Supplementary material for the article: Manzano, J. I.; Konstantinović, J.; Scaccabarozzi, D.; Perea, A.; Pavić, A.; Cavicchini, L.; Basilico, N.; Gamarro, F.; Šolaja, B. A. 4-Aminoquinoline-Based Compounds as Antileishmanial Agents That Inhibit the Energy Metabolism of Leishmania. European Journal of Medicinal Chemistry 2019, 180, 28–40. https://doi.org/10.1016/j.ejmech.2019.07.010

Supplementary material for: [https://doi.org/10.1016/j.ejmech.2019.07.010]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3287

Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy

A mesenchymal stromal cell line resistant to paclitaxel that spontaneously differentiates into osteoblast-like cells

The mesenchymal stromal cell line SR- 4987 has been established in our laboratory from the bone marrow of BDF/1 mice. Recent information on mesenchymal stem cells biology and the need to deal with well-characterized cell lines suggest to critically consider the existent data on this cell line by updating them with new investigations on growth parameters, in vitro plasticity, and drug sensitivity to anti-cancer, anti-inflammatory, and a histone deacetylase inhibitor. SR-4987 cells show a population doubling time of 24.5±5.4 h, a plating efficiency of 2.87±1.19%, and under stimulation maintain only in part their multipotency by differentiating towards chondro-osteogenic lineages but not into adipogenic. Surprisingly, these mesenchymal stromal cells differentiate spontaneously into osteoblast-like cells and this is significantly stimulated by valproic acid. SR-4987 cells show a dramatic resistance to paclitaxel (PTX) with a resistance index of 39.6 times (evaluated versus MOLT-4 leukemia) and of 68.2 (versus HT-29 colorectal carcinoma). SR-4987 resistance is reversed by verapamil and correlates with high expression of Pglycoprotein that is down-modulated by PTX. Taken together, our results indicated that SR-4987 line is a very interesting cell model useful to investigate both drug sensitivity resistance and physiopathological aspects related to mesenchymal cell function.JRC.I.4-Molecular biology and genomic

Quinolizidine-Derived Lucanthone and Amitriptyline Analogues Endowed with Potent Antileishmanial Activity

Leishmaniases are neglected diseases that are endemic in many tropical and sub-tropical Countries. Therapy is based on different classes of drugs which are burdened by severe side effects, occurrence of resistance and high costs, thereby creating the need for more efficacious, safer and inexpensive drugs. Herein, sixteen 9-thioxanthenone derivatives (lucanthone analogues) and four compounds embodying the diarylethene substructure of amitriptyline (amitriptyline analogues) were tested in vitro for activity against Leishmania tropica and L. infantum promastigotes. All compounds were characterized by the presence of a bulky quinolizidinylalkyl moiety. All compounds displayed activity against both species of Leishmania with IC50 values in the low micromolar range, resulting in several fold more potency than miltefosine, comparable to that of lucanthone, and endowed with substantially lower cytotoxicity to Vero-76 cells, for the best of them. Thus, 4-amino-1-(quinolizidinylethyl)aminothioxanthen-9-one (14) and 9-(quinolizidinylmethylidene)fluorene (17), with selectivity index (SI) in the range 16\u201324, represent promising leads for the development of improved antileishmanial agents. These two compounds also exhibited comparable activity against intramacrophagic amastigotes of L. infantum. Docking studies have suggested that the inhibition of trypanothione reductase (TryR) may be at the basis (eventually besides other mechanisms) of the observed antileishmanial activity. Therefore, these investigated derivatives may deserve further structural improvements and more in-depth biological studies of their mechanisms of action in order to develop more efficient antiparasitic agents

CD45+/CD133+ Positive Cells Expanded from Umbilical Cord Blood Expressing PDX-1 and Markers of Pluripotency

Human umbilical cord blood (UCB) contains cells able to differentiate into nonhaematopoietic cell lineages. It also contains cells similar to primitive embryonic stem cells (ESCs) that can differentiate into pancreatic-like cells. However, little data has been reported regarding the possibility of expanding these cells or their differential gene expression occurring in vitro. We expanded previously frozen UCB cells by treatment with Stem Cell Factor (SCF) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) in the presence of valproic acid (VPA). Gene expression profiles for beta-cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT-PCR and immunocytochemistry. There was a dramatic expansion ( >150 fold) of hematopoietic progenitors (CD45+/CD133+) that also expressed embryo markers of pluripotency (nanog, kfl-4,sox-2,oct3/4 and c-myc) , nestin, pancreatic markers as pax-4, ngn-3, pdx-1 and syt-1 . UCB cells can be expanded to produce large numbers of cells of hematopoietic lineage that naturally (without retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Our results confirm that frozen UCB cells can be dramatically expanded along the hematopoietic cell lineage (CD45+/CD133+) which express both markers of pluripotency and a gene pattern compatible with endocrine pancreatic precursors.JRC.I.3-Molecular Biology and Genomic

Quinolizidine-Derived Lucanthone and Amitriptyline Analogues Endowed with Potent Antileishmanial Activity

