated in relation to histopathological parameters, PHACTR3 and E2F4 gene alterations and survival. Alterations in aforementioned genes were previously detected and identified based on AP-PCR profiles of paired normal and tumor DNA samples, selection of DNA bands with altered mobility in tumor samples and their characterization by the reamplification, cloning and sequencing. Results MYC expression was significantly increased in NSCLC samples and its overexpression significantly associated with squamous cell carcinoma subtype. Most importantly, MYC overexpression significantly coincided with mutations in PHACTR3 and E2F4 genes, in group of all patients and in squamous cell carcinoma subtype. Moreover, patients with jointly overexpressed MYC and altered PHACTR3 or E2F4 showed trend of shorter survival. Conclusions Overall, MYC is frequently overexpressed in NSCLC and it is associated with mutated PHACTR3 gene, as well as mutated E2F4 gene. These joint gene alterations could be considered as potential molecular markers of NSCLC and its specific subtypes.

Background:Dual-targeting in cancer treatment by a single drug is an unconventional approach in relation to drug combinations. The rationale for the development of dualtargeting agents is to overcome incomplete efficacy and drug resistance frequently present when applying individual targeting agents. Consequently, -a more favorable outcome of cancer treatment is expected with dual-targeting strategies.Methods:We reviewed the literature, concentrating on the association between clinically relevant and/or novel dual inhibitors with the potential to modulate multidrug resistant phenotype of cancer cells, particularly the activity of P-glycoprotein. A balanced analysis of content was performed to emphasize the most important findings and optimize the structure of this review.Results:Two-hundred and forty-five papers were included in the review. The introductory part was interpreted by 9 papers. Tyrosine kinase inhibitors’ role in the inhibition of Pglycoprotein and chemosensitization was illustrated by 87 papers. The contribution of naturalbased compounds in overcoming multidrug resistance was reviewed using 92 papers, while specific dual inhibitors acting against microtubule assembling and/or topoisomerases were described with 55 papers. Eleven papers gave an insight into a novel and less explored approach with hybrid drugs. Their influence on P-glycoprotein and multidrug resistance was also evaluated.Conclusion:These findings bring into focus rational anticancer strategies with dual-targeting agents. Most evaluated synthetic and natural drugs showed a great potential in chemosensitization. Further steps in this direction are needed for the optimization of anticancer treatment.

Cancer-initiating cells referred to as cancer stem cells (CSCs) retain the essential property of self-renewal and protection. The protective mechanisms enable tumour regrowth even after the application of chemotherapy that was believed to be successful. Among the protective mechanisms of CSCs, the overexpression of ATP binding cassette (ABC) membrane transporters is highly important. ABC transporters involved in the development of cancer multidrug resistance (MDR) such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are considered as particular features of CSCs. They provide a shield for CSCs and protect them from the adverse effects of chemotherapeutics. Hence, combating MDR would be one of the strategies for the elimination of CSCs. In order to investigate this phenomenon many model systems comprising MDR cancer cells have been established. Some of them were developed by a selection process through exposure to various anticancer drugs, others by transfection of genes for ABC transporters, while some were obtained by sorting the side population considered to possess stemness and resistant phenotypes. Herein we review the potential of cancer MDR models for studying CSCs because gaining a better insight into the mechanisms of CSC resistance to chemotherapy may lead to the discovery of new therapeutic targets and the development of better anticancer strategies.

Resistance to chemotherapeautic drugs is one of the main obstacles to effective cancer treatment. Multidrug resistance (MDR) is defined as resistance to structurally and/or functionally unrelated drugs, and has been extensively investigated for the last three decades. There are two types of MDR: intrinsic and acquired. Tumor microenvironment selection pressure leads to the development of intrinsic MDR, while acquired resistance is a consequence of the administered chemotherapy. A central issue in chemotherapy failure is the existence of heterogeneous populations of cancer cells within one patient and patient-to-patient variability within each type of cancer. Numerous genes and pathways contribute to the development of MDR in cancer. Point mutations, gene amplification or other genetic or epigenetic changes all affect biological functions and may lead to the occurrence of MDR phenotype. Similar to the characteristics of cancerogenesis, the main features of MDR include abnormal tumor vasculature, regions of hypoxia, aerobic glycolysis, and a lower susceptibility to apoptosis. In order to achieve a lethal effect on cancer cells, drugs need to reach their intracellular target molecules. The overexpression of the efflux transporter P-glycoprotein (P-gp) in MDR cancer cells leads to decreased uptake of the drug and intracellular drug accumulation, minimising drug-target interactions. New agents being or inspired by natural products that sucessfully target these mechanisms are the main subject of this review. Two key approaches in combating MDR in cancer are discussed (i) finding agents that preserve citotoxicity toward MDR cancer cells; (ii) developing compounds that restore the cytotoxic activity of classic anticancer drugs. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}III41031]; COST Actions {[}CM1106, CM1407] Related to: [https://ibiss-r.rcub.bg.ac.rs/handle/123456789/3882].

