Cellular Effects of Selected Unsymmetrical Bisacridines on the Multicellular Tumor Spheroids of HCT116 Colon and A549 Lung Cancer Cells in Comparison to Monolayer Cultures.

Multicellular tumor spheroids are a good tool for testing new anticancer drugs, including those that may target cancer stem cells (CSCs), which are responsible for cancer progression, metastasis, and recurrence. Therefore, we applied this model in our studies of highly active antitumor unsymmetrical bisacridines (UAs). We investigated the cellular response induced by UAs in 2D and 3D cultures of HCT116 colon and A549 lung cancer cells, with an additional focus on their impact on the CSC-like population. We showed that UAs affected the viability of the studied cells, as well as their spherogenic potential in the 2D and 3D cultures. Furthermore, we proved that the most promising UAs (C-2045 and C-2053) induced apoptosis in the HCT116 and A549 spheres to a similar, or even higher, extent than what was found in monolayer conditions. Next, we identified the population of the CSC-like cells in the 2D and 3D cultures of the studied cell lines by determining the levels of CD166, CD133, CD44, and EpCAM markers. We showed that the selected UAs affected the CSC-like population in both of the cell lines, and that A549 was affected more profoundly in 3D than in 2D cultures. Thus, the UAs exhibited high antitumor properties in both the 2D and 3D conditions, which makes them promising candidates for future therapeutic applications.

An influence of molecular weight, deacetylation degree of chitosan xerogels on their antimicrobial activity and cytotoxicity. Comparison of chitosan materials obtained using lactic acid and CO2 saturation.

This paper presents a comparison of the antimicrobial activity and cytotoxicity against L929 cells of chitosan xerogels prepared by dissolving the polymer in a solution of lactic acid (LA) or carbonic acid (CO2) and then freeze-drying. There was no simple relationship between the antimicrobial activity and cytotoxicity of the samples obtained using both techniques (LA and CO2). Chitosan materials obtained by the LA method in a 1:1 dilution were characterized by the highest cytotoxicity against L929 cells (∼20%). For the same diluted samples prepared using the CO2 saturation method, the viability of L929 cells was approximately 2.5 times greater. Some of the tested chitosan materials obtained by the innovative method were characterized by significantly lower antimicrobial activity, for example, reduction of E. coli bacteria for MMW-LA and MMW-CO2 samples by 6.00 and 0.75 logarithmic order, respectively. This clearly indicates that in many applications, the presence of the acid necessary to dissolve chitosan is responsible for the antimicrobial activity of the polymer solution and its products.

Investigation on Filaments for 3D Printing of Nasal Septum Cartilage Implant.

Septoplasty is a widely used method in treating deviated septum. Although it is successfully implemented, there are problems with excessive bleeding, septal perforation, or infections. The use of anatomically shaped implants could help overcome these problems. This paper focuses on assessing the possibility of the usage of a nasal septum cartilage implant 3D printed from various market-available filaments. Five different types of laments were used, two of which claim to be suitable for medical use. A combination of modeling, mechanical (bending, compression), structural (FTIR), thermal (DSC, MFR), surface (contact angle), microscopic (optical), degradation (2 M HCl, 5 M NaOH, and 0.01 M PBS), printability, and cell viability (MTT) analyses allowed us to assess the suitability of materials for manufacturing implants. Bioflex had the most applicable properties among the tested materials, but despite the overall good performance, cell viability studies showed toxicity of the material in MTT test. The results of the study show that selected filaments were not suitable for nasal cartilage implants. The poor cell viability of Bioflex could be improved by surface modification. Further research on biocompatible elastic materials for 3D printing is needed either by the synthesis of new materials or by modifying existing ones.

pH-Responsive Drug Delivery Nanoplatforms as Smart Carriers of Unsymmetrical Bisacridines for Targeted Cancer Therapy.

