Exploring the Mechanisms behind the Anti-Tumoral Effects of Model C-Scorpionate Complexes.

The growing worldwide cancer incidence, coupled to the increasing occurrence of multidrug cancer resistance, requires a continuous effort towards the identification of new leads for cancer management. In this work, two C-scorpionate complexes, [FeCl2(κ3-Tpm)] (1) and [Co(κ3-TpmOH)2](NO3)2 (2), (Tpm = hydrotris(pyrazol-1-yl)methane and TpmOH = 2,2,2-tris(pyrazol-1-yl)ethanol), were studied as potential scaffolds for future anticancer drug development. Their cytotoxicity and cell migration inhibitory activity were analyzed, and an untargeted metabolomics approach was employed to elucidate the biological processes significantly affected by these two complexes, using two tumoral cell lines (B16 and HCT116) and a non-tumoral cell line (HaCaT). While [FeCl2(κ3-Tpm)] did not display a significant cytotoxicity, [Co(κ3-TpmOH)2](NO3)2 was particularly cytotoxic against the HCT116 cell line. While [Co(κ3-TpmOH)2](NO3)2 significantly inhibited cell migration in all tested cell lines, [FeCl2(κ3-Tpm)] displayed a mixed activity. From a metabolomics perspective, exposure to [FeCl2(κ3-Tpm)] was associated with changes in various metabolic pathways involving tyrosine, where iron-dependent enzymes are particularly relevant. On the other hand, [Co(κ3-TpmOH)2](NO3)2 was associated with dysregulation of cell adhesion and membrane structural pathways, suggesting that its antiproliferative and anti-migration properties could be due to changes in the overall cellular adhesion mechanisms.

The 2-hydroxy-nevirapine metabolite as a candidate for boosting apolipoprotein A1 and for modulating anti-HDL antibodies.

The antiretroviral nevirapine (NVP) is associated to a reduction of atherosclerotic lesions and increases in high-density lipoprotein (HDL)-cholesterol. Despite being a hepatotoxic drug, which forbids its re-purposing to other therapeutic areas, not all NVP metabolites have the same potential to induce toxicity. Our aim was to investigate the effects of NVP and its metabolites in an exploratory study, towards the identification of a candidate to boost HDL. A pilot prospective (n = 11) and a cross-sectional (n = 332) clinical study were performed with the following endpoints: HDL-cholesterol and apolipoprotein A1 (ApoA1) levels, anti-HDL and anti-ApoA1 antibodies titers, paraoxonase, arylesterase and lactonase activities of paraoxonase-1, and NVP's metabolite profile. NVP treatment increased HDL-cholesterol, ApoA1 and paraoxonase-1 activities, and lowered anti-HDL and anti-ApoA1 titers. In the prospective study, the temporal modulation induced by NVP was different for each HDL-related endpoint. The first observation was a decrease in the anti-HDL antibodies titers. In the cross-sectional study, the lower titers of anti-HDL antibodies were associated to the proportion of 2-hydroxy-NVP (p = 0.03). In vitro models of hepatocytes were employed to clarify the individual contribution of NVP's metabolites for ApoA1 modulation. Long-term incubations of NVP and 2-hydroxy-NVP in the metabolically competent 3D model caused an increase in ApoA1 reaching 43 % (p < 0.05) and 86 % (p < 0.001), respectively. These results support the contribution of drug biotransformation for NVP-induced HDL modulation, highlighting the role of 2-hydroxy-NVP as ApoA1 booster and its association to lower anti-HDL titers. This biotransformation-guided approach allowed us to identify a non-toxic NVP metabolite as a candidate for targeting HDL.

The Secretome of Human Neonatal Mesenchymal Stem Cells Modulates Doxorubicin-Induced Cytotoxicity: Impact in Non-Tumor Cells.

