Salinomycin disturbs Golgi function and specifically affects cells in epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) gives rise to cells with properties similar to cancer stem cells (CSCs). Targeting the EMT program to selectively eliminate CSCs is a promising way to improve cancer therapy. Salinomycin (Sal), a K+/H+ ionophore, was identified as highly selective towards CSC-like cells, but its mechanism of action and selectivity remains elusive. Here, we show that Sal, similar to monensin and nigericin, disturbs the function of the Golgi. Sal alters the expression of Golgi-related genes and leads to marked changes in Golgi morphology, particularly in cells that have undergone EMT. Moreover, Golgi-disturbing agents severely affect post-translational modifications of proteins, including protein processing, glycosylation and secretion. We discover that the alterations induced by Golgi-disturbing agents specifically affect the viability of EMT cells. Collectively, our work reveals a novel vulnerability related to the EMT, suggesting an important role for the Golgi in the EMT and that targeting the Golgi could represent a novel therapeutic approach against CSCs.

Harmicens, Novel Harmine and Ferrocene Hybrids: Design, Synthesis and Biological Activity.

Cancer and malaria are both global health threats. Due to the increase in the resistance to the known drugs, research on new active substances is a priority. Here, we present the design, synthesis, and evaluation of the biological activity of harmicens, hybrids composed of covalently bound harmine/ß-carboline and ferrocene scaffolds. Structural diversity was achieved by varying the type and length of the linker between the ß-carboline ring and ferrocene, as well as its position on the ß-carboline ring. Triazole-type harmicens were prepared using Cu(I)-catalyzed azide-alkyne cycloaddition, while the synthesis of amide-type harmicens was carried out by applying a standard coupling reaction. The results of in vitro biological assays showed that the harmicens exerted moderate antiplasmodial activity against the erythrocytic stage of P. falciparum (IC50 in submicromolar and low micromolar range) and significant and selective antiproliferative activity against the MCF-7 and HCT116 cell lines (IC50 in the single-digit micromolar range, SI > 5.9). Cell localization experiments showed different localizations of nonselective harmicene 36 and HCT116-selective compound 28, which clearly entered the nucleus. A cell cycle analysis revealed that selective harmicene 28 had already induced G1 cell cycle arrest after 24 h, followed by G2/M arrest with a concomitant drastic reduction in the percentage of cells in the S phase, whereas the effect of nonselective compound 36 on the cell cycle was much less pronounced, which agreed with their different localizations within the cell.

Interaction of crown ethers with the ABCG2 transporter and their implication for multidrug resistance reversal.

Overexpression of ABC transporters, such as ABCB1 and ABCG2, plays an important role in mediating multidrug resistance (MDR) in cancer. This feature is also attributed to a subpopulation of cancer stem cells (CSCs), having enhanced tumourigenic potential. ABCG2 is specifically associated with the CSC phenotype, making it a valuable target for eliminating aggressive and resistant cells. Several natural and synthetic ionophores have been discovered as CSC-selective drugs that may also have MDR-reversing ability, whereas their interaction with ABCG2 has not yet been explored. We previously reported the biological activities, including ABCB1 inhibition, of a group of adamantane-substituted diaza-18-crown-6 (DAC) compounds that possess ionophore capabilities. In this study, we investigated the mechanism of ABCG2-inhibitory activity of DAC compounds and the natural ionophores salinomycin, monensin and nigericin. We used a series of functional assays, including real-time microscopic analysis of ABCG2-mediated fluorescent substrate transport in cells, and docking studies to provide comparative aspects for the transporter-compound interactions and their role in restoring chemosensitivity. We found that natural ionophores did not inhibit ABCG2, suggesting that their CSC selectivity is likely mediated by other mechanisms. In contrast, DACs with amide linkage in the side arms demonstrated noteworthy ABCG2-inhibitory activity, with DAC-3Amide proving to be the most potent. This compound induced conformational changes of the transporter and likely binds to both Cavity 1 and the NBD-TMD interface. DAC-3Amide reversed ABCG2-mediated MDR in model cells, without affecting ABCG2 expression or localization. These results pave the way for the development of new crown ether compounds with improved ABCG2-inhibitory properties.

Design, synthesis, biological evaluation and QSAR analysis of novel N-substituted benzimidazole derived carboxamides.

As a result of our previous research focussed on benzimidazoles, herein we present design, synthesis, QSAR analysis and biological activity of novel N-substituted benzimidazole derived carboxamides. Carboxamides were designed to study the influence of the number of methoxy groups, the type of the substituent placed at the benzimidazole core on biological activity. Pronounced antioxidative activity displayed unsubstituted 28 (IC50 ≈ 3.78 mM, 538.81 mmolFe2+/mmolC) and dimethoxy substituted derivative 34 (IC50 ≈ 5.68 mM, 618.10 mmolFe2+/mmolC). Trimethoxy substituted 43 and unsubstituted compound 40 with isobutyl side chain at N atom showed strong activity against HCT116 (IC50 ≈ 0.6 µM, both) and H 460 cells (IC50 ≈ 2.5 µM; 0.4 µM), being less cytotoxic towards non-tumour cell. Antioxidative activity in cell generally confirmed relatively modest antioxidant capacity obtained in DPPH/FRAP assays of derivatives 34 and 40. The 3D-QSAR models were generated to explore molecular properties that have the highest influence on antioxidative activity.

Novel amino substituted tetracyclic imidazo[4,5-b]pyridine derivatives: Design, synthesis, antiproliferative activity and DNA/RNA binding study.