Selective therapy and controlled drug release at an intracellular level remain key challenges for effective cancer treatment. Here, we employed folic acid (FA) as a self-navigating molecule in nanoconjugates containing quantum dots (QDs) and ß-cyclodextrin (ß-CD) for the delivery of antitumor unsymmetrical bisacridine compound (C-2028) to lung and prostate cancers as well as normal cells. The bisacridine derivative can form the inclusion complex with ß-cyclodextrin molecule, due to the presence of a planar fragment in its structure. The stability of such a complex is pH-dependent. The drug release profile at different pH values and the mechanism of C-2028 release from QDs-ß-CD-FA nanoconjugates were investigated. Next, the intracellular fate of compounds and their influence on lysosomal content in the cells were also studied. Confocal Laser Scanning Microscopy studies proved that all investigated compounds were delivered to acidic organelles, the pH of which promoted an increased release of C-2028 from its nanoconjugates. Since the pH in normal cells is higher than in cancer cells, the release of C-2028 from its nanoconjugates is decreased in these cells. Additionally, we obtained the concentration profiles of C-2028 in the selected cells treated with unbound C-2028 or nanoconjugate by the HPLC analysis.

A Novel Method of Endotoxins Removal from Chitosan Hydrogel as a Potential Bioink Component Obtained by CO2 Saturation.

The article presents a new approach in the purification of chitosan (CS) hydrogel in order to remove a significant amount of endotoxins without changing its molecular weight and viscosity. Two variants of the method used to purify CS hydrogels from endotoxins were investigated using the PyroGene rFC Enzymatic Cascade assay kit. The effect of the CS purification method was assessed in terms of changes in the dynamic viscosity of its hydrogels, the molecular weight of the polymer, microbiological purity after refrigerated storage and cytotoxicity against L929 cells based on the ISO 10993-5:2009(E) standard. The proposed purification method 1 (M1) allows for the removal of significant amounts of endotoxins: 87.9-97.6% in relation to their initial concentration in the CS hydrogel without affecting the solution viscosity. Moreover, the final solutions were sterile and microbiologically stable during storage. The M1 purification method did not change the morphology of the L929 cells.

c-Myc Protein Level Affected by Unsymmetrical Bisacridines Influences Apoptosis and Senescence Induced in HCT116 Colorectal and H460 Lung Cancer Cells.

Unsymmetrical bisacridines (UAs) are highly active antitumor compounds. They contain in their structure the drugs previously synthesized in our Department: C-1311 and C-1748. UAs exhibit different properties than their monomer components. They do not intercalate to dsDNA but stabilize the G-quadruplex structures, particularly those of the MYC and KRAS genes. Since MYC and KRAS are often mutated and constitutively expressed in cancer cells, they can be used as therapeutic targets. Herein, we investigate whether UAs can affect the expression and protein level of c-Myc and K-Ras in HCT116 and H460 cancer cells, and if so, what are the consequences for the UAs-induced cellular response. UAs did not affect K-Ras, but they strongly influenced the expression and translation of the c-Myc protein, and in H460 cells, they caused its full inhibition. UAs treatment resulted in apoptosis, as confirmed by the morphological changes, the presence of sub-G1 population and active caspase-3, cleaved PARP, annexin-V/PI staining and a decrease in mitochondrial potential. Importantly, apoptosis was induced earlier and to a greater extent in H460 compared to HCT116 cells. Moreover, accelerated senescence occurred only in H460 cells. In conclusion, the strong inhibition of c-Myc by UAs in H460 cells may participate in the final cellular response (apoptosis, senescence).

Foliate-Targeting Quantum Dots-ß-Cyclodextrin Nanocarrier for Efficient Delivery of Unsymmetrical Bisacridines to Lung and Prostate Cancer Cells.

Targeted drug delivery by nanocarriers molecules can increase the efficiency of cancer treatment. One of the targeting ligands is folic acid (FA), which has a high affinity for the folic acid receptors, which are overexpressed in many cancers. Herein, we describe the preparation of the nanoconjugates containing quantum dots (QDs) and ß-cyclodextrin (ß-CD) with foliate-targeting properties for the delivery of anticancer compound C-2028. C-2028 was bound to the nanoconjugate via an inclusion complex with ß-CD. The effect of using FA in QDs-ß-CD(C-2028)-FA nanoconjugates on cytotoxicity, cellular uptake, and the mechanism of internalization in cancer (H460, Du-145, and LNCaP) and normal (MRC-5 and PNT1A) cells was investigated. The QDs-ß-CD(C-2028)-FA were characterized using DLS (dynamic light scattering), ZP (zeta potential), quartz crystal microbalance with dissipation (QCM-D), and UV-vis spectroscopy. The conjugation of C-2028 with non-toxic QDs or QDs-ß-CD-FA did not change the cytotoxicity of this compound. Confocal microscopy studies proved that the use of FA in nanoconjugates significantly increased the amount of delivered compound, especially to cancer cells. QDgreen-ß-CD(C-2028)-FA enters the cells through multiple endocytosis pathways in different levels, depending on the cell line. To conclude, the use of FA is a good self-navigating molecule in the QDs platform for drug delivery to cancer cells.

