Phenethyl isothiocyanate and dasatinib combination synergistically reduces hepatocellular carcinoma growth via cell cycle arrest and oxeiptosis.

Introduction: Hepatocellular carcinoma (HCC) is the most common type of liver cancer, which is among the most lethal tumours. Combination therapy exploits multiple drugs to target key pathways synergistically to reduce tumour growth. Isothiocyanates have been shown to possess anticancer potential and to complement the anticancer activity of other compounds. This study aimed to investigate the potential of phenethyl isothiocyanate (PEITC) to synergise with dasatinib, improving its anticancer potential in HCC. Methods: MTT, 3D spheroids and clonogenic assays were used to assess the combination anti-tumour effect in vitro, whereas a murine syngeneic model was employed to evaluate the combination efficacy in vivo. DCFDA staining was employed to evaluate the production of reactive oxygen species (ROS), while flow cytometry and Western blot assays were used to elucidate the molecular mechanism of the synergistic activiy. Results: PEITC and dasatinib combination exhibited a synergistic effect in vitro and in vivo. The combination induced DNA damage and oxidative stress through the production of ROS, which led to the formation of a premature CDK1/Cyclin B1 complex associated with induction of mitotic catastrophe. Furthermore, ROS activated oxeiptosis, a caspase-independent form of programmed cell death. Conclusion: PEITC showed to enhance dasatinib action in treating HCC with increased production of ROS that induced cell cycle arrest followed by mitotic catastrophe, and to induce oxeiptosis. These results highlight the role that ITCs may have in cancer therapy as a complement of clinically approved chemotherapeutic drugs.

Blocking glycosphingolipid production alters autophagy in osteoclasts and improves myeloma bone disease.

Glycosphingolipids (GSLs) are key constituents of membrane bilayers playing a role in structural integrity, cell signalling in microdomains, endosomes and lysosomes, and cell death pathways. Conversion of ceramide into GSLs is controlled by GCS (glucosylceramide synthase) and inhibitors of this enzyme for the treatment of lipid storage disorders and specific cancers. With a diverse range of functions attributed to GSLs, the ability of the GSC inhibitor, eliglustat, to reduce myeloma bone disease was investigated. In pre-clinical models of multiple myeloma, osteoclast-driven bone loss was reduced by eliglustat in a mechanistically separate manner to zoledronic acid, a bisphosphonate that prevents osteoclast-mediated bone destruction. Autophagic degradation of TNF receptor-associated factor 3 (TRAF3), a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. By altering GSL composition, eliglustat prevented lysosomal degradation whilst exogenous addition of missing GSLs rescued TRAF3 degradation to restore osteoclast formation in bone marrow cells from myeloma patients. This work highlights the clinical potential of eliglustat as a therapy for myeloma bone disease. Furthermore, using eliglustat as a lysosomal inhibitor in osteoclasts may widen its therapeutic uses to other bone disorders such as bone metastasis, osteoporosis and inflammatory bone loss.

A Mouse Model with a Frameshift Mutation in the Nuclear Factor I/X (NFIX) Gene Has Phenotypic Features of Marshall-Smith Syndrome.

The nuclear factor I/X (NFIX) gene encodes a ubiquitously expressed transcription factor whose mutations lead to two allelic disorders characterized by developmental, skeletal, and neural abnormalities, namely, Malan syndrome (MAL) and Marshall-Smith syndrome (MSS). NFIX mutations associated with MAL mainly cluster in exon 2 and are cleared by nonsense-mediated decay (NMD) leading to NFIX haploinsufficiency, whereas NFIX mutations associated with MSS are clustered in exons 6-10 and escape NMD and result in the production of dominant-negative mutant NFIX proteins. Thus, different NFIX mutations have distinct consequences on NFIX expression. To elucidate the in vivo effects of MSS-associated NFIX exon 7 mutations, we used CRISPR-Cas9 to generate mouse models with exon 7 deletions that comprised: a frameshift deletion of two nucleotides (Nfix Del2); in-frame deletion of 24 nucleotides (Nfix Del24); and deletion of 140 nucleotides (Nfix Del140). Nfix +/Del2, Nfix +/Del24, Nfix +/Del140, Nfix Del24/Del24, and Nfix Del140/Del140 mice were viable, normal, and fertile, with no skeletal abnormalities, but Nfix Del2/Del2 mice had significantly reduced viability (p < 0.002) and died at 2-3 weeks of age. Nfix Del2 was not cleared by NMD, and NfixDel2/Del2 mice, when compared to Nfix +/+ and Nfix +/Del2 mice, had: growth retardation; short stature with kyphosis; reduced skull length; marked porosity of the vertebrae with decreased vertebral and femoral bone mineral content; and reduced caudal vertebrae height and femur length. Plasma biochemistry analysis revealed Nfix Del2/Del2 mice to have increased total alkaline phosphatase activity but decreased C-terminal telopeptide and procollagen-type-1-N-terminal propeptide concentrations compared to Nfix +/+ and Nfix +/Del2 mice. Nfix Del2/Del2 mice were also found to have enlarged cerebral cortices and ventricular areas but smaller dentate gyrus compared to Nfix +/+ mice. Thus, Nfix Del2/Del2 mice provide a model for studying the in vivo effects of NFIX mutants that escape NMD and result in developmental abnormalities of the skeletal and neural tissues that are associated with MSS. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Prostaglandin E2/EP4 axis is upregulated in Spondyloarthritis and contributes to radiographic progression.

Ankylosing spondylitis (AS) is an inflammatory disease leading to spine ankylosis; however, the mechanisms behind new bone formation are still not fully understood. Single Nucleotide Polymorphisms (SNPs) in PTGER4, encoding for the receptor EP4 of prostaglandin E2 (PGE2), are associated with AS. Since the PGE2-EP4 axis participates in inflammation and bone metabolism, this work aims at investigating the influence of the prostaglandin-E2 axis on radiographic progression in AS. In 185 AS (97 progressors), baseline serum PGE2 predicted progression, and PTGER4 SNP rs6896969 was more frequent in progressors. Increased EP4/PTGER4 expression was observed in AS circulating immune cells, synovial tissue, and bone marrow. CD14highEP4 + cells frequency correlated with disease activity, and when monocytes were cocultured with mesenchymal stem cells, the PGE2/EP4 axis induced bone formation. In conclusion, the Prostaglandin E2 axis is involved in bone remodelling and may contribute to the radiographic progression in AS due to genetic and environmental upregulation.

Modulating glycosphingolipid metabolism and autophagy improves outcomes in pre-clinical models of myeloma bone disease.

Patients with multiple myeloma, an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions that severely impact quality of life and clinical outcomes. Eliglustat, a U.S. Food and Drug Administration-approved glucosylceramide synthase inhibitor, reduced osteoclast-driven bone loss in preclinical in vivo models of myeloma. In combination with zoledronic acid, a bisphosphonate that treats myeloma bone disease, eliglustat provided further protection from bone loss. Autophagic degradation of TRAF3, a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. Eliglustat blocked autophagy by altering glycosphingolipid composition whilst restoration of missing glycosphingolipids rescued autophagy markers and TRAF3 degradation thus restoring osteoclastogenesis in bone marrow cells from myeloma patients. This work delineates both the mechanism by which glucosylceramide synthase inhibition prevents autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the clinical translational potential of eliglustat for the treatment of myeloma bone disease.

Rodent Models of Spondyloarthritis Have Decreased White and Bone Marrow Adipose Tissue Depots.

Bone marrow adipose tissue (BMAT) has recently been recognized as a distinct fat depot with endocrine functions. However, if and how it is regulated by chronic inflammation remains unknown. Here, we investigate the amount of white fat and BMAT in HLA-B27 transgenic rats and curdlan-challenged SKG mice, two well-established models of chronic inflammatory spondyloarthritis (SpA). Subcutaneous and gonadal white adipose tissue and BMAT was reduced by 65-70% and by up to 90% in both experimental models. Consistently, B27 rats had a 2-3-fold decrease in the serum concentrations of the adipocyte-derived cytokines adiponectin and leptin as well as a 2-fold lower concentration of triglycerides. The bone marrow of B27 rats was further characterized by higher numbers of neutrophils, lower numbers of erythroblast precursors, and higher numbers of IL-17 producing CD4+ T cells. IL-17 concentration was also increased in the serum of B27 rats. Using a cell culture model, we show that high levels of IL-17 in the serum of B27 rats negatively impacted adipogenesis (-76%), an effect that was reversed in the presence of neutralizing anti-IL-17 antibody. In summary, these findings show BMAT is severely reduced in two experimental models of chronic inflammatory SpA and suggest that IL-17 is involved in this process.

Loss and gain of bone in spondyloarthritis: what drives these opposing clinical features?

The breadth of bone lesion types seen in spondyloarthritis is unprecedented in medicine and includes increased bone turnover, bone loss and fragility, osteitis, osteolysis and erosion, osteosclerosis, osteoproliferation of soft tissues adjacent to bone and spinal skeletal structure weakness. Remarkably, these effects can be present simultaneously in the same patient. The search for a potential unifying cause of effects on the skeleton necessarily focuses on inflammation arising from the dysregulation of immune response to microorganisms, particularly dysregulation of TH17 lymphocytes, and the dysbiosis of established gut and other microbiota. The compelling notion that a common antecedent pathological mechanism affects existing bone and tissues with bone-forming potential (entheses), simultaneously with variable effect in the former but bone-forming in the latter, drives basic research forward and focuses our awareness on the effects on these bone mechanisms of the increasing portfolio of targeted immunotherapies used in the clinic.

GM-CSF drives dysregulated hematopoietic stem cell activity and pathogenic extramedullary myelopoiesis in experimental spondyloarthritis.

Dysregulated hematopoiesis occurs in several chronic inflammatory diseases, but it remains unclear how hematopoietic stem cells (HSCs) in the bone marrow (BM) sense peripheral inflammation and contribute to tissue damage in arthritis. Here, we show the HSC gene expression program is biased toward myelopoiesis and differentiation skewed toward granulocyte-monocyte progenitors (GMP) during joint and intestinal inflammation in experimental spondyloarthritis (SpA). GM-CSF-receptor is increased on HSCs and multipotent progenitors, favoring a striking increase in myelopoiesis at the earliest hematopoietic stages. GMP accumulate in the BM in SpA and, unexpectedly, at extramedullary sites: in the inflamed joints and spleen. Furthermore, we show that GM-CSF promotes extramedullary myelopoiesis, tissue-toxic neutrophil accumulation in target organs, and GM-CSF prophylactic or therapeutic blockade substantially decreases SpA severity. Surprisingly, besides CD4+ T cells and innate lymphoid cells, mast cells are a source of GM-CSF in this model, and its pathogenic production is promoted by the alarmin IL-33.

Fully reduced HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to GAlert.

A major discovery of recent decades has been the existence of stem cells and their potential to repair many, if not most, tissues. With the aging population, many attempts have been made to use exogenous stem cells to promote tissue repair, so far with limited success. An alternative approach, which may be more effective and far less costly, is to promote tissue regeneration by targeting endogenous stem cells. However, ways of enhancing endogenous stem cell function remain poorly defined. Injury leads to the release of danger signals which are known to modulate the immune response, but their role in stem cell-mediated repair in vivo remains to be clarified. Here we show that high mobility group box 1 (HMGB1) is released following fracture in both humans and mice, forms a heterocomplex with CXCL12, and acts via CXCR4 to accelerate skeletal, hematopoietic, and muscle regeneration in vivo. Pretreatment with HMGB1 2 wk before injury also accelerated tissue regeneration, indicating an acquired proregenerative signature. HMGB1 led to sustained increase in cell cycling in vivo, and using Hmgb1-/- mice we identified the underlying mechanism as the transition of multiple quiescent stem cells from G0 to GAlert HMGB1 also transitions human stem and progenitor cells to GAlert Therefore, exogenous HMGB1 may benefit patients in many clinical scenarios, including trauma, chemotherapy, and elective surgery.

Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment.

Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling.

Strain dependent differences in glucocorticoid-induced bone loss between C57BL/6J and CD-1 mice.