Recently, we demonstrated that ferrocene-containing compounds with a cinchona moiety displayed marked anticancer activity. Here we report on the effects of the most promising isomers encompassing quinine- (compounds 4 and 5) and quinidine-epimers (compounds 6 and 7) – synthesized using improved methods providing controlled diastereoselectivity – in three different human multidrug resistant (MDR) cancer cell lines and their sensitive counterparts (non-small cell lung carcinoma NCI-H460/R/NCI-H460, colorectal carcinoma DLD1-TxR/DLD1 and glioblastoma U87-TxR/U87). We observed that the presence of the MDR phenotype did not diminish the activity of the compounds suggesting that ferrocene quinine- and quinidine-epimers are not substrates for P-glycoprotein, which has been indicated as a major mechanism of MDR in the cell lines used. Considering that metal-based anticancer agents mainly act by increasing ROS production, we investigated the potential of ferrocene–quinidine epimers to generate ROS. We found that 6 and 7 more readily increased ROS production and induced mitochondrial damage in MDR cancer cells. According to cell death analysis, 6 and 7 were more active against MDR cancer cells showing collateral sensitivity. In addition, our data suggest that these compounds could act as inhibitors of autophagy. Importantly, simultaneous treatments of 6 and 7 with paclitaxel (PTX) increased the sensitivity of MDR cancer cells to PTX. In conclusion, the ferrocene–quinidine epimers, besides being selective towards MDR cancer cells, could also possess potential to overcome PTX resistance. Metallomics (2017), 9(8): 1132-1141

The genus Centaurium includes about 50 plant species found throughout the northern hemisphere. Plant species Centaurium erythraea Rafn., commonly known as common centaury, has been used for centuries for medical purposes. Centaury is used to treat anemia, jaundice, and gout and to cure febrile conditions and regulate blood sugar. So far, centaury species were genetically transformed mostly using Agrobacterium rhizogenes. Only one report has described A. tumefaciens-mediated gene delivery for the production of transgenic centaury plants. Genetic transformation of centaury using AtCKX genes did not influence the quality but influenced the quantity of xanthones in shoots and roots. The majority of AtCKX transformed centaury lines grown in vitro produced increased eustomin and/or demethyleustomin content than untransformed control plants. This work clearly demonstrates, for the first time, the effect of centaury secoiridoids and xanthones on colorectal cancer cell line (DLD1) and its resistant counterpart (DLD1-TxR). The xanthone eustomin showed the most significant cell growth inhibition effects. Since xanthones are increasingly being used for their pharmacological properties, AtCKX transgenic centaury plants could be used as a useful source of plant material for the production of novel drugs. Sumita Jha, editor. Transgenesis and Secondary Metabolism. Springer International Publishing; 2016. p. 1-26. (Reference Series in Phytochemistry).

An extended study of minor diarylheptanoids from the bark of black alder has resulted in the isolation of twenty diarylheptanoids, ten of which have not previously been reported (14-18, 20-24). The structures and configurations of all compounds were elucidated by NMR, HRESIMS, UV, IR, and CD. The anti-cancer potency of twenty diarylheptanoids and four previously isolated compounds (7, 10, 12, 13) was investigated in human non-small cell lung carcinoma cell lines (sensitive and multi-drug resistant variants) as well as in normal human keratinocytes. Diarylheptanoids with a p-coumaroyl group, 14 and 18, platyphylloside (1), platyphyllonol-5-O-beta-D-xylopyranoside (2), alnuside B (4) and hirsutenone (9) exhibited strong anti-cancer activity, considerably higher than diarylheptanoid curcumin, which served as a positive control. Compounds 4, 9, 14, and 18 displayed significant selectivity towards the cancer cells. Structure/activity analysis of twenty-four closely related diarylheptanoids revealed a high dependence of cytotoxic action on the presence of a carbonyl group at C-3. Substitution of a heptane chain on C-5 and a number of hydroxyl groups in the aromatic rings also emerged as a significant structural feature that influenced their cytotoxic potential. (C) 2013 Elsevier Ltd. All rights reserved. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3662]

New 6-triazolyl-substituted sulfocoumarins were described as potent inhibitors of the transmembrane human carbonic anhydrase isoforms, CAIX and CAXII. These membrane associated enzymes that maintain pH and CO2 homeostasis are involved in cancer progression, invasion, and resistance to therapy. Recently, it was shown that CAXII expression associates with the expression of P-glycoprotein in multidrug resistant cancer cells. CAXII regulates P-glycoprotein activity by maintaining high intracellular pHi. In this study, the activity of three new sulfocoumarins was evaluated in three sensitive and corresponding multidrug resistant cancer cell lines with increased P-glycoprotein expression (non-small cell lung carcinoma, colorectal carcinoma and glioblastoma). Compound 3 showed the highest potential for cancer cell growth inhibition in all tested cell lines. Flow cytometric analyses showed that compound 3 induced intracellular acidification, cell cycle arrest in G2/M phase and necrosis in non-small cell lung carcinoma cells. Compound 3 demonstrated irreversible, concentration- and time-dependent inhibition of P-glycoprotein activity in multidrug resistant non-small cell lung carcinoma cells. The suppression of P-glycoprotein activity was accompanied with increased P-glycoprotein expression suggesting a compensatory mechanism of multidrug resistant cancer cells. In addition, compound 3 was able to sensitize multidrug resistant non-small cell lung carcinoma cells to doxorubicin. Overall, results imply that compound 3 has multidrug resistance modulating effect through intracellular acidification and subsequent inhibition of P-glycoprotein activity.

Most chemotherapeutics harm normal cells causing severe side effects and induce the development of resistance in cancer cells. Antimicrobial peptides (AMPs), recognized as anti-cancer agents, may overcome these limitations. The most studied mechanism underlying multi-drug resistance (MDR) is the over-expression of cell membrane transporter P-glycoprotein (P-gp), which extrudes a variety of hydrophobic drugs. Additionally, P-gp contributes to cell membrane composition and increases the net negative charge on cell surface. We postulated that NK-lysin derived cationic peptide NK-2 might discriminate and preferentially eliminate P-gp over-expressing cancer cells. To test this hypothesis, we employed MDR non-small cell lung carcinoma (NCI-H460/R) and colorectal carcinoma (DLD1-TxR) cell lines with high P-gp expression. MDR cancer cells that survived NK-2 treatment had decreased P-gp expression and were more susceptible to doxorubicin. We found that NK-2 more readily eliminated P-gp high-expressing cells. Acting in 'carpet-like' manner NK-2 co-localized with P-gp on the MDR cancer cell membrane. The inhibition of P-gp reduced the NK-2 effect in MDR cancer cells and, vice versa, NK-2 decreased P-gp transport activity. In conclusion, NK-2 could modulate MDR in unique way, eliminating the P-gp high-expressing cells from heterogeneous cancers and making them more vulnerable to classical drug treatment.

Thirteen jatrophane diterpenoids (1-10, 13-15), three previously isolated (11, 12, 16) and a known tigliane (17) were isolated from the latex of Euphorbia dendroides. The structures and relative configurations of compounds were elucidated by spectroscopic techniques. The P-glycoprotein (P-gp) inhibiting activities of the representative set of jatrophanes (1-6 and 11-16) have been assessed. Jatrophanes 2 and 5 demonstrated the most powerful inhibition of P-gp, higher than R(+)-verapamil and tariquidar in colorectal multi-drug resistant (MDR) cells (DLD1-TxR). Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2902]

