Supplementary material for the article: Jeremić, M.; Pešić, M.; Dinić, J.; Banković, J.; Novakovićc, I.; Šegan, D.; Sladić, D. Simple Avarone Mimetics as Selective Agents against Multidrug Resistant Cancer Cells. European Journal of Medicinal Chemistry 2016, 118, 107–120. https://doi.org/10.1016/j.ejmech.2016.04.011

Supplementary material for: [https://doi.org/10.1016/j.ejmech.2016.04.011]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2256

Decreased TSPAN14 Expression Contributes to NSCLC Progression

Tspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients' samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression

Supplementary material for the article: Jeremić, M.; Pešić, M.; Dinić, J.; Banković, J.; Novakovićc, I.; Šegan, D.; Sladić, D. Simple Avarone Mimetics as Selective Agents against Multidrug Resistant Cancer Cells. European Journal of Medicinal Chemistry 2016, 118, 107–120. https://doi.org/10.1016/j.ejmech.2016.04.011

Supplementary material for: [https://doi.org/10.1016/j.ejmech.2016.04.011]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2256

No-modified saquinavir is equally efficient against doxorubicin sensitive and resistant non-small cell lung carcinoma cells

Background: The NO-modified form of the HIV inhibitor saquinavir (Saq-NO) inhibited the growth of a variety of cancer cell lines in vitro and in vivo more potently than the original compound in a nontoxic fashion. In addition, chemo- and immunosensitizing properties were observed. The aim of the present study was to evaluate its anticancer action against non-small cell lung carcinoma cells in their doxorubicin (DOXO) sensitive and resistant phenotype (NCI-H460 and NCI-H460/R). Methods: The viability of cells was analyzed by MTT and crystal violet assays. DR5 expression was estimated by real time RT-PCR and flow cytometry. Activity of P-glycoprotein (P-gp) pumps was evaluated by the Rho123 accumulation assay. Results: Saq-NO diminished the viability of lung cancer cells through induction of cell cycle arrest in the G0/G1 phase independently of the overexpression of the P-gp pumps. In addition, Saq-NO elevated or completely reconstituted the doxorubicin efficacy in NCI-H460 and NCI-H460/R, respectively. The chemosensitizing effect in DOXO resistant cells was a consequence of P-gp inhibition which was found to be more potent than that observed with dex-verapamil, a conventional inhibitor of P-gp. Sensitization to DOXO upon Saq-NO was accompanied by elevated DR5 expression, but the resistance to TRAIL was not abrogated. Conclusions: The NO-modified HIV inhibitor saquinavir displayed equal antiproliferative and chemosensitizing properties in DOXO sensitive and resistant non-small cell lung carcinoma cells, suggesting the importance of the evaluation of this drug as an antineoplastic agent.Uvod: Inhibitor HIV proteaze - sakvinavir nakon modifikacije kovalentnim vezivanjem NO (Saq-NO) gubi toksična svojstva dok potentnije od originalnog jedinjenja inhibira in vitro i in vivo rast brojnih ćelijskih linija kancera. Pored direktnog antitumorskog delovanja, Saq-NO povećava osetljivost ćelija kancera na antitumorski imunski odgovor i konvencionalnu hemioterapiju. Ova studija je imala za cilj ispitivanje antitumorskog potencijala Saq-NO na ćelijskim linijama nesitnoćelijskog karcinoma pluća, senzitivnim (NCI-H460), odnosno rezistentnim (NCI-H460/R) na doksorubicin. Metode: Vijabilitet ćelija je evaluiran testovima MTT i 'kristal violet'. Ekspresija receptora DR5 je procenjivana me-odom RT-PCR u realnom vremenu i protočnom citofluorimetrijom. Aktivnost P-gp pumpi određivana je akumulacionim testom Rho123. Rezultati: Saq-NO inhibira rast ćelija kancera pluća zaustavljanjem ćelija u fazi G0/G1 ćelijskog ciklusa a zapaženi efekat nije oslabljen povećanjem ekspresije P-gp pumpi. Pored toga, Saq-NO povećava osetljivost NCI-H460 ćelija, dok u slučaju rezistentne forme, NCI-H460/R, potpuno rekonstituiše njihovu osetljivost na doksorubicin. Efekat hemosenzitizacije je posledica inhibicije P-gp pumpi, što Saq-NO čini potentnijim od deksverapamila, uobičajenog inhibitora P-gp. Opisani fenomen je praćen povećanjem ekspresije receptora DR5 na genskom i membranskom nivou, ali time rezistencija na molekul TRAIL nije ukinuta. Zaključak: Saq-NO pokazuje značajan antiproliferativan i hemosenzitizujući potencijal na ćelijama nesitnoćelijskog kancera pluća nezavisno od njihove osetljivosti odnosno rezistencije na doksorubicin, ukazujući na potrebu daljeg ispitivanja ovog jedinjenja u svojstvu potencijalnog antineoplastičnog agensa.Projekat ministarstva br. 173013 i br. III4103

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

Anti-invasive effects of CXCR4 and FAK inhibitors in non-small cell lung carcinomas with mutually inactivated p53 and PTEN tumor suppressors

