Triple-threat activity of PEDF in bone tumors: Tumor inhibition, tissue preservation and cardioprotection against doxorubicin.

Pigment epithelium-derived factor (PEDF) is known for its osteogenic properties, but its effects against primary and secondary bone tumors have not comprehensively been demonstrated. We show the ubiquitous expression of PEDF in murine embryonic tissue. Continuous administration of PEDF in pregnant mice for five days did not adversely affect foetal health, despite PEDF's known potent antiangiogenic properties. In the case of the devastating childhood bone cancer osteosarcoma, PEDF has direct anticancer activity per se, and protects against the toxicity of doxorubicin in the heart, small intestine and testes. PEDF demonstrated anti-proliferative and pro-apoptotic effects against human prostate and breast cancer cells, tumors which are known to metastasize to bone as the preferred secondary site. Caspase-2 was activated in both tumor cell types by PEDF. In models of prostate and breast cancer in bone, PEDF significantly reduced tumor volumes. When combined with zoledronic acid, continuously-administered PEDF significantly reduced breast tumor volume at the bone, and was able to preserve the quality of bone better than the combination therapy. These multiple positive findings make PEDF an ideal endogenous and safe biological for possible future clinical testing.

Nucleotropic doxorubicin nanoparticles decrease cancer cell viability, destroy mitochondria, induce autophagy and enhance tumour necrosis.

OBJECTIVE: Doxorubicin (Dox) is used clinically against various neoplasias, but suffers from serious side effects, and for the past three decades, this shortcoming has spurred research towards finding better drug delivery systems (DDSs) for this frontline drug. METHODS: A non-targeted nucleotropic Dox-loaded nanoparticle (DNP) DDS is described, which has a simple chemical design, is easy to formulate and administer, is inexpensive, non-biohazardous and may prove to be useful clinically. KEY FINDINGS: The DNP formulated via vortex-assisted complex coarcevation enhanced (300-fold) cell-inhibitory activity of the drug in a panel of human cancer cells (osteosarcoma, breast, prostate and colorectal cancer) and enhanced (10-fold) efficacy against osteosarcoma (OS) in vivo. The slow-release DNPs localised to the endoplasmic reticulum disrupted the mitochondria and entered the nucleus. Prominent cytosolic vacuolisation, budding off of portions of the cytoplasm, both suggestive of autophagy, were observed. Mice that were administered with DNPs intratumorally had the smallest tumours at the end of the study, with more necrotic hotspots. CONCLUSION: This promising nucleotropic DDS enhances the cell delivery and activity of Dox against a variety of human cancer cell lines and in OS tumours in mice.

Cardiomyocyte apoptosis vs autophagy with prolonged doxorubicin treatment: comparison with osteosarcoma cells.

OBJECTIVE: Doxorubicin (Dox) is a frontline chemotherapeutic against osteosarcoma (OS) that is plagued by side effects, particularly in the heart. The specific objective of this article is to investigate whether low-dose Dox treatment had pro-autophagic effects in cardiomyocytes as well as osteosarcoma cells. METHODS: This study characterises apoptotic (Bax) and autophagic (Beclin-1) biomarker levels in human OS and cardiomyocyte cell lines as well as in various tissues when mice are exposed to low (1 mg/kg, thrice weekly) and high (3 mg/kg thrice weekly) dose Dox for a month. KEY FINDINGS: There was a decrease in Bax and increase in Beclin-1 in cardiac tissue in the high-dose group. Dox decreased Beclin-1 in the skin and liver, with no clear indication in the stomach, small intestine and testis. At low Dox doses of 10 and 100 nm in cardiomyocytes and OS cells, there is a pro-apoptotic effect, with a quicker response in the 100-nm condition, and a slower but steady increase of a pro-apoptotic response at the lower 10-nm dose. However, electron microscopy images revealed changes to human OS cells that resembled autophagy. Human prostate, breast and colorectal cells treated with 10-nm Dox showed ∼ 40% reduction in cell viability after 24 h. CONCLUSION: In culture, cells of both cardiomyocytes and OS revealed a predominant pro-apoptotic response at the expense of autophagy, although both seemed to be occurring in vivo.

The potential role of free chitosan in bone trauma and bone cancer management.

Bone defects caused by fractures or cancer-mediated destruction are debilitating. Chitosan is commonly used in scaffold matrices for bone healing, but rarely as a free drug. We demonstrate that free chitosan promotes osteoblast proliferation and osteogenesis in mesenchymal stem cells, increases osteopontin and collagen I expression, and reduces osteoclastogenesis. Chitosan inhibits invasion of endothelial cells, downregulating uPA/R, MT1-MMP, cdc42 and Rac1. Better healing of bone fractures with greater trabecular bone formation was observed in mice treated with chitosan. Chitosan induces apoptosis in osteotropic prostate and breast cancer cells via caspase-2 and -3 activation, and reduces their establishment in bone. Chitosan is pro-apoptotic in osteosarcoma cells, but not their normal counterpart, osteoblasts, or chondrosarcoma cells. Systemic delivery of chitosan does not perturb angiogenesis, bone volume or instinctive behaviour in pregnant mice, but decreases foetal length and changes pancreatic secretory acini. With certain controls in place, chitosan could be useful for bone trauma management.

Co-nanoencapsulated doxorubicin and Dz13 control osteosarcoma progression in a murine model.

OBJECTIVES: Chitosan is a green (natural, abundant, biodegradable, biocompatible) biopolymer that can be formulated to encapsulate a variety of therapeutic compounds. This study aimed to investigate chitosan nanoparticles (NPs) as a means of improving delivery of the clinically used anti-cancer agent doxorubicin (Dox) and the preclinical lead compound Dz13 oligonucleotide together. METHODS: A novel chitosan NP system encapsulating Dox and Dz13 was designed, biophysically characterised and tested in a clinically relevant model of the metastasising bone tumour, osteosarcoma (OS). KEY FINDINGS: By careful alteration of the concentration of the individual components, a final formulation of Dz13-Dox NPs (DDNPs) was achieved, with high (>91%) loading of both compounds, which consisted of individual 50-nm particles forming aggregates as large as 500 nm, with a large positive ζ-potential. The DDNPs could be stored at various temperatures for a week without loss in activity but were prone to degradation in serum. DDNPs successfully inhibited OS tumour growth more effectively than treatment with NPs of Dz13 and Dox-chitosan, as well as Dox administered intraperitoneally. Apart from inhibiting tumour growth, DDNPs protected the affected bone from substantial destruction by aggressive tumour growth and reduced the incidence of metastasis to the lungs without causing adverse effects in mice. CONCLUSION: This NP is a promising formulation that could be useful for clinical management of OS.

Leukemia inhibitory factor protects axons in experimental autoimmune encephalomyelitis via an oligodendrocyte-independent mechanism.

Leukemia inhibitory factor (LIF) and Ciliary Neurotrophic factor (CNTF) are members of the interleukin-6 family of cytokines, defined by use of the gp130 molecule as an obligate receptor. In the murine experimental autoimmune encephalomyelitis (EAE) model, antagonism of LIF and genetic deletion of CNTF worsen disease. The potential mechanism of action of these cytokines in EAE is complex, as gp130 is expressed by all neural cells, and could involve immuno-modulation, reduction of oligodendrocyte injury, neuronal protection, or a combination of these actions. In this study we aim to investigate whether the beneficial effects of CNTF/LIF signalling in EAE are associated with axonal protection; and whether this requires signalling through oligodendrocytes. We induced MOG35₋55 EAE in CNTF, LIF and double knockout mice. On a CNTF null background, LIF knockout was associated with increased EAE severity (EAE grade 2.1±0.14 vs 2.6±0.19; P<0.05). These mice also showed increased axonal damage relative to LIF heterozygous mice, as indicated by decreased optic nerve parallel diffusivity on MRI (1540±207 µm²-/s vs 1310±175 µm²-/s; P<0.05), and optic nerve (-12.5%) and spinal cord (-16%) axon densities; and increased serum neurofilament-H levels (2.5 fold increase). No differences in inflammatory cell numbers or peripheral auto-immune T-cell priming were evident. Oligodendrocyte-targeted gp130 knockout mice showed that disruption of CNTF/LIF signalling in these cells has no effect on acute EAE severity. These studies demonstrate that endogenous CNTF and LIF act centrally to protect axons from acute inflammatory destruction via an oligodendrocyte-independent mechanism.

Small amphipathic molecules modulate secondary structure and amyloid fibril-forming kinetics of Alzheimer disease peptide Aß(1-42).

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aß peptide occur. Aß is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aß(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and ß-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aß(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aß may provide insight for developing therapeutic strategies aimed at stabilizing Aß in a favorable conformation.

Low intrinsic running capacity is associated with reduced skeletal muscle substrate oxidation and lower mitochondrial content in white skeletal muscle.

Chronic metabolic diseases develop from the complex interaction of environmental and genetic factors, although the extent to which each contributes to these disorders is unknown. Here, we test the hypothesis that artificial selection for low intrinsic aerobic running capacity is associated with reduced skeletal muscle metabolism and impaired metabolic health. Rat models for low- (LCR) and high- (HCR) intrinsic running capacity were derived from genetically heterogeneous N:NIH stock for 20 generations. Artificial selection produced a 530% difference in running capacity between LCR/HCR, which was associated with significant functional differences in glucose and lipid handling by skeletal muscle, as assessed by hindlimb perfusion. LCR had reduced rates of skeletal muscle glucose uptake (∼30%; P = 0.04), glucose oxidation (∼50%; P = 0.04), and lipid oxidation (∼40%; P = 0.02). Artificial selection for low aerobic capacity was also linked with reduced molecular signaling, decreased muscle glycogen, and triglyceride storage, and a lower mitochondrial content in skeletal muscle, with the most profound changes to these parameters evident in white rather than red muscle. We show that a low intrinsic aerobic running capacity confers reduced insulin sensitivity in skeletal muscle and is associated with impaired markers of metabolic health compared with high intrinsic running capacity. Furthermore, selection for high running capacity, in the absence of exercise training, endows increased skeletal muscle insulin sensitivity and oxidative capacity in specifically white muscle rather than red muscle. These data provide evidence that differences in white muscle may have a role in the divergent aerobic capacity observed in this generation of LCR/HCR.

Mitochondrial dysfunction in CD4+ lymphocytes from stavudine-treated HIV patients.

HIV therapy with nucleoside analogue reverse transcriptase inhibitors (NRTI) such as stavudine remains in widespread use in resource-limited nations due to potent efficacy, convenience of formulation and lack of practical alternatives. However, it remains unclear whether adverse side effects with NRTI include reduced mitochondrial respiratory function, particularly in peripheral blood lymphocytes (PBLs). The aim of this study was to determine whether stavudine-based highly active antiretroviral therapy (HAART) is associated with impaired mitochondrial respiratory transport chain function in patient derived CD4+PBLs. CD4+PBLs were isolated from asymptomatic HIV-infected patients treated with stavudine-HAART for 3 months (n=10), HIV-infected patients not on treatment (n=9) and uninfected controls (n=18). The basal mitochondrial oxygen consumption of CD4+PBLs from stavudine-treated patients was reduced relative to that of untreated HIV-infected patients and controls (stavudine treated group, 4.22 (25% 2.16, 75% 8.84); control uninfected, 11.2 (25% 3.95, 75% 16.6); untreated 18.1 (25% 11.8, 75% 37.9)ng oxygen atoms/min/ml). Maximal oxygen consumption (stimulated with the proton ionophore FCCP) in cells from stavudine treated patients was also reduced relative to that of untreated patients and controls (stavudine treated, 24.4+/-10.5; control uninfected, 50.6+/-39.5; untreated, 68.8+/-41.1 ng oxygen atoms/min/ml). Citrate synthase activities, relative mitochondrial volume (by electron microscopy) and mtDNA copy numbers per cell were not different between groups. Therapy with stavudine results in impaired mitochondrial function in CD4+PBLs that does not appear to be due to reduced mitochondrial volume or DNA content and cannot be attributed to infection with HIV.

A nanoparticulate system that enhances the efficacy of the tumoricide Dz13 when administered proximal to the lesion site.

We demonstrate that Dz13, a DNA enzyme that cleaves c-Jun mRNA, and is capable of inhibiting cancer cell growth in vitro, can be encapsulated into chitosan nanoparticles. For optimisation of this chitosan-based formulation, pH 6, 0.02% chitosan concentration, and 55 degrees C were found to be best among the variables tested. Particles were 50-300nm in diameter and encapsulated Dz13 was active when particles were exposed to cancer cells. Nanoparticles were stable during storage even for a month, but were not stable in mouse and human serum. In two different clinically-relevant disease models, and using a clinically-adoptable dosing regimen, these Dz13-nanoparticles were shown to be efficacious against a bone tumour (osteosarcoma), for which no real cure exists currently. However, no toxicity against other bone-dwelling cells was observed with the formulation, and no side-effects were noted in vivo in lymphatic and reticuloendothelial tissues proximal and distal to the administration site.

The performance of doxorubicin encapsulated in chitosan-dextran sulphate microparticles in an osteosarcoma model.