The Influence of Antitumor Unsymmetrical Bisacridines on 3D Cancer Spheroids Growth and Viability.

The culture of 3D spheroids is a promising tool in drug development and testing. Recently, we synthesized a new group of compounds, unsymmetrical bisacridines (UAs), which exhibit high cytotoxicity against various human cell lines and antitumor potency against several xenografts. Here, we describe the ability of four UAs-C-2028, C-2041, C-2045, and C-2053-to influence the growth of HCT116 and H460 spheres and the viability of HCT116 cells in 3D culture compared with that in 2D standard monolayer culture. Spheroids were generated using ultra-low-attachment plates. The morphology and diameters of the obtained spheroids and those treated with UAs were observed and measured under the microscope. The viability of cells exposed to UAs at different concentrations and for different incubation times in 2D and 3D cultures was assessed using 7-AAD staining. All UAs managed to significantly inhibit the growth of HCT116 and H460 spheroids. C-2045 and C-2053 caused the death of the largest population of HCT116 spheroid cells. Although C-2041 seemed to be the most effective in the 2D monolayer experiments, in 3D conditions, it turned out to be the weakest compound. The 3D spheroid culture seems to be a suitable method to examine the efficiency of new antitumor compounds, such as unsymmetrical bisacridines.

Metabolic Profiles of New Unsymmetrical Bisacridine Antitumor Agents in Electrochemical and Enzymatic Noncellular Systems and in Tumor Cells.

New unsymmetrical bisacridines (UAs) demonstrated high activity not only against a set of tumor cell lines but also against human tumor xenografts in nude mice. Representative UA compounds, named C-2028, C-2045 and C-2053, were characterized in respect to their physicochemical properties and the following studies aimed to elucidate the role of metabolic transformations in UAs action. We demonstrated with phase I and phase II enzymes in vitro and in tumors cells that: (i) metabolic products generated by cytochrome P450 (P450), flavin monooxygenase (FMO) and UDP-glucuronosyltransferase (UGT) isoenzymes in noncellular systems retained the compound's dimeric structures, (ii) the main transformation pathway is the nitro group reduction with P450 isoenzymes and the metabolism to N-oxide derivative with FMO1, (iii), the selected UGT1 isoenzymes participated in the glucuronidation of one compound, C-2045, the hydroxy derivative. Metabolism in tumor cells, HCT-116 and HT-29, of normal and higher UGT1A10 expression, respectively, also resulted in the glucuronidation of only C-2045 and the specific distribution of all compounds between the cell medium and cell extract was demonstrated. Moreover, P4503A4 activity was inhibited by C-2045 and C-2053, whereas C-2028 affected UGT1A and UGT2B action. The above conclusions indicate the optimal strategy for the balance among antitumor therapeutic efficacy and drug resistance in the future antitumor therapy.

Quantum Dots as a Good Carriers of Unsymmetrical Bisacridines for Modulating Cellular Uptake and the Biological Response in Lung and Colon Cancer Cells.