We have investigated the effect of long-term glucocorticoid (GC) administration on bone turnover in two frequently used mouse strains; C57BL/6J and CD1, in order to assess the influence of their genetic background on GC-induced osteoporosis (GIO). GIO was induced in 12 weeks old female C57BL/6J and CD1 mice by subcutaneous insertion of long-term release prednisolone or placebo pellets. Biomechanical properties as assessed by three point bent testing revealed that femoral elasticity and strength significantly decreased in CD1 mice receiving GC, whereas C57BL/6J mice showed no differences between placebo and prednisolone treatment. Bone turnover assessed by microcomputer tomography revealed that contrary to C57BL/6J mice, prednisolone treated CD1 mice developed osteoporosis. In vitro experiments have underlined that, at a cellular level, C57BL/6J mice osteoclasts and osteoblasts were less responsive to GC treatment and tolerated higher doses than CD1 cells. Whilst administration of long-term release prednisolone pellets provided a robust GIO animal model in 12 weeks old CD1 mice, age matched C57BL/6J mice were not susceptible to the bone changes associated with GIO. This study indicates that for the induction of experimental GIO, the mouse strain choice together with other factors such as age should be carefully evaluated.

Macrophage Polarization and Bone Formation: A review.

The contribution of inflammation to bone loss is well documented in arthritis and other diseases with an emphasis on how inflammatory cytokines promote osteoclastogenesis. Macrophages are the major producers of cytokines in inflammation, and the factors they produce depend upon their activation state or polarization. In recent years, it has become apparent that macrophages are also capable of interacting with osteoblasts and their mesenchymal precursors. This interaction provides growth and differentiation factors from one cell that act on the other and visa versa-a concept akin to the requirement for a feeder layer to grow hemopoietic cells or the coupling that occurs between osteoblasts and osteoclasts to maintain bone homeostasis. Alternatively, activated macrophages are the most likely candidates to promote bone formation and have also been implicated in the tissue repair process in other tissues. In bone, a number of factors, including oncostatin M, have been shown to promote osteoblast formation both in vitro and in vivo. This review discusses the different cell types involved, cellular mediators, and how this can be used to direct new bone anabolic approaches.

Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease.

Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell-derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.

Low-dose TNF augments fracture healing in normal and osteoporotic bone by up-regulating the innate immune response.

The mechanism by which trauma initiates healing remains unclear. Precise understanding of these events may define interventions for accelerating healing that could be translated to the clinical arena. We previously reported that addition of low-dose recombinant human TNF (rhTNF) at the fracture site augmented fracture repair in a murine tibial fracture model. Here, we show that local rhTNF treatment is only effective when administered within 24 h of injury, when neutrophils are the major inflammatory cell infiltrate. Systemic administration of anti-TNF impaired fracture healing. Addition of rhTNF enhanced neutrophil recruitment and promoted recruitment of monocytes through CCL2 production. Conversely, depletion of neutrophils or inhibition of the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death. Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair. If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization.

OX40L blockade is therapeutic in arthritis, despite promoting osteoclastogenesis.

An immune response is essential for protection against infection, but, in many individuals, aberrant responses against self tissues cause autoimmune diseases such as rheumatoid arthritis (RA). How to diminish the autoimmune response while not augmenting infectious risk is a challenge. Modern targeted therapies such as anti-TNF or anti-CD20 antibodies ameliorate disease, but at the cost of some increase in infectious risk. Approaches that might specifically reduce autoimmunity and tissue damage without infectious risk would be important. Here we describe that TNF superfamily member OX40 ligand (OX40L; CD252), which is expressed predominantly on antigen-presenting cells, and its receptor OX40 (on activated T cells), are restricted to the inflamed joint in arthritis in mice with collagen-induced arthritis and humans with RA. Blockade of this pathway in arthritic mice reduced inflammation and restored tissue integrity predominantly by inhibiting inflammatory cytokine production by OX40L-expressing macrophages. Furthermore, we identify a previously unknown role for OX40L in steady-state bone homeostasis. This work shows that more targeted approaches may augment the "therapeutic window" and increase the benefit/risk in RA, and possibly other autoimmune diseases, and are thus worth testing in humans.

Enhanced osseous implant fixation with strontium-substituted bioactive glass coating.