Doxorubicin (Dox) is one of the most widely used treatments for breast cancer, although limited by the well-documented cardiotoxicity and other off-target effects. Mesenchymal stem cell (MSC) secretome has shown immunomodulatory and regenerative properties, further potentiated under 3D conditions. This work aimed to uncover the effect of the MSC-derived secretome from 3D (CM3D) or 2D (CM2D) cultures, in human malignant breast cells (MDA-MB-231), non-tumor breast epithelial cells (MCF10A) and differentiated AC16 cardiomyocytes, co-treated with Dox. A comprehensive proteomic analysis of CM3D/CM2D was also performed to unravel the underlying mechanism. CM3D/CM2D co-incubation with Dox revealed no significant differences in MDA-MB-231 viability when compared to Dox alone, whereas MCF10A and AC16 viability was consistently improved in Dox+CM3D-treated cells. Moreover, neither CM2D nor CM3D affected Dox anti-migratory and anti-invasive effects in MDA-MB-231. Notably, Ge-LC-MS/MS proteomic analysis revealed that CM3D displayed protective features that might be linked to the regulation of cell proliferation (CAPN1, CST1, LAMC2, RANBP3), migration (CCN3, MMP8, PDCD5), invasion (TIMP1/2), oxidative stress (COX6B1, AIFM1, CD9, GSR) and inflammation (CCN3, ANXA5, CDH13, GDF15). Overall, CM3D decreased Dox-induced cytotoxicity in non-tumor cells, without compromising Dox chemotherapeutic profile in malignant cells, suggesting its potential use as a chemotherapy adjuvant to reduce off-target side effects.

Nevirapine Biotransformation Insights: An Integrated In Vitro Approach Unveils the Biocompetence and Glutathiolomic Profile of a Human Hepatocyte-Like Cell 3D Model.

The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7. Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.

Singularities of nevirapine metabolism: from sex-dependent differences to idiosyncratic toxicity.

Nevirapine (NVP) is a first-generation non-nucleoside reverse transcriptase inhibitor widely used for the treatment and prophylaxis of human immunodeficiency virus infection. The drug is taken throughout the patient's life and, due to the availability of an extended-release formulation, it is administered once daily. This antiretroviral is one of the scarce examples of drugs with prescription criteria based on sex, in order to prevent adverse reactions. The therapy with NVP has been associated with potentially life-threatening liver and idiosyncratic skin toxicity. Multiple evidence has emerged regarding the formation of electrophilic NVP metabolites as crucial for adverse idiosyncratic reactions. The formation of reactive metabolites that yield covalent adducts with proteins has been demonstrated in patients under NVP-based treatment. Interestingly, several pharmacogenetic- and sex-related factors associated with NVP toxicity can be mechanistically explained by an imbalance toward increased formation of NVP-derived reactive metabolites and/or impaired detoxification capability. Moreover, the haptenation of self-proteins by these reactive species provides a plausible link between NVP bioactivation and immunotoxicity, further supporting the relevance of this toxicokinetics hypothesis. In the current paper, we review the existing knowledge and recent developments on NVP metabolism and their relation to NVP toxicity.

Revisiting the metabolic syndrome: the emerging role of aquaglyceroporins.

The metabolic syndrome (MetS) includes a group of medical conditions such as insulin resistance (IR), dyslipidemia and hypertension, all associated with an increased risk for cardiovascular disease. Increased visceral and ectopic fat deposition are also key features in the development of IR and MetS, with pathophysiological sequels on adipose tissue, liver and muscle. The recent recognition of aquaporins (AQPs) involvement in adipose tissue homeostasis has opened new perspectives for research in this field. The members of the aquaglyceroporin subfamily are specific glycerol channels implicated in energy metabolism by facilitating glycerol outflow from adipose tissue and its systemic distribution and uptake by liver and muscle, unveiling these membrane channels as key players in lipid balance and energy homeostasis. Being involved in a variety of pathophysiological mechanisms including IR and obesity, AQPs are considered promising drug targets that may prompt novel therapeutic approaches for metabolic disorders such as MetS. This review addresses the interplay between adipose tissue, liver and muscle, which is the basis of the metabolic syndrome, and highlights the involvement of aquaglyceroporins in obesity and related pathologies and how their regulation in different organs contributes to the features of the metabolic syndrome.

Pyridine-Containing Macrocycles Display MMP-2/9 Inhibitory Activity and Distinct Effects on Migration and Invasion of 2D and 3D Breast Cancer Models.