Nanotechnology-based drug delivery provides a promising area for improving the efficacy of cancer treatments. Therefore, we investigate the potential of using quantum dots (QDs) as drug carriers for antitumor unsymmetrical bisacridine derivatives (UAs) to cancer cells. We examine the influence of QD-UA hybrids on the cellular uptake, internalization (Confocal Laser Scanning Microscope), and the biological response (flow cytometry and light microscopy) in lung H460 and colon HCT116 cancer cells. We show the time-dependent cellular uptake of QD-UA hybrids, which were more efficiently retained inside the cells compared to UAs alone, especially in H460 cells, which could be due to multiple endocytosis pathways. In contrast, in HCT116 cells, the hybrids were taken up only by one endocytosis mechanism. Both UAs and their hybrids induced apoptosis in H460 and HCT116 cells (to a greater extent in H460). Cells which did not die underwent senescence more efficiently following QDs-UAs treatment, compared to UAs alone. Cellular senescence was not observed in HCT116 cells following treatment with both UAs and their hybrids. Importantly, QDgreen/red themselves did not provoke toxic responses in cancer or normal cells. In conclusion, QDs are good candidates for targeted UA delivery carriers to cancer cells while protecting normal cells from toxic drug activities.

Design, synthesis and high antitumor potential of new unsymmetrical bisacridine derivatives towards human solid tumors, specifically pancreatic cancers and their unique ability to stabilize DNA G-quadruplexes.

New promising unsymmetrical bisacridine derivatives (UAs), have been developed. Three groups including 36 compounds were synthesized by the condensation of 4-nitro or 4-methylacridinone, imidazoacridinone and triazoloacridinone derivatives with 1-nitroacridine compounds linked with an aminoalkyl chain. Cytotoxicity screening revealed the high potency of these compounds against several tumor cell lines. Particularly, imidazoacridinone-1-nitroacridine dimers strongly inhibited pancreatic Panc-1, Mia-Pa-Ca-2, Capan-2 and prostate cancer DU-145 cell growth. The studied compounds showed very strong antitumor activity (T/C> 300%) against Walker 256 rat adenocarcinoma. The selected 26 UAs were tested against 12 human tumor xenografts in nude mice, including colon, breast, prostate and pancreatic cancers. The studies on the molecular mechanism of action demonstrated that these unsymmetrical dimers significantly responded to the presence of G-quadruplex not to dsDNA. Structure-activity relationships for UAs potency to G-quadruplex stabilization indicated that thermal stability of this drug-G-quadruplex complex depended not only on the structure of heterocyclic rings, but also on the properties of dialkylamino chains of the ring linkers. In conclusion, the presented studies identified the new group of effective antitumor agents against solid human tumors, particularly pancreatic Panc-1, BxPC-3 and Mia-Pa-Ca-2 and strongly indicated their distinctive interactions with DNA. In contrast to monomers, G-quadruplex not dsDNA is proposed to be the first molecular target for these compounds.

Enhanced Activity of P4503A4 and UGT1A10 Induced by Acridinone Derivatives C-1305 and C-1311 in MCF-7 and HCT116 Cancer Cells: Consequences for the Drugs' Cytotoxicity, Metabolism and Cellular Response.

Activity modulation of drug metabolism enzymes can change the biotransformation of chemotherapeutics and cellular responses induced by them. As a result, drug-drug interactions can be modified. Acridinone derivatives, represented here by C-1305 and C-1311, are potent anticancer drugs. Previous studies in non-cellular systems showed that they are mechanism-based inhibitors of cytochrome P4503A4 and undergo glucuronidation via UDP-glucuronosyltranspherase 1A10 isoenzyme (UGT1A10). Therefore, we investigated the potency of these compounds to modulate P4503A4 and UGT1A10 activity in breast MCF-7 and colon HCT116 cancer cells and their influence on cytotoxicity and cellular response in cells with different expression levels of studied isoenzymes. We show that C-1305 and C-1311 are inducers of not only P4503A4 but also UGT1A10 activity. MCF-7 and HCT116 cells with high P4503A4 activity are more sensitive to acridinone derivatives and undergo apoptosis/necrosis to a greater extent. UGT1A10 was demonstrated to be responsible for C-1305 and C-1311 glucuronidation in cancer cells and glucuronide products were excreted outside the cell very fast. Finally, we show that glucuronidation of C-1305 antitumor agent enhances its pro-apoptotic properties in HCT116 cells, while the cytotoxicity and cellular response induced by C-1311 did not change after drug glucuronidation in both cell lines.

New Unsymmetrical Bisacridine Derivatives Noncovalently Attached to Quaternary Quantum Dots Improve Cancer Therapy by Enhancing Cytotoxicity toward Cancer Cells and Protecting Normal Cells.