A novel series of tetracyclic imidazo[4,5-b]pyridine derivatives was designed and synthesized as potential antiproliferative agents. Their antiproliferative activity against human cancer cells was influenced by the introduction of chosen amino side chains on the different positions on the tetracyclic skeleton and particularly, by the position of N atom in the pyridine nuclei. Thus, the majority of compounds showed improved activity in comparison to standard drug etoposide. Several compounds showed pronounced cytostatic effect in the submicromolar range, especially on HCT116 and MCF-7 cancer cells. The obtained results have confirmed the significant impact of the position of N nitrogen in the pyridine ring on the enhancement of antiproliferative activity, especially for derivatives bearing amino side chains on position 2. Thus, regioisomers 6, 7 and 9 showed noticeable enhancement of activity in comparison to their counterparts 10, 11 and 13 with IC50 values in a nanomolar range of concentration (0.3-0.9 µM). Interactions with DNA (including G-quadruplex structure) and RNA were influenced by the position of amino side chains on the tetracyclic core of imidazo[4,5-b]pyridine derivatives and the ligand charge. Moderate to high binding affinities (logKs = 5-7) obtained for selected imidazo[4,5-b]pyridine derivatives suggest that DNA/RNA are potential cell targets.

Synthesis, antiproliferative activity and DNA/RNA-binding properties of mono- and bis-(1,2,3-triazolyl)-appended benzimidazo[1,2-a]quinoline derivatives.

The three series of 5-mono- and 2,5-bis-1,2,3-triazolyl-substituted benzimidazo[1,2-a]quinolines as potential antitumor agents were synthesized. Their growth-inhibitory activity is influenced by the introduction of fluorine at C-2 and the mono-triazolyl nuclei at C-5 of the tetracyclic skeleton, particularly if the 1,2,3-triazole moiety contains a short aliphatic side-chain. Thus, the chloropropyl side-chain in all three series had the highest impact on the inhibitory effect. 1,2,3-Triazolyl-2-fluorobenzimidazo[1,2-a]quinoline conjugates 8a and 8b with 3-chloropropyl and 2-hydroxyethyl substituents, respectively, exhibited the most pronounced cytostatic effect on colon cancer (HCT116) cells in the submicromolar range. The compound 8a emerged as the most promising candidate because of its higher potency and some selectivity in the non-tumor aneuploid immortal keratinocyte (HaCaT) cells. Fluorescence and CD spectroscopy, as well as the thermal denaturation assays, revealed moderate to high DNA/RNA binding affinities of the selected compounds and identified intercalation as a dominant binding mode to both polynucleotides. However, results of intracellular distribution assay in human lung carcinoma (H460) cells suggest that both 8a and 8b do not target nuclear DNA and that their non-specific cytotoxic effect may be attributed to the damage of intercellular membranes.

Experimental and Computational Study of the Antioxidative Potential of Novel Nitro and Amino Substituted Benzimidazole/Benzothiazole-2-Carboxamides with Antiproliferative Activity.

We present the synthesis of a range of benzimidazole/benzothiazole-2-carboxamides with a variable number of methoxy and hydroxy groups, substituted with nitro, amino, or amino protonated moieties, which were evaluated for their antiproliferative activity in vitro and the antioxidant capacity. Antiproliferative features were tested on three human cancer cells, while the antioxidative activity was measured using 1,1-diphenyl-picrylhydrazyl (DPPH) free radical scavenging and ferric reducing antioxidant power (FRAP) assays. Trimethoxy substituted benzimidazole-2-carboxamide 8 showed the most promising antiproliferative activity (IC50 = 0.6-2.0 µM), while trihydroxy substituted benzothiazole-2-carboxamide 29 was identified as the most promising antioxidant, being significantly more potent than the reference butylated hydroxytoluene BHT in both assays. Moreover, the latter also displays antioxidative activity in tumor cells. The measured antioxidative capacities were rationalized through density functional theory (DFT) calculations, showing that 29 owes its activity to the formation of two [O•∙∙∙H-O] hydrogen bonds in the formed radical. Systems 8 and 29 were both chosen as lead compounds for further optimization of the benzazole-2-carboxamide scaffold in order to develop more efficient antioxidants and/or systems with the antiproliferative activity.

Crown ethers reverse P-glycoprotein-mediated multidrug resistance in cancer cells.

Multidrug resistance (MDR) is a widespread phenomenon exhibited by many cancers and represents a fundamental obstacle for successful cancer treatments. Tumour cells commonly achieve MDR phenotype through overexpression and/or increased activity of ABC transporters. P-glycoprotein transporter (P-gp, ABCB1) is a major cause of MDR and therefore represents a valuable target for MDR reversal. Several naturally occurring potassium ionophores (e.g. salinomycin) were shown to inhibit P-gp effectively. We have previously shown antitumour activity of a number of 18-crown-6 ether compounds that transport potassium ions across membranes. Here we present data on P-gp inhibitory activity of 16 adamantane-substituted monoaza- and diaza-18-crown-6 ether compounds, and their effect on MDR reversal in model cell lines. We show that crown ether activity depends on their lipophilicity as well as on the linker to adamantane moiety. The most active crown ethers were shown to be more effective in sensitising MDR cells to paclitaxel and adriamycin than verapamil, a well-known P-gp inhibitor. Altogether our data demonstrate a novel use of crown ethers for inhibition of P-gp and reversal of MDR phenotype.