The use of endosseous implants is firmly established in skeletal reconstructive surgery, with rapid and permanent fixation of prostheses being a highly desirable feature. Implant coatings composed of hydroxyapatite (HA) have become the standard and have been used with some success in prolonging the time to revision surgery, but aseptic loosening remains a significant issue. The development of a new generation of more biologically active coatings is a promising approach for tackling this problem. Bioactive glasses are an ideal candidate material due to the osteostimulative properties of their dissolution products. However, to date, they have not been formulated with stability to devitrification or thermal expansion coefficients (TECs) that are suitable for stable coating onto metal implants while still retaining their bioactive properties. Here, we present a strontium-substituted bioactive glass (SrBG) implant coating which has been designed to encourage peri-implant bone formation and with a TEC similar to that of HA. The coating can be successfully applied to roughened Ti6Al4V and after implantation into the distal femur and proximal tibia of twenty-seven New Zealand White rabbits for 6, 12, or 24 weeks, it produced no adverse tissue reaction. The glass dissolved over a 6 week period, stimulating enhanced peri-implant bone formation compared with matched HA coated implants in the contralateral limb. Furthermore, superior mechanical fixation was evident in the SrBG group after 24 weeks of implantation. We propose that this coating has the potential to enhance implant fixation in a variety of orthopedic reconstructive surgery applications.

Cells of the immune system orchestrate changes in bone cell function.

There is a complex interplay between the cells of the immune system and bone. Immune cells, such as T and NK cells, are able to enhance osteoclast formation via the production of RANKL. Yet there is increasing evidence to show that during the resolution of inflammation or as a consequence of increased osteoclastogenesis there is an anabolic response via the formation of more osteoblasts. Furthermore, osteoblasts themselves are involved in the control of immune cell function, thus promoting the resolution of inflammation. Hence, the concept of "coupling"-how bone formation is linked to resorption-needs to be more inclusive rather than restricting our focus to osteoblast-osteoclast interactions as in a whole organism these cells are never in isolation. This review will investigate the role of immune cells in normal bone homeostasis and in inflammatory diseases where the balance between resorption and formation is lost.

Potential of human fetal chorionic stem cells for the treatment of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic bone pathology with prenatal onset, characterized by brittle bones in response to abnormal collagen composition. There is presently no cure for OI. We previously showed that human first trimester fetal blood mesenchymal stem cells (MSCs) transplanted into a murine OI model (oim mice) improved the phenotype. However, the clinical use of fetal MSC is constrained by their limited number and low availability. In contrast, human fetal early chorionic stem cells (e-CSC) can be used without ethical restrictions and isolated in high numbers from the placenta during ongoing pregnancy. Here, we show that intraperitoneal injection of e-CSC in oim neonates reduced fractures, increased bone ductility and bone volume (BV), increased the numbers of hypertrophic chondrocytes, and upregulated endogenous genes involved in endochondral and intramembranous ossification. Exogenous cells preferentially homed to long bone epiphyses, expressed osteoblast genes, and produced collagen COL1A2. Together, our data suggest that exogenous cells decrease bone brittleness and BV by directly differentiating to osteoblasts and indirectly stimulating host chondrogenesis and osteogenesis. In conclusion, the placenta is a practical source of stem cells for the treatment of OI.

Immune cells and bone: coupling goes both ways.

There is a mounting body of research describing how cells of the immune system direct bone cell function whilst bone cells are involved in the generation and retention of immune cells and their precursors. Recent works into regulation of the haemopoietic stem cell niche have firmly implicated osteoblasts and osteoclasts. On the other hand, virtually all of the mature immune cells have been described to influence bone formation in vitro and in vivo. This review will summarize the latest developments and discuss the importance of the coupling of bone formation to resorption when considering the contributions from cells of the immune system.

Effect of glycosphingolipids on osteoclastogenesis and osteolytic bone diseases.

Alterations in glycosphingolipid (GSL) production results in lysosomal storage disorders associated with neurodegenerative changes. In Gaucher's disease, the patients also develop osteoporosis that is ameliorated upon treatment for the underlying defect in GSL metabolism. The role of GSLs in osteoclast and osteoblast formation is discussed here as well as the potential therapeutic uses of already approved drugs that limit GSL production in bone loss disorders such as multiple myeloma and periodontal disease.