The use of nanoparticles for the controlled drug delivery to cells has emerged as a good alternative to traditional systemic delivery. Quantum dots (QDs) offer potentially invaluable societal benefits such as drug targeting and in vivo biomedical imaging. In contrast, QDs may also pose risks to human health and the environment under certain conditions. Here, we demonstrated that a unique combination of nanocrystals core components (Ag-In-Zn-S) would eliminate the toxicity problem and increase their biomedical applications. The alloyed quaternary nanocrystals Ag-In-Zn-S (QDgreen, Ag1.0In1.2Zn5.6S9.4; QDred, Ag1.0In1.0Zn1.0S3.5) were used to transport new unsymmetrical bisacridine derivatives (UAs, C-2028 and C-2045) into lung H460 and colon HCT116 cancer cells for improving the cytotoxic and antitumor action of these compounds. UAs were coupled with QD through physical adsorption. The obtained results clearly indicate that the synthesized nanoconjugates exhibited higher cytotoxic activity than unbound compounds, especially toward lung H460 cancer cells. Importantly, unsymmetrical bisacridines noncovalently attached to QD strongly protect normal cells from the drug action. It is worth pointing out that QDgreen or QDred without UAs did not influence the growth of cancer and normal cells, which is consistent with in vivo results. In noncellular systems, at pH 5.5 and 4.0, which relates to the conditions of endosomes and lysosomes, the UAs were released from QD-UAs nanoconjugates. An increase of total lysosomes content was observed in H460 cells treated with QDs-UAs which can affect the release of the UAs from the conjugates. Moreover, confocal laser scanning microscopy analyses revealed that QD-UAs nanoconjugates enter H460 cells more efficiently than to HCT116 and normal cells, which may be the reason for their higher cytotoxicity against lung cancer. Summarizing, the noncovalent attachment of UAs to QDs increases the therapeutic efficiency of UAs by improving cytotoxicity toward lung H460 cancer cells and having protecting effects on normal cells.

Obtaining and Characterization of the PLA/Chitosan Foams with Antimicrobial Properties Achieved by the Emulsification Combined with the Dissolution of Chitosan by CO2 Saturation.

A new method of obtaining functional foam material has been proposed. The materials were created by mixing the poly lactic acid (PLA) solution in chloroform, chitosan (CS) dissolved in water saturated with CO2 and polyethylene glycol (PEG), and freeze-dried for removal of the solvents. The composite foams were characterized for their structural (SEM, FT-IR, density, porosity), thermal (DSC), functional (hardness, elasticity, swelling capacity, solubility), and biological (antimicrobial and cytotoxic) properties. Chitosan in the composites was a component for obtaining their foamed form with 7.4 to 22.7 times lower density compared to the neat PLA and high porosity also confirmed by the SEM. The foams had a hardness in the range of 70-440 kPa. The FT-IR analysis confirmed no new chemical bonds between the sponge ingredients. Other results showed low sorption capacity (2.5-7.2 g/g) and solubility of materials (less than 0.2%). The obtained foams had the lower Tg value and improved ability of crystallization compared to neat PLA. The addition of chitosan provides the bacteriostatic and bactericidal properties against Escherichia coli and Staphylococcus aureus. Biocompatibility studies have shown that the materials obtained are not cytotoxic to the L929 cell line.

Stable nanoconjugates of transferrin with alloyed quaternary nanocrystals Ag-In-Zn-S as a biological entity for tumor recognition.

One way to limit the negative effects of anti-tumor drugs on healthy cells is targeted therapy employing functionalized drug carriers. Here we present a biocompatible and stable nanoconjugate of transferrin anchored to Ag-In-Zn-S quantum dots modified with 11-mercaptoundecanoic acid (Tf-QD) as a drug carrier versus typical anticancer drug, doxorubicin. Detailed investigations of Tf-QD nanoconjugates without and with doxorubicin by fluorescence studies and cytotoxic measurements showed that the biological activity of both the transferrin and doxorubicin was fully retained in the nanoconjugate. In particular, the intercalation capabilities of free doxorubicin versus ctDNA remained essentially intact upon its binding to the nanoconjugate. In order to evaluate these capabilities, we studied the binding constant of doxorubicin attached to Tf-QDs with ctDNA as well as the binding site size on the ctDNA molecule. The binding constant slightly decreased compared to that of free doxorubicin while the binding site size, describing the number of consecutive DNA lattice residues involved in the binding, increased. It was also demonstrated that the QDs alone and in the form of a nanoconjugate with Tf were not cytotoxic towards human non-small cell lung carcinoma (H460 cell line) and the tumor cell sensitivity of the DOX-Tf-QD nanoconjugate was comparable to that of doxorubicin alone.