The role of metalloproteinases (MMPs) on the migration and invasion of cancer cells has been correlated with tumor aggressiveness, namely with the up-regulation of MMP-2 and 9. Herein, two pyridine-containing macrocyclic compounds, [15]pyN5 and [16]pyN5, were synthesized, chemically characterized and evaluated as potential MMP inhibitors for breast cancer therapy using 3D and 2D cellular models. [15]pyN5 and [16]pyN5 (5-20 µM) showed a marked inhibition of MMPs activity (100% at concentrations ≥ 7.5 µM) when compared to ARP-100, a known MMP inhibitor. The inhibitory activity of [15]pyN5 and [16]pyN5 was further supported through in silico docking studies using Goldscore and ChemPLP scoring functions. Moreover, although no significant differences were observed in the invasion studies in the presence of all MMPs inhibitors, cell migration was significantly inhibited by both pyridine-containing macrocycles at concentrations above 5 µM in 2D cells (p < 0.05). In spheroids, the same effect was observed, but only with [16]pyN5 at 20 µM and ARP-100 at 40 µM. Overall, [15]pyN5 and [16]pyN5 led to impaired breast cancer cell migration and revealed to be potential inhibitors of MMPs 2 and 9.

Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing.

BACKGROUND AIMS: The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX®. METHODS: Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia. RESULTS: Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo. DISCUSSION: This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products.

Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro.

3D cultures of human stem cell-derived hepatocyte-like cells (HLCs) have emerged as promising models for short- and long-term maintenance of hepatocyte phenotype in vitro cultures by better resembling the in vivo environment of the liver and consequently increase the translational value of the resulting data. In this study, the first stage of hepatic differentiation of human neonatal mesenchymal stem cells (hnMSCs) was performed in 2D monolayer cultures for 17 days. The second stage was performed by either maintaining cells in 2D cultures for an extra 10 days, as control, or alternatively cultured in 3D as self-assembled spheroids or in multicompartment membrane bioreactor system. All systems enabled hnMSC differentiation into HLCs as shown by positive immune staining of hepatic markers CK-18, HNF-4α, albumin, the hepatic transporters OATP-C and MRP-2 as well as drug-metabolizing enzymes like CYP1A2 and CYP3A4. Similarly, all models also displayed relevant glucose, phase I and phase II metabolism, the ability to produce albumin and to convert ammonia into urea. However, EROD activity and urea production were increased in both 3D systems. Moreover, the spheroids revealed higher bupropion conversion, whereas bioreactor showed increased albumin production and capacity to biotransform diclofenac. Additionally, diclofenac resulted in an IC50 value of 1.51 ± 0.05 and 0.98 ± 0.03 in 2D and spheroid cultures, respectively. These data suggest that the 3D models tested improved HLC maturation showing a relevant biotransformation capacity and thus provide more appropriate reliable models for mechanistic studies and more predictive systems for in vitro toxicology applications.

Hepatocyte spheroids as a competent in vitro system for drug biotransformation studies: nevirapine as a bioactivation case study.

The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12 days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with N-acetyl-L-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.

Mesenchymal stem cell secretome for regenerative medicine: Where do we stand?

BACKGROUND: Mesenchymal stem cell (MSC)-based therapies have yielded beneficial effects in a broad range of preclinical models and clinical trials for human diseases. In the context of MSC transplantation, it is widely recognized that the main mechanism for the regenerative potential of MSCs is not their differentiation, with in vivo data revealing transient and low engraftment rates. Instead, MSCs therapeutic effects are mainly attributed to its secretome, i.e., paracrine factors secreted by these cells, further offering a more attractive and innovative approach due to the effectiveness and safety of a cell-free product. AIM OF REVIEW: In this review, we will discuss the potential benefits of MSC-derived secretome in regenerative medicine with particular focus on respiratory, hepatic, and neurological diseases. Both free and vesicular factors of MSC secretome will be detailed. We will also address novel potential strategies capable of improving their healing potential, namely by delivering important regenerative molecules according to specific diseases and tissue needs, as well as non-clinical and clinical studies that allow us to dissect their mechanisms of action. KEY SCIENTIFIC CONCEPTS OF REVIEW: MSC-derived secretome includes both soluble and non-soluble factors, organized in extracellular vesicles (EVs). Importantly, besides depending on the cell origin, the characteristics and therapeutic potential of MSC secretome is deeply influenced by external stimuli, highlighting the possibility of optimizing their characteristics through preconditioning approaches. Nevertheless, the clarity around their mechanisms of action remains ambiguous, whereas the need for standardized procedures for the successful translation of those products to the clinics urges.

Mechanistic insights into the cytotoxicity and genotoxicity induced by glycidamide in human mammary cells.

Acrylamide (AA) is a well-known industrial chemical classified as a probable human carcinogen. Benign and malignant tumours at different sites, including the mammary gland, have been reported in rodents exposed to AA. This xenobiotic is also formed in many carbohydrate-rich foods prepared at high temperatures. For this reason, AA is an issue of concern in terms of human cancer risk. The epoxide glycidamide (GA) is thought to be the ultimate genotoxic AA metabolite. Despite extensive experimental and epidemiological data focused on AA-induced breast cancer, there is still lack of information on the deleterious effects induced by GA in mammary cells. The work reported here addresses the characterisation and modulation of cytotoxicity, generation of reactive oxygen species, formation of micronuclei (MN) and quantification of specific GA-DNA adducts in human MCF10A epithelial cells exposed to GA. The results show that GA significantly induces MN, impairs cell proliferation kinetics and decreases cell viability at high concentrations by mechanisms not involving oxidative stress. KU55933, an inhibitor of ataxia telangiectasia mutated kinase, enhanced the cytotoxicity of GA (P < 0.05), supporting a role of this enzyme in regulating the repair of GA-induced DNA lesions. Moreover, even at low GA levels, N7-GA-Gua adducts were generated in a linear dose-response manner in MCF10A cells. These results confirm that human mammary cells are susceptible to GA toxicity and reinforce the need for additional studies to clarify the potential correlation between dietary AA exposure and breast cancer risk in human populations.

Differential effects of methoxyamine on doxorubicin cytotoxicity and genotoxicity in MDA-MB-231 human breast cancer cells.

Pharmacological inhibition of DNA repair is a promising approach to increase the effectiveness of anticancer drugs. The chemotherapeutic drug doxorubicin (Dox) may act, in part, by causing oxidative DNA damage. The base excision repair (BER) pathway effects the repair of many DNA lesions induced by reactive oxygen species (ROS). Methoxyamine (MX) is an indirect inhibitor of apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional BER protein. We have evaluated the effects of MX on the cytotoxicity and genotoxicity of Dox in MDA-MB-231 metastatic breast cancer cells. MX has little effects on the viability and proliferation of Dox-treated cells. However, as assessed by the cytokinesis-block micronucleus assay (CBMN), MX caused a significant 1.4-fold increase (P<0.05) in the frequency of micronucleated binucleated cells induced by Dox, and also altered the distribution of the numbers of micronuclei. The fluorescence probe dihydroethidium (DHE) indicated little production of ROS by Dox. Overall, our results suggest differential outcomes for the inhibition of APE1 activity in breast cancer cells exposed to Dox, with a sensitizing effect observed for genotoxicity but not for cytotoxicity.

Synthesis and biological activity of 6-selenocaffeine: potential modulator of chemotherapeutic drugs in breast cancer cells.

We report the development of a new microwave-based synthetic methodology mediated by Woollins' reagent that allowed an efficient conversion of caffeine into 6-selenocaffeine. A preliminary evaluation on the modulation of antioxidant activity upon selenation of caffeine, using the DPPH assay, indicated a mild antioxidant activity for 6-selenocaffeine, contrasting with caffeine, that exhibited no antioxidant activity under the same experimental conditions. Interestingly, whereas 6-selenocaffeine has revealed to have a low cytotoxic potential in both MCF10A and MCF-7 breast cells (24 h, up to 100 µM, MTT assay), a differential effect was observed when used in combination with the anticancer agents doxorubicin and oxaliplatin in MCF-7 breast cancer cells. The co-treatment of doxorubicin (1 µM) and 6-selenocaffeine (100 µM) resulted in a slight decrease in cellular viability when compared to doxorubicin (1 µM) alone. Conversely, the seleno-caffeine derivative at the same concentration markedly increased the viability of oxaliplatin (100 µM)-treated cells (p < 0.01). Overall, this work highlights an emerging methodology to synthesize organoselenium compounds and points out the differential roles of 6-selenocaffeine in the modulation of the cytotoxicity of anticancer agents.

The Redox-Active Manganese(III) Porphyrin, MnTnBuOE-2-PyP5+, Impairs the Migration and Invasion of Non-Small Cell Lung Cancer Cells, Either Alone or Combined with Cisplatin.