Chemoresistance and invasion properties are severe limitations to efficient glioma therapy. Therefore, development of glioma in vivo models that more accurately resemble the situation observed in patients emerges. Previously, we established RC6 rat glioma cell line resistant to DNA damaging agents including antiglioma approved therapies such as 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ). Herein, we evaluated the invasiveness of RC6 cells in vitro and in a new orthotopic animal model. For comparison, we used C6 cells from which RC6 cells originated. Differences in cell growth properties were assessed by real-time cell analyzer. Cells’ invasive potential in vitro was studied in fluorescently labeled gelatin and by formation of multicellular spheroids in hydrogel. For animal studies, fluorescently labeled cells were inoculated into adult male Wistar rat brains. Consecutive coronal and sagittal brain sections were analyzed 10 and 25 days post-inoculation, while rats’ behavior was recorded during three days in the open field test starting from 25th day post-inoculation. We demonstrated that development of chemoresistance induced invasive phenotype of RC6 cells with significant behavioral impediments implying usefulness of orthotopic RC6 glioma allograft in preclinical studies for the examination of new approaches to counteract both chemoresistance and invasion of glioma cells.

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' 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.

Chemoresistance is a severe limitation to glioblastoma (GBM) therapy and there is a strong need to understand the underlying mechanisms that determine its response to different chemotherapeutics. Therefore, we induced resistance in C6 rat glioma cell line, which considerably resembles the characteristics of human GBM. The resistant phenotype was developed by 3-bis (2-chloroethyl)-1-nitrosourea (BCNU), one of the most commonly used therapeutic drug in the course of GBM treatment. After confirmation of the cross-resistance to cisplatin (CPt) and temozolomide (TMZ) in newly established RC6 cell line, we examined cell death induction and DNA damage by these drugs. Resistance to apoptosis and deficiency in forming DNA double-strand breaks was followed by significant decrease in the mRNA expression of pro-apoptotic and anti-apoptotic genes. The development of drug resistance was associated with significant increase in reactive oxygen species (ROS) and decrease in oxidized to reduced gluthatione ratio in RC6 cell line indicating a reduced level of oxidative stress. The mRNA expression levels of manganese superoxid dismutase (MnSOD), inducible nitric oxide synthase (iNOS) and gluthatione peroxidase (GPx) were increased while hypoxia-inducible factor 1-alpha (HIF-1 alpha) was decreased in RC6 compared to C6 cells. This was in line with obtained changes in ROS content and increased antioxidative capacity of RC6 cells. Importantly, RC6 cells demonstrated collateral sensitivity to doxorubicin (DOX). The analysis of this phenomenon revealed increased accumulation of DOX in RC6 cells due to their adaptation to high ROS content and acidification of cytoplasm. In conclusion, newly established RC6 rat glioma cell line could be used as a starting material for the development of allogenic animal model and preclinical evaluation of new antiglioma agents. Collateral sensitivity to DOX obtained after BCNU treatment may prompt new studies aimed to find efficient delivery of DOX to the glioma site in brain. (C) 2015 Elsevier Inc. All rights reserved. Ministry of Education, Science and Technological Development of Serbia {[}III 41031, III 41025]

Data on medical applications of cerium oxide nanoparticles CeO2 (CONP) are promising, yet information regarding their action in cells is incomplete and there are conflicting reports about in vitro toxicity. Herein, we have studied cytotoxic effect of CONP in several cancer and normal cell lines and their potential to change intracellular redox status. The IC50 was achieved only in two of eight tested cell lines, melanoma 518A2 and colorectal adenocarcinoma HT-29. Self-propagating room temperature method was applied to produce CONP with an average crystalline size of 4 nm. The results confirmed presence of Ce3+ and O2- vacancies. The induction of cell death by CONP and the production of reactive oxygen species (ROS) were analyzed by flow-cytometry. Free radicals related antioxidant capacity of the cells was studied by the reduction of stable free radical TEMPONE using electron spin resonance spectroscopy. CONP showed low or moderate cytotoxicity in cancer cell lines: adenocarcinoma DLD1 and multi-drug resistant DLD1-TxR, non-small cell lung carcinoma NCI-H460 and multi-drug resistant NCI-H460/R, while normal cell lines (keratinocytes HaCaT, lung fetal fibroblasts MRC-5) were insensitive. The most sensitive were 518A2 melanoma and HT-29 colorectal adenocarcinoma cell lines, with the IC50 values being between 100 and 200 mu M. Decreased rate of TEMPONE reduction and increased production of certain ROS species (peroxynitrite and hydrogen peroxide anion) indicates that free radical metabolism, thus redox status was changed, and antioxidant capacity damaged in the CONP treated 518A2 and HT-29 cells. In conclusion, changes in intracellular redox status induced by CONP are partly attributed to the prooxidant activity of the nanoparticles. Further, ROS induced cell damages might eventually lead to the cell death. However, low inhibitory potential of CONP in the other human cell lines tested indicates that CONP may be safe for human usage in industry and medicine. (C) 2015 Elsevier Ireland Ltd. All rights reserved. Ministry of Education, Science and Technology of the Republic of Serbia {[}III45012, III41031, III41005, III45005-4]