Leishmaniases are neglected diseases that are endemic in many tropical and sub-tropical Countries. Therapy is based on different classes of drugs which are burdened by severe side effects, occurrence of resistance and high costs, thereby creating the need for more efficacious, safer and inexpensive drugs. Herein, sixteen 9-thioxanthenone derivatives (lucanthone analogues) and four compounds embodying the diarylethene substructure of amitriptyline (amitriptyline analogues) were tested in vitro for activity against Leishmania tropica and L. infantum promastigotes. All compounds were characterized by the presence of a bulky quinolizidinylalkyl moiety. All compounds displayed activity against both species of Leishmania with IC50 values in the low micromolar range, resulting in several fold more potency than miltefosine, comparable to that of lucanthone, and endowed with substantially lower cytotoxicity to Vero-76 cells, for the best of them. Thus, 4-amino-1-(quinolizidinylethyl)aminothioxanthen-9-one (14) and 9-(quinolizidinylmethylidene)fluorene (17), with selectivity index (SI) in the range 16–24, represent promising leads for the development of improved antileishmanial agents. These two compounds also exhibited comparable activity against intramacrophagic amastigotes of L. infantum. Docking studies have suggested that the inhibition of trypanothione reductase (TryR) may be at the basis (eventually besides other mechanisms) of the observed antileishmanial activity. Therefore, these investigated derivatives may deserve further structural improvements and more in-depth biological studies of their mechanisms of action in order to develop more efficient antiparasitic agents

Bcl-2 down modulation in WEHI-3B/CTRES cells resistant to Cholera Toxin (CT)-induced apoptosis

The very different effects of Cholera Toxin (CT) on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the \"in vitro\" clonogenicity, the Population Doubling Time (PDT), apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone (WEHI-3B/CTRES). In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells (WEHI-3B/CTRES), Bcl-2 expression was down regulated by both CT or drug treatment (eg., ciprofloxacin, CPX) although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase (PKA) in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models (of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX) provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function

Paclitaxel-Loaded Silk Fibroin Nanoparticles: Method Validation by UHPLC-MS/MS to Assess an Exogenous Approach to Load Cytotoxic Drugs

The aim of this work was to load an anticancer drug, paclitaxel (PTX), on Silk Fibroin Nanoparticles (SFNs) by using an exogenous approach. SFNs were produced, freeze-dried and then loaded with PTX. An exogenous method allowed us to reduce both drug loss and environmental impact. In order to quantify PTX loaded in SFNs, a simple and reliable method using reversed phase liquid chromatography coupled to tandem mass spectrometry (rp-UHPLC-MS/MS) was developed. This methodology was validated by the determination of spiked QC samples in three consecutive days. Good accuracy and precision of the method were obtained, while the intra-day and inter-day precisions were less than 10.3%. For PTX, the limit of quantitation (LOQ) was 5.0 ng/mL. Recovery from the matrix (SFNs-PTX pellets) was calculated (81.2% at LOQ value) as PTX was entrapped in a new matrix like the polymer silk fibroin-based. This method was successfully applied to determine the encapsulation efficiency (1.00 ± 0.19%) and the nanoparticle loading (0.12 ± 0.02% w/w). The in vitro anticancer activity of SFNs-PTX was tested against CFPAC-1 cancer cells; results demonstrated a very high cytotoxic activity of SFNs-PTX, with a dose dependent inhibition of CFPAC-1 proliferation, confirmed by the IC50 value of 3450 ± 750 ng/mL

Human amniotic mesenchymal stromal cells (hAMSCs) as potential vehicles for drug delivery in cancer therapy: an in vitro study

INTRODUCTION: In the context of drug delivery, mesenchymal stromal cells (MSCs) from bone marrow and adipose tissue have emerged as interesting candidates due to their homing abilities and capacity to carry toxic loads, while at the same time being highly resistant to the toxic effects. Amongst the many sources of MSCs which have been identified, the human term placenta has attracted particular interest due to its unique, tissue-related characteristics, including its high cell yield and virtually absent expression of human leukocyte antigens and co-stimulatory molecules. Under basal, non-stimulatory conditions, placental MSCs also possess basic characteristics common to MSCs from other sources. These include the ability to secrete factors which promote cell growth and tissue repair, as well as immunomodulatory properties. The aim of this study was to investigate MSCs isolated from the amniotic membrane of human term placenta (hAMSCs) as candidates for drug delivery in vitro. METHODS: We primed hAMSCs from seven different donors with paclitaxel (PTX) and investigated their ability to resist the cytotoxic effects of PTX, to upload the drug, and to release it over time. We then analyzed whether the uptake and release of PTX was sufficient to inhibit proliferation of CFPAC-1, a pancreatic tumor cell line sensitive to PTX. RESULTS: For the first time, our study shows that hAMSCs are highly resistant to PTX and are not only able to uptake the drug, but also release it over time. Moreover, we show that PTX is released from hAMSCs in a sufficient amount to inhibit tumor cell proliferation, whilst some of the PTX is also retained within the cells. CONCLUSION: Taken together, for the first time our results show that placental stem cells can be used as vehicles for the delivery of cytotoxic agents

Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week; the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases