The establishing of resistant cancer cell lines as a model for testing of new chemotherapeutics: molecular characterization of resistance developed after continuous treatment with paclitaxel

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

Potential of Melittin to induce apoptosis and overcome multidrug resistance in human colon cancer cell line

In this study, the anticancer effect of Melittin was demonstrated through its antiproliferative and proapoptotic effects on HT-29 cells, as well as its ability to reverse multidrug resistance. Melittin directly affects the death receptor-dependent apoptotic pathway via increase of the Fas receptor protein expression, Caspase 8 gene expression and activity of Caspase 8. Results of decreased Caspase 9 gene and protein expression, and multi-fold increased expression of Bcl-2 gene suggest that mitochondria and the inner apoptotic pathway are not involved in the execution of Melittin induced apoptosis, as well as redox regulation of apoptosis based on decreased concentration of superoxide anion radicals and no effect glutathione levels. Specially significance of this work are results on ability of Melittin to modulate the metabolism and export system in cancer cells. Based on the increased expression of all the investigated genes related to the biotransformation process, it can be assumed that CYP1A1, CYP1B1, GSTP1 and MRP2 are involved in metabolism of Melittin in HT-29 cells. P-glycoprotein is associated with the occurrence of resistance in anticancer therapy, so its reduced gene and protein expression by Melittin represents significant result in terms of possible therapeutic application and examination.Publishe

Src Inhibitors Pyrazolo[3,4-d]pyrimidines, Si306 and Pro-Si306, Inhibit Focal Adhesion Kinase and Suppress Human Glioblastoma Invasion In Vitro and In Vivo

Glioblastoma (GBM), as the most aggressive brain tumor, displays a high expression of Src tyrosine kinase, which is involved in the survival, migration, and invasiveness of tumor cells. Thus, Src emerged as a potential target for GBM therapy. The effects of Src inhibitors pyrazolo[3,4-d]pyrimidines, Si306 and its prodrug pro-Si306 were investigated in human GBM cell lines (U87 and U87-TxR) and three primary GBM cell cultures. Primary GBM cells were more resistant to Si306 and pro-Si306 according to the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. However, the ability of all GBM cells to degrade the extracellular matrix was considerably compromised after Si306 and pro-Si306 applications. Besides reducing the phosphorylation of Src and its downstream signaling pathway components, both compounds decreased the phosphorylated form of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) expression, showing the potential to suppress the aggressiveness of GBM. In vivo, Si306 and pro-Si306 displayed an anti-invasive effect against U87 xenografts in the zebrafish embryo model. Considering that Si306 and pro-Si306 are able to cross the blood-brain barrier and suppress the spread of GBM cells, we anticipate their clinical testing in the near future. Moreover, the prodrug showed similar efficacy to the drug, implying the rationality of its use in clinical settings

Supplementary data for the article: Dinić, J.; Novaković, M.; Podolski-Renić, A.; Stojković, S.; Mandić, B.; Tešević, V.; Vajs, V.; Isaković, A.; Pešić, M. Antioxidative Activity of Diarylheptanoids from the Bark of Black Alder (Alnus Glutinosa) and Their Interaction with Anticancer Drugs. Planta Medica 2014, 80 (13), 1088–1096. https://doi.org/10.1055/s-0034-1382993

Supplementary material for: [https://doi.org/10.1055/s-0034-1382993]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1845

Association of Overexpressed MYC Gene with Altered PHACTR3 and E2F4 Genes Contributes to Non-Small Cell Lung Carcinoma Pathogenesis

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

Supplementary material for the article: Dinić, J.; Novaković, M.; Podolski-Renić, A.; Vajs, V.; Tešević, V.; Isaković, A.; Pešić, M. Structural Differences in Diarylheptanoids Analogues from Alnus Viridis and Alnus Glutinosa Influence Their Activity and Selectivity towards Cancer Cells. Chemico-Biological Interactions 2016, 249, 36–45. https://doi.org/10.1016/j.cbi.2016.02.019

Supplementary material for: [https://doi.org/10.1016/j.cbi.2016.02.019]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2066

Design, synthesis, and biological evaluation of novel derivatives of dithiodiglycolic acid prepared via oxidative coupling of thiols.

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

DTA0100, dual topoisomerase II and microtubule inhibitor, evades paclitaxel resistance in P-glycoprotein overexpressing cancer cells

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 beta-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 beta-tubulin isoforms expression observed in multi drug resistant cancer cells may influence the efficacy of DTA0100. Importantly, DTA0100 blocked the Pglycoprotein function in both multi-drug resistant cancer cell lines without inducing the increase in Pglycoprotein 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.Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1079]This is the peer reviewed version of the paper: Podolski-Renić, A., Banković, J., Dinić, J., Rios-Luci, C., Fernandes, M. X., Ortega, N., Kovačević Grujičić, N., Martin, V. S., Padron, J. M., & Pesić, M. (2017). DTA0100, dual topoisomerase II and microtubule inhibitor, evades paclitaxel resistance in P-glycoprotein overexpressing cancer cells. European Journal of Pharmaceutical Sciences, 105, 159–168. [https://doi.org/10.1016/j.ejps.2017.05.011

New anti-glioblastoma strategy with natural compounds sclareol and doxorubicin

Background: Doxorubicin (DOX) has been very effective against glioblastoma invitro. Its application in vivo is hampered because it cannot pass the blood–brainbarrier (BBB). Significant research efforts are invested to overcome this limitation.Sclareol (SC) is an aromatic compound naturally found in clary sage. Thecombination of SC and DOX showed promising effects in different tumor types invitro and in vivo. Therefore, we tested their combination and innovative hybridmolecules (SC:DOX) on glioblastoma cells with the expression of P-glycoprotein, amajor component of BBB and cancer multidrug resistance marker. Methods:Cytotoxicity and selectivity towards glioblastoma cells of SC, DOX, theircombination, and SC:DOX were examined by MTT assay. The effect of SC on DOXaccumulation was determined by flow cytometry. We also studied SC:DOXaccumulation, cellular uptake, localization imaging, and DNA damage induction.Results: The effects of simultaneous SC and DOX treatments demonstrated theconsiderable potential of SC to reverse DOX resistance in glioblastoma cells andincrease DOX accumulation. SC:DOX hybrids, named CON1 and CON2 were lesscytotoxic than DOX, but with reduced resistance and increased selectivity towardsglioblastoma cells. Cellular uptake of CON1 and CON2 was increased in glioblastomacells compared to DOX. Perinuclear localization of CON1 and CON2 vs. nuclearlocalization of DOX as well as no DNA damaging effects suggest a differentmechanism of action for SC:DOX. Conclusion: The combination of SC and DOX, andtheir innovative hybrids, could be considered a promising strategy that can overcomethe limitations of DOX application in glioblastoma.Kanazir S, Savić D, editors. Book of abstracts: 8th Congress of Serbian neuroscience society with international participation; 2023 May 31 - Jun 2; Belgrade, Serbia. Belgrade : Serbian Neuroscience Society; 2023. p. 71

Supplementary data for the article: Novaković, M.; Pešić, M.; Trifunović, S.; Vučković, I.; Todorović, N.; Podolski-Renić, A.; Dinić, J.; Stojković, S.; Tešević, V.; Vajs, V.; et al. Diarylheptanoids from the Bark of Black Alder Inhibit the Growth of Sensitive and Multi-Drug Resistant Non-Small Cell Lung Carcinoma Cells. Phytochemistry 2014, 97, 46–54. https://doi.org/10.1016/j.phytochem.2013.11.001

Supplementary material for: [https://doi.org/10.1016/j.phytochem.2013.11.001]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1492