Purine nucleoside analogs have been in clinical use for almost 50 years. At the beginning developed as antiviral agents, later their efficacy was demonstrated in cancer treatment, especially hematological malignances. The approval of new purine nucleoside analogs by US Food and Drug Administration (FDA) over the past decade implies that the interest for these drugs still exists. Here, we review new nucleoside analogs that are currently in preclinical or clinical development as anticancer agents. In addition, we highlight the potential for implementation of these drugs in other pathological conditions, particularly in neuroinflammation. Molecular inhibitors in targeted therapy (2015), 1(1): 3-14

Glavni uzrok neuspeha hemioterapije u lečenju kancera je pojava višestruke (engl. „multi-drug“) rezistencije (MDR). Efikasnost paklitaksela (PTX) je često ograničena pojavom rezistencije. Cilj ove doktorske disertacije je bio ispitivanje molekularnih i fenotipskih promena u toku razvoja MDR-a indukovanih PTX-om kod ćelijskih linija humanog karcinoma debelog creva (DLD1) i glioblastoma (U87). Takođe je testirana upotrebljivost dobijenih MDR modela u evaluaciji četiri anti-kancer agensa. Kontinuirani tretman PTX-om doveo je do razvoja MDR-a kod obe ispitivane ćelijske linije koje su postale rezistentne na strukturno i funkcionalno različite hemioterapeutike. Nakon potvrde prisustva ukrštene rezistencije kod novouspostavljenih ćelijskih linija DLD1-TxR i U87-TxR, analizirana je ekspresija membranskih trasportera uključenih u razvoj MDR-a na nivou iRNK. Ćelije su imale povišen nivo ekspresije mdr1 gena i smanjen nivo ekspresije mrp1 gena. Prekomerna ekspresija P-glikoproteina (P-gp), koji kodira mdr1 gen, je uočena kod obe MDR ćelijske linije. Analiza na protočnom citofluorimetru je pokazala da je akumulacija Pgp supstrata (rodamina 123 i doksorubicina) kod DLD1-TxR i U87-TxR ćelija značajno manja u poređenju sa odgovarajućim parentalnim ćelijama, DLD1 i U87. Značajno smanjenje ekspresije gst-π gena i koncentracije glutationa (GSH) je uočeno kod U87- TxR ćelija. Sekrecija vaskularnog endotelijalnog faktora rasta (VEGF) je inhibirana u jednokratnom tretmanu kod ćelijskih linija karcinoma debelog creva i u kontinuiranom tretmanu kod ćelijske linije glioblastoma. Analiza ćelijskog ciklusa je pokazala da je jednokratni tretman PTX-om kod ćelijskih linija humanog karcinoma debelog creva praćen porastom subG0 faze, odnosno povećanjem procenta mrtvih ćelija, dok je kod ćelija glioblastoma došlo do umiranja tokom interfaze (G1, S ili G2). MDR tumorske ćelijske linije su stekle nove strukturne i numerićke hromozomske aberacije. Sticanje MDR fenotipa kod U87-TxR ćelija je praćeno smanjenem jednog nivoa ploidije. Takođe je uočen gubitak hromozomskog regiona 6q kod obe rezistentne ćelijske linije, kao i inaktivacija p53 tumor spresor gena kod U87-TxR ćelija i PTEN tumor supresor gena kod DLD1-TxR ćelija. Multi-drug resistance (MDR) is a major obstacle to successful cancer treatment. The efficacy of paclitaxel (PTX) is often limited by appearance of drug resistance. The aim of this study was to explore molecular and phenotypic alterations during development of MDR induced by PTX in human colon carcinoma (DLD1) and glioblastoma (U87) cell lines. We also tested the usefulness of developed MDR models in the evaluation of four anti-cancer agents. Continuous treatment with PTX led to the development of MDR in both tested cancer cell lines that became resistant to structurally and functionally unrelated chemotherapeutics. After confirmation of the cross-resistance in newly established DLD1-TxR and U87-TxR, we analyzed the mRNA expression of membrane transporters involved in MDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased. Over-expression of P-glycoprotein (P-gp), coded by mdr1, was observed in both MDR cancer cell lines. Flow cytometry analyzes showed that the accumulation of P-gp substrates (rhodamine 123 and doxorubicin) in DLD1-TxR and U87-TxR was significantly lower compared to DLD1 and U87, respectively. The significant depletion of gst-π gene expression and glutathione (GSH) concentration was observed in U87-TxR. Vascular Endothelial Growth Factor (VEGF) secretion was inhibited by single PTX treatment of colon cancer and in continuous treatment of glioblastoma cell lines. The analysis of cell cycle kinetics revealed extensive cell death in colon cancer cells that were accumulated in subG0 phase after PTX treatment, while glioblastoma cells died through interphase (G1, S or G2). The MDR cancer cell lines acquired novel structural or numerical chromosomal aberrations. Polyploidy reduction was observed after development of MDR in U87-TxR. Losses of 6q in both resistant cancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were also revealed. We evaluated the anti-cancer activities and MDR reversal potential of the Akt inhibitor (GSK690693), the Ras inhibitor (Tipifarnib) and two P-gp inhibitors (jatrophane diterpenoids Euphodendrophane H-Euph H and Euphodendrophane S -Euph S). Their effects vary due to the cell-type differences, existence of MDR phenotype or tumor suppressors’ alterations. Tipifarnib, Euph H and S, significantly sensitized MDR cancer cells to PTX.

Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-β-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI–H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC50 values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic.

Dimers tomoroside A (1) and tomoroside B (2) of the co-occuring known chalcone monomer (3), along with the seven known flavonoid glucosides (4-10), were isolated from the aerial parts of Helichrysum zivojinii Cernjavski & Soska. The structures of the isolated compounds were elucidated by spectroscopic techniques. Compound 1 inhibited topo Ha and hif-1 alpha expression and stimulated doxorubicin anticancer effect, while 2 increased the expression of hif-1 alpha a, probably acting as antioxidant and redox status modulator. Notably, 2 synergized with Tipifarnib showing potential to improve the action of this new chemotherapeutic involved in the modulation of mitogene activated protein (MAP) kinase signaling pathway. (C) 2013 Elsevier Ltd. All rights reserved.

Achieving an effective treatment of cancer is difficult, particularly when resistance to conventional chemotherapy is developed. P-glycoprotein (P-gp) activity governs multi-drug resistance (MDR) development in different cancer cell types. Identification of anti-cancer agents with the potential to kill cancer cells and at the same time inhibit MDR is important to intensify the search for novel therapeutic approaches. We examined the effects of sulfinosine (SF), a quite unexplored purine nucleoside analog, in MDR (P-gp over-expressing) non-small cell lung carcinoma (NSCLC) and glioblastoma cell lines (NCI-H460/R and U87-TxR, respectively). SF showed the same efficacy against MDR cancer cell lines and their sensitive counterparts. However, it was non-toxic for normal human keratinocytes (HaCaT). SF induced caspase-dependent apoptotic cell death and autophagy in MDR cancer cells. After SF application, reactive oxygen species (ROS) were generated and glutathione (GSH) concentration was decreased. The expression of key enzyme for GSH synthesis, gamma Glutamyl-cysteine-synthetase (γGCS) was decreased as well as the expression of gst-π mRNA. Consequently, SF significantly decreased the expression of hif-1α, mdr1 and vegf mRNAs even in hypoxic conditions. SF caused the inhibition of P-gp (coded by mdr1) expression and activity. The accumulation of standard chemotherapeutic agent – doxorubicin (DOX) was induced by SF in concentration- and time-dependent manner. The best effect of SF was obtained after 72 h when it attained the effect of known P-gp inhibitors (Dex-verapamil and tariquidar). Accordingly, SF sensitized the resistant cancer cells to DOX in subsequent treatment. Furthermore, SF decreased the experssion of vascular endothelial growth factor (VEGF) on mRNA and protein level and modulated its secretion. In conclusion, the effects on P-gp (implicated in pharmacokinetics and MDR), GSH (implicated in detoxification) and VEGF (implicated in tumor-angiogenesis and progression) qualify SF as multi-potent anti-cancer agent, which use must be considered, in particular for resistant malignancies.