Manganese(III) porphyrin MnTnBuOE-2-PyP5+ (MnBuOE, BMX-001) is a third-generation redox-active cationic substituted pyridylporphyrin-based drug with a good safety/toxicity profile that has been studied in several types of cancer. It is currently in four phase I/II clinical trials on patients suffering from glioma, head and neck cancer, anal squamous cell carcinoma and multiple brain metastases. There is yet an insufficient understanding of the impact of MnBuOE on lung cancer. Therefore, this study aims to fill this gap by demonstrating the effects of MnBuOE on non-small cell lung cancer (NSCLC) A549 and H1975 cell lines. The cytotoxicity of MnBuOE alone or combined with cisplatin was evaluated by crystal violet (CV) and/or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-Tetrazolium (MTS) reduction assays. Intracellular ROS levels were assessed using two fluorescent probes. Furthermore, the impact of MnBuOE alone or in combination with cisplatin on collective cell migration, individual chemotactic migration and chemoinvasion was assessed using the wound-healing and transwell assays. The expression of genes related to migration and invasion was assessed through RT-qPCR. While MnBuOE alone decreased H1975 cell viability at high concentrations, when combined with cisplatin it markedly reduced the viability of the more invasive H1975 cell line but not of A549 cell line. However, MnBuOE alone significantly decreased the migration of both cell lines. The anti-migratory effect was more pronounced when MnBuOE was combined with cisplatin. Finally, MnBuOE alone or combined with cisplatin significantly reduced cell invasion. MnBuOE alone or combined with cisplatin downregulated MMP2, MMP9, VIM, EGFR and VEGFA and upregulated CDH1 in both cell lines. Overall, our data demonstrate the anti-metastatic potential of MnBuOE for the treatment of NSCLC.

Quality, Safety and Biological Studies on Campylanthus glaber Aerial Parts.

In Cabo Verde, several endemic species are used in traditional medicine. However, no scientific studies have been conducted on the quality, efficacy, and safety of most of these plants. This study focused on establishing the botanical and chemical identification parameters required for a quality monograph of Campylanthus glaber Benth. aerial parts, a medicinal plant of Cabo Verde traditionally used to treat fever and muscular pain. In addition, in vitro antioxidant and antihyperglycemic activity, cytotoxicity, and genotoxicity were assessed for this medicinal plant. Optical microscopy, LC/UV-DAD-ESI/MS, and colorimetric assays were used for botanical, chemical, and biological studies, respectively. Cytotoxicity was assessed by the MTT assay with HepG2 cells, and genotoxicity by the Ames test. Microscopically, the xeromorphic leaf of C. glaber presents a thick cuticle (13.6-25.5 µm), thick-walled epidermal cells, anomocytic-type stomata, glandular trichomes (stalk length = 49.4-120.8 µm), and idioblasts containing calcium oxalate microcrystals. The chemical screening of aqueous and hydroethanolic extracts of this medicinal plant revealed the presence of organic acids, iridoids, phenylethanoids, and flavonoids as the main classes of marker compounds, with malic acid, citric acid, and verbascoside being the main marker compounds identified. Both extracts showed similar LC/UV-DAD/ESI-MS qualitative profiles and DPPH radical scavenger activity (IC50 = 130.9 ± 1.4; 134.3 ± 3.1 µg/mL). The hydroethanolic extract inhibited both α-amylase and α-glucosidase enzymes in a dose-dependent manner. Both extracts showed no cytotoxicity (up to 1000 µg/mL) by the MTT assay and no genotoxic potential with or without metabolic activation up to 5 mg /plate. The results obtained are an important contribution to the monographic quality assessment of C. glaber aerial parts and suggest that this medicinal plant may be safe and potentially used as an herbal drug raw material for pharmaceutical purposes.

3D-MSCs A151 ODN-loaded exosomes are immunomodulatory and reveal a proteomic cargo that sustains wound resolution.

INTRODUCTION: Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. OBJECTIVES: To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. METHODS: MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. RESULTS: Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro, with upregulation of IL-1α and VEGF-α or increased secretion of TGF-ß, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-ß, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. CONCLUSION: Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines.

MnTnHex-2-PyP5+ Displays Anticancer Properties and Enhances Cisplatin Effects in Non-Small Cell Lung Cancer Cells.

The manganese(III) porphyrin MnTnHex-2-PyP5+ (MnTnHex) is a potent superoxide dismutase mimic and modulator of redox-based transcriptional activity that has been studied in the context of different human disease models, including cancer. Nevertheless, for lung cancer, hardly any information is available. Thus, the present work aims to fill this gap and reports the effects of MnTnHex in non-small cell lung cancer (NSCLC) cells, more specifically, A549 and H1975 cells, in vitro. Both cell lines were initially characterized in terms of innate levels of catalase, glutathione peroxidase 1, and peroxiredoxins 1 and 2. To assess the effect of MnTnHex in NSCLC, alone or in combination with cisplatin, endpoints related to the cell viability, cell cycle distribution, cell motility, and characterization of the volatile carbonyl compounds (VCCs) generated in the extracellular medium (i.e., exometabolome) were addressed. The results show that MnTnHex as a single drug markedly reduced the viability of both NSCLC cell lines, with some IC50 values reaching sub-micromolar levels. This redox-active drug also altered the cell cycle distribution, induced cell death, and increased the cytotoxicity pattern of cisplatin. MnTnHex also reduced collective cell migration. Finally, the metabolomics study revealed an increase in the levels of a few VCCs associated with oxidative stress in MnTnHex-treated cells. Altogether these results suggest the therapeutic potential of MnTnHex to be further explored, either alone or in combination therapy with cisplatin, in NSCLC.

Perfusion of 3D encapsulated hepatocytes--a synergistic effect enhancing long-term functionality in bioreactors.

Long-term primary cultures of hepatocytes are essential for bioartificial liver (BAL) devices and to reduce and replace animal tests in lead candidate optimization in drug discovery and toxicology tests. The aim of this work was to improve bioreactor cultures of hepatocyte spheroids by adding a more physiological perfusion feeding regime to these bioreactor systems. A continuous perfusion feeding was compared with 50% medium replacement (routinely used for in vitro tests) at the same dilution rate, 0.125 day(-1), for three operative weeks. Perfusion feeding led to a 10-fold improvement in albumin synthesis in bioreactors containing non-encapsulated hepatocyte spheroids; no significant improvement was observed in phase I drug metabolizing activity. When ultra high viscous alginate encapsulated spheroids were cultured in perfusion, urea synthesis, phase I drug metabolizing activity and oxygen consumption had a threefold improvement over the 50% medium replacement regime; albumin production was the same for both feeding regimes. The effective diffusion of albumin in the alginate capsules was 7.75.10(-9) cm(2) s(-1) and no diffusion limitation for this protein was observed using these alginate capsules under our operational conditions. In conclusion, perfusion feeding coupled with alginate encapsulation of hepatocyte spheroids showed a synergistic effect with a threefold improvement in three independent liver-specific functions of long-term hepatocyte spheroid cultures.

A Critical Perspective on 3D Liver Models for Drug Metabolism and Toxicology Studies.

The poor predictability of human liver toxicity is still causing high attrition rates of drug candidates in the pharmaceutical industry at the non-clinical, clinical, and post-marketing authorization stages. This is in part caused by animal models that fail to predict various human adverse drug reactions (ADRs), resulting in undetected hepatotoxicity at the non-clinical phase of drug development. In an effort to increase the prediction of human hepatotoxicity, different approaches to enhance the physiological relevance of hepatic in vitro systems are being pursued. Three-dimensional (3D) or microfluidic technologies allow to better recapitulate hepatocyte organization and cell-matrix contacts, to include additional cell types, to incorporate fluid flow and to create gradients of oxygen and nutrients, which have led to improved differentiated cell phenotype and functionality. This comprehensive review addresses the drug-induced hepatotoxicity mechanisms and the currently available 3D liver in vitro models, their characteristics, as well as their advantages and limitations for human hepatotoxicity assessment. In addition, since toxic responses are greatly dependent on the culture model, a comparative analysis of the toxicity studies performed using two-dimensional (2D) and 3D in vitro strategies with recognized hepatotoxic compounds, such as paracetamol, diclofenac, and troglitazone is performed, further highlighting the need for harmonization of the respective characterization methods. Finally, taking a step forward, we propose a roadmap for the assessment of drugs hepatotoxicity based on fully characterized fit-for-purpose in vitro models, taking advantage of the best of each model, which will ultimately contribute to more informed decision-making in the drug development and risk assessment fields.