Madalena Cipriano

Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing

IntroductionThe secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds.MethodsA UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively.ResultsUCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system already showing glands and hair follicles.ConclusionsThis work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products by providing a proof of concept that a reproducible system for the production of UCX®-conditioned medium can be used to prime a secretome for eventual clinical applications.

Development of pyridine-containing macrocyclic copper(II) complexes: potential role in the redox modulation of oxaliplatin toxicity in human breast cells

Abstract The unique redox and catalytic chemistry of Cu has justified the development of novel Cu complexes for different therapeutic uses including cancer therapy. In this work, four pyridine-containing aza-macrocyclic copper(II) complexes were prepared (CuL1–CuL4) varying in ring size and/or substituents and their superoxide scavenging activity evaluated. CuL3, the most active superoxide scavenger, was further studied as a modulator of the cytotoxicity of oxaliplatin in epithelial breast MCF10A cells and in MCF7 breast cancer cells. Our results show that CuL3 enhances the therapeutic window of oxaliplatin, by both protecting non-tumour cells and increasing its cytotoxic effect in breast carcinoma cells. CuL3 is thus a promising complex to be further studied and to be used as a lead compound for the optimization of novel chemotherapy sensitizers.

Role of the Copper(II) Complex Cu[15]pyN5 in Intracellular ROS and Breast Cancer Cell Motility and Invasion.

Multiple mechanisms related to metastases undergo redox regulation. Cu[15]pyN5 is a redox‐active copper(II) complex previously studied as a chemotherapy sensitizer in mammary cells. The effects of a cotreatment with Cu[15]pyN5 and doxorubicin (dox) were evaluated in two human breast cancer cell lines: MCF7 (low aggressiveness) and MDA‐MB‐231 (highly aggressive). Cu[15]pyN5 decreased MCF7‐directed cell migration. In addition, a cotreatment with dox and Cu[15]pyN5 reduced the proteolytic invasion of MDA‐MB‐231 cells. Cell detachment was not affected by exposure to these agents. Cu[15]pyN5 and dox significantly increased intracellular ROS in both cell lines. This increase could be at least partially due to H2O2 accumulation. The combination of Cu[15]pyN5 with dox may be beneficial in breast cancer treatment as it could help reduce cancer cell migration and invasion. Moreover, the ligand [15]pyN5 has a high affinity for copper(II) and displays potential anti‐angiogenic properties. Overall, we present a potential drug that might arrest the progression of breast cancer by different and complementary mechanisms.

Cytotoxic effects of cadmium in mammary epithelial cells: protective role of the macrocycle [15]pyN5.

Human exposure to cadmium (Cd) occurs via different routes, including diet. The increasing amount of data linking Cd with different cellular effects in the mammary gland justifies additional toxicological assessments using human mammary epithelial cells. This work aimed therefore to assess the cytotoxic effects of Cd in MCF10A cells and to characterize the cytoprotective role of the macrocycle [15]pyN(5) in the form of calcium salt. Cadmium chloride revealed to be cytotoxic to MCF10A cells, decreasing cell viability and proliferation in a concentration-dependent manner. Comparable dose-response curves and IC50 values (57-63 μM, 24h treatment) were obtained using the MTT reduction, crystal violet and BrdU assays. In terms of reactive oxygen species formation, only a slight increase in superoxide radical anion was observed at very high Cd concentrations (≥100 μM). Chelation should thus constitute the primary strategy to mitigate the cytotoxic effects induced by Cd in mammary cells. In this context, [15]pyN(5) which presents appropriate chemical and thermodynamic features was studied as a Cd chelator. This macrocycle (25 and 50 μM) significantly reduced or even abolished Cd-induced cytotoxicity. Protective effects were observed in terms of cell viability, cell proliferation and morphological alterations, being the protection mostly attributed to a chelating-based mechanism.

Unmasking efavirenz neurotoxicity: Time matters to the underlying mechanisms

ABSTRACT Efavirenz is an anti‐HIV drug that presents relevant short‐ and long‐term central nervous system adverse reactions. Its main metabolite (8‐hydroxy‐efavirenz) was demonstrated to be a more potent neurotoxin than efavirenz itself. This work was aimed to understand how efavirenz biotransformation to 8‐hydroxy‐efavirenz is related to its short‐ and long‐term neuro‐adverse reactions. To access those mechanisms, the expression and activity of Cyp2b enzymes as well as the thiolomic signature (low molecular weight thiols plus S‐thiolated proteins) were longitudinally evaluated in the hepatic and brain tissues of rats exposed to efavirenz during 10 and 36 days. Efavirenz and 8‐hydroxy‐efavirenz plasma concentrations were monitored at the same time points. Cyp2b induction had a delayed onset in liver (p < 0.001), translating into increases in Cyp2b activity in liver and 8‐hydroxy‐efavirenz plasma concentration (p < 0.001). Moreover, an increase in S‐cysteinyl‐glycinylated proteins (p < 0.001) and in free low molecular weight thiols was also observed in liver. A distinct scenario was observed in hippocampus, which showed an underexpression of Cyp2b as well as a decrease in S‐cysteinylated and S‐glutathionylated proteins. Additionally, the observed changes in tissues were associated with a marked increase of S‐glutathionylation in plasma. Our data suggest that the time course of efavirenz biotransformation results from different mechanisms for its short‐ and long‐term neurotoxicity. The difference in the redox profile between liver and hippocampus might explain why, despite being mostly metabolized by the liver, this drug is neurotoxic. If translated to clinical practice, this evidence will have important implications in efavirenz short‐ and long‐term neurotoxicity prevention and management. Graphical abstract Summary of the major tissue‐dependent long‐term effects of efavirenz exposure. 8‐OH‐EFV: 8‐hydroxy‐efavirenz; Cyp2b1/2: Cytochrome P450 2b1/2; GSH: reduced glutathione; GSSP: S‐glutathionylated proteins; CysSSP: S‐cysteinylated proteins; PSSCysGly S‐cysteinylglycinylated proteins. Figure. No Caption available.