Investigation of an elutable N-propylphosphonic acid chitosan derivative composition with a chitosan matrix prepared from carbonic acid solution.

Porous chitosan composites using CO2 dissolution procedure and including water soluble N-propylphosphonic chitosan derivative (p-CHI) were obtained and characterized. In contrast to the control material, composites containing modified chitosan distinguished by a rapid moisture absorption and good adhesion to the skin. The FTIR analysis confirmed the presence of propylphosphonic group in the structure of the polymer. The porosity of the materials was in the range 55-77% and decreased with increasong amount of modified chitosan in materials. Solubility of composites was dependent on the content of p-CHI in scaffolds (40%, 25% and 15%) and reached values 11%, 9% and 6,5%, respectively. The values of other parameters like swelling degree (30g/g) good antioxidant and antimicrobial properties (almost 100% reduction of S.aureus, E.coli and C. albicans growth) and low in vitro cytotoxicity against fibroblasts were highly advantageous for possible biomedical applications of the composites.

The overexpression of CPR and P450 3A4 in pancreatic cancer cells changes the metabolic profile and increases the cytotoxicity and pro-apoptotic activity of acridine antitumor agent, C-1748.

Drug resistance is one of the major causes of pancreatic cancer treatment failure. Thus, it is still imperative to develop new active compounds and novel approach to improve drug efficacy. Here we present 9-amino-1-nitroacridine antitumor agent, C-1748, developed in our laboratory, as a candidate for pancreatic cancer treatment. We examined (i) the cellular response of pancreatic cancer cell lines: Panc-1, MiaPaCa-2, BxPC-3 and AsPC-1, differing in expression levels of commonly mutated genes for this cancer type, to C-1748 treatment and (ii) the role of P450 3A4 isoenzyme and cytochrome P450 reductase (CPR) in the modulation of this response. C-1748 exhibited the highest cytotoxic activity against MiaPaCa-2, while AsPC-1 cells were the most resistant (IC50: 0.015, 0.075µM, respectively). A considerable amount of apoptosis was detected in Panc-1 and MiaPaCa-2 cells but only limited apoptosis was observed in AsPC-1 and BxPC-3 cells as indicated by morphological changes and biochemical markers. Furthermore, only AsPC-1 cells underwent senescence. Since AsPC-1 cells were the most resistant to C-1748 as evidenced by the lowest P450 3A4 and CPR protein levels, this cell line was subjected to transient transfection either with P450 3A4 or CPR gene. The overexpression of P450 3A4 or CPR changed the pro-apoptotic activity of C-1748 and sensitized AsPC-1 cells to this drug compared to wild-type cells. However, metabolism was changed significantly only for CPR overexpressing cells. In conclusion, the antitumor effectiveness of C-1748 would be improved by multi-drug therapy with chemotherapeutics, that are able to induce P450 3A4 and/or CPR gene expression.

Improved cytotoxicity and preserved level of cell death induced in colon cancer cells by doxorubicin after its conjugation with iron-oxide magnetic nanoparticles.

A promising strategy for overcoming the problem of limited efficacy in antitumor drug delivery and in drug release is the use of a nanoparticle-conjugated drug. Doxorubicin (Dox) anticancer chemotherapeutics has been widely studied in this respect, because of severe cardiotoxic side effects. Here, we investigated the cytotoxic effects, the uptake process, the changes in cell cycle progression and the cell death processes in the presence of iron-oxide magnetic nanoparticles (Nps) and doxorubicin conjugates (Dox-Nps) in human colon HT29 cells. The amount of Dox participated in biological action of Dox-Nps was determined by cyclic voltammetry and thermogravimetric measurements. The cytotoxicity of Dox-Nps was shown to be two/three times higher than free Dox, whereas Nps alone did not inhibit cell proliferation. Dox-Nps penetrated cancer cells with higher efficacy than free Dox, what could be a consequence of Dox-Nps aggregation with proteins in culture medium and/or with cell surface. The treatment of HT29 cells with Dox-Nps and Dox at IC50 concentration resulted in G2/M arrest followed by late apoptosis and necrosis. Summing up, the application of iron-oxide magnetic nanoparticles improved Dox-Nps cell penetration compared to free Dox and achieved the cellular response to Dox-Nps conjugates similar to that of Dox alone.

The role of glucuronidation in drug resistance.

The final therapeutic effect of a drug candidate, which is directed to a specific molecular target strongly depends on its absorption, distribution, metabolism and excretion (ADME). The disruption of at least one element of ADME may result in serious drug resistance. In this work we described the role of one element of this resistance: phase II metabolism with UDP-glucuronosyltransferases (UGTs). UGT function is the transformation of their substrates into more polar metabolites, which are better substrates for the ABC transporters, MDR1, MRP and BCRP, than the native drug. UGT-mediated drug resistance can be associated with (i) inherent overexpression of the enzyme, named intrinsic drug resistance or (ii) induced expression of the enzyme, named acquired drug resistance observed when enzyme expression is induced by the drug or other factors, as food-derived compounds. Very often this induction occurs via ligand binding receptors including AhR (aryl hydrocarbon receptor) PXR (pregnane X receptor), or other transcription factors. The effect of UGT dependent resistance is strengthened by coordinate action and also a coordinate regulation of the expression of UGTs and ABC transporters. This coupling of UGT and multidrug resistance proteins has been intensively studied, particularly in the case of antitumor treatment, when this resistance is "improved" by differences in UGT expression between tumor and healthy tissue. Multidrug resistance coordinated with glucuronidation has also been described here for drugs used in the management of epilepsy, psychiatric diseases, HIV infections, hypertension and hypercholesterolemia. Proposals to reverse UGT-mediated drug resistance should consider the endogenous functions of UGT.

Targeting of FLT3-ITD kinase contributes to high selectivity of imidazoacridinone C-1311 against FLT3-activated leukemia cells.

Drugs targeting receptor tyrosine kinase FLT3 are of particular interest since activating FLT3-internal tandem duplication (ITD) mutations abundantly occur in fatal acute myeloid leukemias (AMLs). Imidazoacridinone C-1311, a DNA-reactive inhibitor of topoisomerase II, has been previously shown to be a potent and selective inhibitor of recombinant FLT3. Here, we expand those findings by studying its effect on leukemia cells with wild-type FLT3, FLT3-ITD mutant and no FLT3 receptor. While brief C-1311 exposure blocked wild-type and FLT3-ITD activity, profound and sustained inhibition was achieved only for FLT3-ITD mutants. C-1311 inhibited FLT3 downstream pathways (MAPK and AKT) independent of FLT3 status, yet translation to decreased viability was significant in FLT3-ITD cells. RNA interference against FLT3-ITD reduced cytotoxic effect and apoptosis induced by C-1311, indicating selective inhibition of FLT3-ITD crucial for high efficacy of drug against activated leukemia cells. Cellular responses in treated FLT3-ITD mutants included G1 and G2/M phase arrest, moderate inhibition of Bcl-2, caspase-3 activation, PARP cleavage, and depolarization of mitochondria. Consistent with selective decrease in FLT3-ITD activity, C-1311 remarkably reduced antiapoptotic survivin mRNA and protein expression, correlating well with enhanced apoptosis of FLT3-ITD cells. No survivin decrease and respectively lower level of apoptosis was found in wild-type and null-FLT3 cells. Combination of C-1311 with cytarabine or doxorubicin again showed distinct synergistic activity in FLT3-ITD-positive cells. The ability of C-1311 to selectively target constitutively active FLT3, suggests a favorable therapeutic index for AML carrying FLT3-ITD mutations. Thus further preclinical and clinical studies addressing its potency against FLT3-ITD kinase is well justified.