The PTP1B Inhibitor Trodusquemine (MSI-1436) Improves Glucose Uptake in Equine Metabolic Syndrome Affected Liver through Anti-Inflammatory and Antifibrotic Activity.

Background: Hyperactivation of protein tyrosine phosphatase (PTP1B) has been associated with several metabolic malfunctions ranging from insulin resistance, metaflammation, lipotoxicity, and hyperglycaemia. Liver metabolism failure has been proposed as a core element in underlying endocrine disorders through persistent inflammation and highly fibrotic phenotype. Methods: In this study, the outcomes of PTP1B inhibition using trodusquemine (MSI-1436) on key equine metabolic syndrome (EMS)-related alterations including inflammation, fibrosis, and glucose uptake have been analyzed in liver explants collected from EMS-affected horses using various analytical techniques, namely, flow cytometry, RT-qPCR, and Western blot. Results: PTP1B inhibition using trodusquemine resulted in decreased proinflammatory cytokines (IL-1ß, TNF-α, and IL-6) release from liver and PBMC affected by EMS and regulated expression of major proinflammatory microRNAs such as miR-802 and miR-211. Moreover, MSI-1436 enhanced the anti-inflammatory profile of livers by elevating the expression of IL-10 and IL-4 and activating CD4+CD25+Foxp3+ regulatory T cells in treated PBMC. Similarly, the inhibitor attenuated fibrogenic pathways in the liver by downregulating TGF-ß/NOX1/4 axis and associated MMP-2/9 overactivation. Interestingly, PTP1B inhibition ameliorated the expression of TIMP-1 and Smad7, both important antifibrotic mediators. Furthermore, application of MSI-1436 was found to augment the abundance of glycosylated Glut-2, which subsequently expanded the glucose absorption in the EMS liver, probably due to an enhanced Glut-2 stability and half-life onto the plasma cell membranes. Conclusion: Taken together, the presented data suggest that the PTP1B inhibition strategy and the use of its specific inhibitor MSI-1436 represents a promising option for the improvement of liver tissue integrity and homeostasis in the course of EMS and adds more insights for ongoing clinical trials for human MetS management.

Sex Hormone-Binding Globulin (SHBG) Maintains Proper Equine Adipose-Derived Stromal Cells (ASCs)' Metabolic Functions and Negatively Regulates their Basal Adipogenic Potential.

BACKGROUND: Sex hormone binding globulin (SHBG) deteriorated expression has been recently strongly correlated to increased level of circulating pro-inflammatory cytokines and insulin resistance, which are typical manifestations of equine metabolic syndrome (EMS). Despite previous reports demonstrated the potential therapeutic application of SHBG for liver-related dysfunctions, whether SHBG might modulate equine adipose-derived stem/stromal cells (EqASCs) metabolic machinery remains unknown. Therefore, we evaluated for the first time the impact of SHBG protein on metabolic changes in ASCs isolated from healthy horses. METHODS: Beforehand, SHBG protein expression has been experimentally lowered using a predesigned siRNA in EqASCs to verify its metabolic implications and potential therapeutic value. Then, apoptosis profile, oxidative stress, mitochondrial network dynamics and basal adipogenic potential have been evaluated using various molecular and analytical techniques. RESULTS: The SHBG knockdown altered the proliferative and metabolic activity of EqASCs, while dampening basal apoptosis via Bax transcript suppression. Furthermore, the cells treated with siRNA were characterized by senescent phenotype, accumulation of reactive oxygen species (ROS), nitric oxide, as well as decreased mitochondrial potential that was shown by mitochondrial membrane depolarization and lower expression of key mitophagy factors: PINK, PARKIN and MFN. The addition of SHBG protein reversed the impaired and senescent phenotype of EMS-like cells that was proven by enhanced proliferative activity, reduced apoptosis resistance, lower ROS accumulation and greater mitochondrial dynamics, which is proposed to be related to a normalization of Bax expression. Crucially, SHBG silencing enhanced the expression of key pro-adipogenic effectors, while decreased the abundance of anti-adipogenic factors namely HIF1-α and FABP4. The addition of exogenous SHBG further depleted the expression of PPARγ and C/EBPα and restored the levels of FABP4 and HIF1-α evoking a strong inhibitory potential toward ASCs adipogenesis. CONCLUSION: Herein, we provide for the first time the evidence that SHBG protein in importantly involved in various key metabolic pathways governing EqASCs functions, and more importantly we showed that SHBG negatively affect the basal adipogenic potential of tested ASCs through a FABP4-dependant pathway, and provide thus new insights for the development of potential anti-obesity therapeutic approach in both animals and humans.