Non-small cell lung carcinoma (NSCLC) is the most common type of lung cancer. At the time of diagnosis, a large percentage of NSCLC patients have already developed metastasis, responsible for extremely high mortality rates. CXCR4 receptor and focal adhesion kinase (FAK) are known to regulate such invasive cancer behavior. Their expression is downregulated by p53 and PTEN tumor suppressors which are commonly co-inactivated in NSCLC patients and contribute to metastasis. Therefore, targeting CXCR4 or FAK seems to be a promising strategy in suppressing metastatic spread of p53/PTEN deficient NSCLCs. In this study, we first examined the invasive characteristics of NSCLC cells with suppressed p53 and PTEN activity using wound healing, gelatin degradation and invasion assays. Further, changes in the expression of CXCR4 and FAK were evaluated by RT-qPCR and Western Blot analysis. Finally, we tested the ability of CXCR4 and FAK inhibitors (WZ811 and PF-573228, respectively) to suppress the migratory and invasive potential of p53/PTEN deficient NSCLC cells, in vitro and in vivo using metastatic models of human NSCLC. Our results showed that cells with mutually inactive p53 and PTEN have significantly increased invasive potential associated with hyperactivation of CXCR4 and FAK signaling pathways. Treatments with WZ811 and PF-573228 inhibitors significantly reduced migratory and invasive capacity in vitro and showed a trend of improved survival in vivo. Accordingly, we demonstrated that p53/PTEN deficient NSCLCs have extremely invasive phenotype and provided a rationale for the use of CXCR4 or FAK inhibitors for the suppression of NSCLC dissemination.This is a post-peer-review, pre-copyedit version of an article published in Investigational New Drugs. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s10637-017-0494-4

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

Purine nucleoside analogs in the therapy of cancer and neuroinflammation

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): 3-1

Supplementary data for the article: Jadranin, M.; Pešić, M.; Aljančić, I. S.; Milosavljević, S. M.; Todorović, N. M.; Podolski-Renić, A.; Banković, J.; Tanić, N.; Marković, I.; Vajs, V. E.; et al. Jatrophane Diterpenoids from the Latex of Euphorbia Dendroides and Their Anti-P-Glycoprotein Activity in Human Multi-Drug Resistant Cancer Cell Lines. Phytochemistry 2013, 86, 208–217. https://doi.org/10.1016/j.phytochem.2012.09.003

Supplementary material for: [https://doi.org/10.1016/j.phytochem.2012.09.003]Relate to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1585

No-modified saquinavir is equally efficient against doxorubicin sensitive and resistant non-small cell lung carcinoma cells

Background: The NO-modified form of the HIV inhibitor saquinavir (Saq-NO) inhibited the growth of a variety of cancer cell lines in vitro and in vivo more potently than the original compound in a nontoxic fashion. In addition, chemo- and immunosensitizing properties were observed. The aim of the present study was to evaluate its anticancer action against non-small cell lung carcinoma cells in their doxorubicin (DOXO) sensitive and resistant phenotype (NCI-H460 and NCI-H460/R). Methods: The viability of cells was analyzed by MTT and crystal violet assays. DR5 expression was estimated by real time RT-PCR and flow cytometry. Activity of P-glycoprotein (P-gp) pumps was evaluated by the Rho123 accumulation assay. Results: Saq-NO diminished the viability of lung cancer cells through induction of cell cycle arrest in the G0/G1 phase independently of the overexpression of the P-gp pumps. In addition, Saq-NO elevated or completely reconstituted the doxorubicin efficacy in NCI-H460 and NCI-H460/R, respectively. The chemosensitizing effect in DOXO resistant cells was a consequence of P-gp inhibition which was found to be more potent than that observed with dex-verapamil, a conventional inhibitor of P-gp. Sensitization to DOXO upon Saq-NO was accompanied by elevated DR5 expression, but the resistance to TRAIL was not abrogated. Conclusions: The NO-modified HIV inhibitor saquinavir displayed equal antiproliferative and chemosensitizing properties in DOXO sensitive and resistant non-small cell lung carcinoma cells, suggesting the importance of the evaluation of this drug as an antineoplastic agent.Uvod: Inhibitor HIV proteaze - sakvinavir nakon modifikacije kovalentnim vezivanjem NO (Saq-NO) gubi toksična svojstva dok potentnije od originalnog jedinjenja inhibira in vitro i in vivo rast brojnih ćelijskih linija kancera. Pored direktnog antitumorskog delovanja, Saq-NO povećava osetljivost ćelija kancera na antitumorski imunski odgovor i konvencionalnu hemioterapiju. Ova studija je imala za cilj ispitivanje antitumorskog potencijala Saq-NO na ćelijskim linijama nesitnoćelijskog karcinoma pluća, senzitivnim (NCI-H460), odnosno rezistentnim (NCI-H460/R) na doksorubicin. Metode: Vijabilitet ćelija je evaluiran testovima MTT i 'kristal violet'. Ekspresija receptora DR5 je procenjivana me-odom RT-PCR u realnom vremenu i protočnom citofluorimetrijom. Aktivnost P-gp pumpi određivana je akumulacionim testom Rho123. Rezultati: Saq-NO inhibira rast ćelija kancera pluća zaustavljanjem ćelija u fazi G0/G1 ćelijskog ciklusa a zapaženi efekat nije oslabljen povećanjem ekspresije P-gp pumpi. Pored toga, Saq-NO povećava osetljivost NCI-H460 ćelija, dok u slučaju rezistentne forme, NCI-H460/R, potpuno rekonstituiše njihovu osetljivost na doksorubicin. Efekat hemosenzitizacije je posledica inhibicije P-gp pumpi, što Saq-NO čini potentnijim od deksverapamila, uobičajenog inhibitora P-gp. Opisani fenomen je praćen povećanjem ekspresije receptora DR5 na genskom i membranskom nivou, ali time rezistencija na molekul TRAIL nije ukinuta. Zaključak: Saq-NO pokazuje značajan antiproliferativan i hemosenzitizujući potencijal na ćelijama nesitnoćelijskog kancera pluća nezavisno od njihove osetljivosti odnosno rezistencije na doksorubicin, ukazujući na potrebu daljeg ispitivanja ovog jedinjenja u svojstvu potencijalnog antineoplastičnog agensa.Projekat ministarstva br. 173013 i br. III4103