Paclitaxel (PTX) is used for treatment of wide range of solid tumors, but its efficacy is often limited by appearance of multidrug resistance (MDR). We explored the MDR induced by PTX in human colon cancer DLD1 and glioblastoma U87 cell lines. After confirmation of the cross-resistance to other anticancer agents in newly established DLD1-TxR and U87-TxR, we analyzed the mRNA expression of membrane transporters involved in MDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased. Flow cytometry analyzes showed that the accumulation of P-glycoprotein (P-gp) substrates (rhodamine 123 and doxorubicin) was significantly lower in DLD1-TxR and U87-TxR compared to DLD1 and U87, respectively. The significant depletion of gst-π gene expression and glutathione (GSH) concentration was observed in U87-TxR. The analysis of cell cycle kinetics revealed extensive cell death in colon cancer cells that were accumulated in subG0 phase after PTX treatment, while glioblastoma cells died during interphase (G1, S or G2). The secretion of vascular endothelial growth factor (VEGF) was inhibited by single PTX treatment of colon cancer and in continuous treatment of glioblastoma cell lines. In conclusion, continuous PTX treatment caused the over-expression of P-gp and acquisition of MDR in colon cancer and glioblastoma cell lines, while some mechanisms of MDR and tumor progression such as GSH detoxification system and VEGF secretion were suppressed. Hence, the present results implicate that PTX is an important clinical tool for colon cancer and glioblastoma treatment even in the presence of MDR.

Multidrug resistance (MDR) is the major cause of failure to a successful cancer treatment. Cancer cells manifesting MDR phenotype are cross-resistant to many structurally and functionally unrelated drugs. The purpose of this study was to establish in vitro models, i.e., resistant cell lines, to further investigate the mechanisms of MDR. We used the human colon cancer cell line DLD1 and the glioma cell line U87 to establish paclitaxel-resistant cell lines, DLD1 -TxR and U87-TxR, by continuous exposure to stepwise increasing concentrations of the drug. The newly established, DLD1 -TxR and U87-TxR cell lines showed extensive resistance to paclitaxel (301-fold and 103-fold respectively). They also demonstrated cross-resistance to other anticancer agents such as doxorubicin, epirubicin, vinblastin, and etoposide. Cytogenetic data based on inverted-DAPI Banding, demonstrated that both resistant cell lines retained several karyotypic characteristics of their sensitive counterparts and also acquired novel structural or numerical chromosomal aberrations that may be related to resistance to chemotherapy. Interestingly, while the dividing parental U87 cells were mainly near-tetraploid, chemoresistance in the U87-TxR cell line was accompanied by one level ploidy reduction as verified by the increased prevalence of near-diploid cell populations. In addition, the DLD1 -TxR cells showed an increase tendency to loose the Y chromosome. Gene expression analyses revealed a 4-fold increase in the level of mdr1 mRNA in DLD1 -TxR cells. U87-TxR cells demonstrated prominent mdr1 activity since the expression in parental cells was undetectable. In contrast, the level of mrp1 mRNA was 40% decreased in both resistant cell lines. The expression of other MDR-related genes, topo IIα, and lrp, was unaffected by paclitaxel resistance. Flow cytometry analyses showed that the accumulation of rhodamine in resistant cells DLD1 -TxR and U87-TxR was significantly lower than in their sensitive counterparts, 17-fold and 8-fold, respectively. These results indicate that P-gp, encoded by mdr1 gene, is significantly overexpressed in both resistant cell lines and may account for paclitaxel resistance. In conclusion, the two novel resistant cell lines, DLD1 -TxR and U87-TxR may serve as appropriate models for the study of the mechanisms of paclitaxel resistance offering novel insights into potential targets for overcoming MDR that would improve the response to chemotherapy and patient outcome. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B42.

Background and Objectives: Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method’s applicability.