The PTP1B inhibitor MSI-1436 ameliorates liver insulin sensitivity by modulating autophagy, ER stress and systemic inflammation in Equine metabolic syndrome affected horses.

Background: Equine metabolic syndrome (EMS) is a multifactorial pathology gathering insulin resistance, low-grade inflammation and past or chronic laminitis. Among the several molecular mechanisms underlying EMS pathogenesis, increased negative insulin signalling regulation mediated by protein tyrosine phosphatase 1 B (PTP1B) has emerged as a critical axis in the development of liver insulin resistance and general metabolic distress associated to increased ER stress, inflammation and disrupted autophagy. Thus, the use of PTP1B selective inhibitors such as MSI-1436 might be considered as a golden therapeutic tool for the proper management of EMS and associated conditions. Therefore, the present investigation aimed at verifying the clinical efficacy of MSI-1436 systemic administration on liver metabolic balance, insulin sensitivity and inflammatory status in EMS affected horses. Moreover, the impact of MSI-1436 treatment on liver autophagy machinery and associated ER stress in liver tissue has been analysed. Methods: Liver explants isolated from healthy and EMS horses have been treated with MSI-1436 prior to gene and protein expression analysis of main markers mediating ER stress, mitophagy and autophagy. Furthermore, EMS horses have been intravenously treated with a single dose of MSI-1436, and evaluated for their metabolic and inflammatory status. Results: Clinical application of MSI-1436 to EMS horses restored proper adiponectin levels and attenuated the typical hyperinsulinemia and hyperglycemia. Moreover, administration of MSI-1436 further reduced the circulating levels of key pro-inflammatory mediators including IL-1ß, TNF-α and TGF-ß and triggered the Tregs cells activation. At the molecular level, PTP1B inhibition resulted in a noticeable mitigation of liver ER stress, improvement of mitochondrial dynamics and consequently, a regulation of autophagic response. Similarly, short-term ex vivo treatment of EMS liver explants with trodusquemine (MSI-1436) substantially enhanced autophagy by upregulating the levels of HSC70 and Beclin-1 at both mRNA and protein level. Moreover, the PTP1B inhibitor potentiated mitophagy and associated expression of MFN2 and PINK1. Interestingly, inhibition of PTP1B resulted in potent attenuation of ER stress key mediators' expression namely, CHOP, ATF6, HSPA5 and XBP1. Conclusion: Presented findings shed for the first time promising new insights in the development of an MSI-1436-based therapy for proper equine metabolic syndrome intervention and may additionally find potential translational application to human metabolic syndrome treatment.

In Vitro Generated Equine Hepatic-Like Progenitor Cells as a Novel Potent Cell Pool for Equine Metabolic Syndrome (EMS) Treatment.

Equine metabolic syndrome (EMS) is recognized as one of the leading cause of health threatening in veterinary medicine worldwide. Recently, PTP1B inhibition has been proposed as an interesting strategy for liver insulin resistance reversion in both equines and humans, however as being a multifactorial disease, proper management of EMS horses further necessities additional interventional approaches aiming at repairing and restoring liver functions. In this study, we hypothesized that in vitro induction of Eq_ASCs hepatogenic differentiation will generate a specialized liver progenitor-like cell population exhibiting similar phenotypic characteristics and regenerative potential as native hepatic progenitor cells. Our obtained data demonstrated that Eq_ASCs-derived liver progenitor cells (Eq_HPCs) displayed typical flattened polygonal morphology with packed fragmented mitochondrial net, lowered mesenchymal CD105 and CD90 surface markers expression, and significant high expression levels of specific hepatic lineage genes including PECAM-1, ALB, AFP and HNF4A. therewith, generated Eq_HPCs exhibited potentiated stemness and pluripotency markers expression (NANOG, SOX-2 and OCT-4). Hence, in vitro generation of hepatic progenitor-like cells retaining high differentiation capacity represents a promising new approach for the establishment of cell-based targeted therapies for the restoration of proper liver functions in EMS affected horses.

Synergistic Effect of Toceranib and Nanohydroxyapatite as a Drug Delivery Platform-Physicochemical Properties and In Vitro Studies on Mastocytoma Cells.

A new combination of Toceranib (Toc; 5-[(5Z)-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-N-[2-(pyrrolidin-1-yl)ethyl]-1H-pyrrole-3-carboxamide) with nanohydroxyapatite (nHAp) was proposed as an antineoplastic drug delivery system. Its physicochemical properties were determined as crystallinity, grain size, morphology, zeta potential and hydrodynamic diameter as well as Toceranib release. The crystalline nanorods of nHAp were synthesised by the co-precipitation method, while the amorphous Toceranib was obtained by its conversion from the crystalline form during nHAp-Toc preparation. The surface interaction between both compounds was confirmed using Fourier-transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV-Vis) and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS). The nHAp-Toc showed a slower and prolonged release of Toceranib. The release behaviour was affected by hydrodynamic size, surface interaction and the medium used (pH). The effectiveness of the proposed platform was tested by comparing the cytotoxicity of the drug combined with nHAp against the drug itself. The compounds were tested on NI-1 mastocytoma cells using the Alamar blue colorimetric technique. The obtained results suggest that the proposed platform shows high efficiency (the calculated IC50 is 4.29 nM), while maintaining the specificity of the drug alone. Performed analyses confirmed that nanohydroxyapatite is a prospective drug carrier and, when Toceranib-loaded, may be an idea worth developing with further research into therapeutic application in the treatment of canine mast cell tumour.

Nanohydroxyapatite-Mediated Imatinib Delivery for Specific Anticancer Applications.

In the present study, a nanoapatite-mediated delivery system for imatinib has been proposed. Nanohydroxyapatite (nHAp) was obtained by co-precipitation method, and its physicochemical properties in combination with imatinib (IM) were studied by means of XRPD (X-ray Powder Diffraction), SEM-EDS (Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy), FT-IR (Fourier-Transform Infrared Spectroscopy), absorption spectroscopy as well as DLS (Dynamic Light Scattering) techniques. The obtained hydroxyapatite was defined as nanosized rod-shaped particles with high crystallinity. The amorphous imatinib was obtained by conversion of its crystalline form. The beneficial effects of amorphous pharmaceutical agents have been manifested in the higher dissolution rate in body fluids improving their bioavailability. Imatinib-to-hydroxyapatite interactions on the surface were confirmed by SEM images as well as absorption and FT-IR spectroscopy. The cytotoxicity of the system was tested on NI-1, L929, and D17 cell lines. The effectiveness of imatinib was not affected by nHAp modification. The calculated IC50 values for drug-modified nHAp were similar to those for the drug itself. However, higher cytotoxicity was observed at higher concentrations of imatinib, in comparison with the drug alone.

The Haematococcus pluvialis extract enriched by bioaccumulation process with Mg(II) ions improves insulin resistance in equine adipose-derived stromal cells (EqASCs).

Insulin resistance (IR) is one of the characteristic features of equine metabolic syndrome (EMS). Presently, the only therapies of choice are caloric restrictions combined with mineral supplementation, which might improve insulin sensitivity. In this study we investigated the effect of Haematococcus pluvialis algae water extract enriched in bioaccumulation process in magnesium ions (Hp_Mg(II)) on equine adipose derived mesenchymal stromal stem cells, in which insulin resistance was induced by palmitic acid (IR-EqASCs). For this purpose, chemical characterization of H. pluvialis was performed with special emphasis on the analysis of minerals composition, total phenolic and carotenoids contents, as well as scavenging activity. To examine the influence of H. pluvialis extract on IR-EqASCs, various methods of molecular biology and microscopic observations (i.e., immunofluorescence staining, SEM, gene expression by RT-qPCR, proliferative and metabolic cells activity analysis) were applied to investigate in vitro viability, oxidative stress markers and apoptosis-related factor accumulation, along with insulin resistance-related genes expression. Obtained results show, that Hp_Mg(II) significantly improves proliferative and metabolic activity of IR-EqASCs, shortens their population doubling time, improves their clonogenic potential and reduces expression of apoptosis related genes. Moreover, anti-oxidative effect of extract was presented.

Correction: Serwotka-Suszczak, A. M. et al. A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells. Int. J. Mol. Sci. 2017, 18, 401.

It has been brought to our attention that the affiliation of Dr. Jerzy Pieczykolan at the time when he was responsible for the work described in the paper [...].

A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells.

Antibody-drug conjugates (ADCs) have recently emerged as efficient and selective cancer treatment therapeutics. Currently, alternative forms of drug carriers that can replace monoclonal antibodies are under intensive investigation. Here, a cytotoxic conjugate of an anti-HER2 (Human Epidermal Growth Factor Receptor 2) diaffibody with monomethyl-auristatin E (MMAE) is proposed as a potential anticancer therapeutic. The anti-HER2 diaffibody was based on the ZHER2:4 affibody amino acid sequence. The anti-HER2 diaffibody has been expressed as a His-tagged protein in E. coli and purified by Ni-nitrilotriacetyl (Ni-NTA) agarose chromatography. The molecule was properly folded, and the high affinity and specificity of its interaction with HER2 was confirmed by surface plasmon resonance (SPR) and flow cytometry, respectively. The (ZHER2:4)2DCS-MMAE conjugate was obtained by coupling the maleimide group linked with MMAE to cysteines, which were introduced in a drug conjugation sequence (DCS). Cytotoxicity of the conjugate was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide MTT assay and the xCELLigence Real-Time Cell Analyzer. Our experiments demonstrated that the conjugate delivered auristatin E specifically to HER2-positive tumor cells, which finally led to their death. These results indicate that the cytotoxic diaffibody conjugate is a highly potent molecule for the treatment of various types of cancer overexpressing HER2 receptors.

A Novel Affibody-Auristatin E Conjugate With a Potent and Selective Activity Against HER2+ Cell Lines.

Targeted therapy is a new type of cancer treatment that most often uses biologically active drugs attached to a monoclonal antibody. This so called antibody-drug conjugate strategy allows the use of highly toxic substances that target tumor cells specifically, leaving healthy tissues largely unaffected. Over the last few years, antibody-drug conjugates have become a powerful tool in cancer treatment. We developed and characterized a novel cytotoxic conjugate against HER2 tumors in which the antibody has been substituted with a much smaller molecule: the affibody. The conjugate is composed of the ZHER2:2891 affibody that recognizes HER2 and a highly potent cytotoxic drug auristatin E. The ZHER2:2891 molecule does not contain cysteine(s) in its amino acid sequence. We generated 3 variants of ZHER2:2891, each containing a single cysteine to allow conjugation through the maleimide group that is present in the cytotoxic component. In 2 variants, we introduced single S46C and D53C substitutions. In the third variant, a short Drug Conjugation Sequence (DCS) containing a single cysteine was introduced at the C-terminus of ZHER2:2891, resulting in ZHER2:2891-DCS. The latter variant exhibited a significantly higher conjugation yield, and therefore its cytotoxicity has been studied more thoroughly. The ZHER2:2891-DCS-MMAE conjugate killed the HER2-overexpressing SK-BR-3 and MDA-MB-453 cells efficiently (IC50 values of 5.2 and 24.8 nM, respectively). The T-47-D and MDA-MB-231 cells that express normal levels of HER2 were significantly less sensitive to the conjugate (IC50 values of 135.6 and 161.5 nM, respectively). Overall, we have demonstrated for the first time that proteins other than antibodies/antibody fragments can be successfully combined with a linker-drug module, resulting in conjugates that eliminate cancer cells selectively.