Osteosarcoma (OS) is the most common primary bone cancer affecting children and adolescents. It is potentially debilitating and fatal due to pulmonary metastasis. A common management strategy, chemotherapy, has a 10-year disease-free survival of approximately 60%. However, a targeted approach to OS tumor inhibition is still lacking, calling for improved management strategies. A frontline drug for OS, doxorubicin (DOX), causes multiple side-effects (example myelosuppression, heart failure, hepatic toxicity, alopecia) in patients, especially in high doses required to control tumor growth. A drug delivery system (DDS) was developed to deliver DOX specifically to tumor sites. Through DOX encapsulation into chitosan DDS via the complex coacervation method with dextran sulphate, novel DOX microparticles (DMPs), with a DOX loading content of more than 99%, were formed. Multiple optimisation steps produced DMPs which caused OS cell death through apoptosis, necrosis and autophagic cell death. Treatment of mice bearing orthotopic OS with DMP decreased tumor volume, decreased bone lysis, and reduced secondary metastasis to the lungs. DMP-treated mice also maintained their weight and did not appear to suffer from any visible side-effects such as heart failure or dry skin. Thus, DMP may prove to be a useful DDS platform clinically provided further studies are performed to rigorously validate this technology.

Resolution of platelet function defects with imatinib therapy in a patient with chronic myeloid leukaemia in chronic phase.

Platelet function defects are frequently found in patients with chronic myeloid leukaemia. Major clinical bleeding, however, is a rare and infrequently reported complication. Platelet function abnormalities have also not been previously correlated with molecular monitoring of BCR-ABL in chronic myeloid leukaemia. We report a case of a patient with major clinical bleeding as a presenting feature of chronic myeloid leukaemia. The patient developed compartment syndrome of the thigh secondary to a haematoma developing after minor trauma. Fasciotomy was complicated by severe bleeding requiring massive transfusion. Haemostasis was only obtained after activated recombinant factor VII was administered. Laboratory investigations revealed a platelet function defect with reduced platelet aggregation to collagen, epinephrine and arachidonic acid. As imatinib therapy commenced, molecular response was associated with near-normalization of platelet function, which subsequently became significantly abnormal with molecular relapse. Electron microscopy demonstrated normal platelet ultrastructure. We conclude that dysregulated Abelson kinase plays a pathogenic role in platelet function defects associated with chronic myeloid leukaemia, and discuss the management of clinically significant bleeding in patients with platelet function defects associated with myeloproliferative disorders.

Validation of a novel biomarker for acute axonal injury in experimental autoimmune encephalomyelitis.

In multiple sclerosis, inflammatory axonal injury is a key pathological mechanism responsible for the development of progressive neurological dysfunction. The injured axon represents a therapeutic target in this disease; however, therapeutic trials of neuroprotective candidates will initially require preclinical testing in an animal model of inflammatory axonal injury and subsequently the development of a reliable paraclinical measure of axonal degeneration in humans. In the present study, we demonstrate the validity of serum phosphorylated neurofilament H (pNF-H) as a marker of axonal injury in murine experimental autoimmune encephalomyelitis (EAE). At the time of maximum disease severity (EAE day 22), the average serum pNF-H level reached 5.7 ng/ml, correlating significantly with the EAE paraplegia score (r = 0.75, P < 0.001). On average, 40% of axons in the spinal cord were lost in EAE, and serum pNF-H levels were highly correlated with axon loss (r = 0.8, P < 0.001). Axonal injury was a severe and acute event, insofar as serum pNF-H levels were not significantly elevated at early (EAE day 12) or late (EAE days 35 and 50) disease time points. Our results demonstrate that acute inflammatory axonal injury is a pathological feature of murine MOG(35-55) EAE, indicating that this model may mirror the acute pathological events in active multiple sclerosis lesions. Furthermore, we have validated the serum pNF-H assay as an unbiased measurement of axonal injury in EAE, facilitating rapid screening of potential neuroprotective therapies in this model.

Biocompatible chitosan-DNAzyme nanoparticle exhibits enhanced biological activity.

DNAzymes are oligonucleotides capable of specific catalysis of target mRNA. To date, a delivery vehicle for DNAzymes has not been developed. Chitosan is a biomaterial obtained abundantly in nature. A biocompatible c-jun (an oncogene) DNAzyme nanoparticle formulated from chitosan was found to be more active against osteosarcoma (bone cancer) cells, inducing apoptotic cell death in these cells. The formulation was stable in serum for a week and at room temperature for a month. Clinically, knockdown of c-jun gene expression with chitosan nanobiotechnology may improve treatment outcome for tumours growing in bone.

Downregulation of c-jun results in apoptosis-mediated anti-osteosarcoma activity in an orthotopic model.

c-jun has been found to be upregulated in a variety of cancers including osteosarcoma. DNAzymes are oligonucleotides capable of specific downregulation of target genes. c-jun knockdown-mediated apoptosis in osteosarcoma cells involved caspases-1, -2 and -8, but not the Fas/FasL pathway. A c-jun DNAzyme, encapsulated within a novel cationic multilamellar vesicle liposome, inhibited the growth and metastasis of osteosarcoma in an orthotopic spontaneously metastasising model of the disease. The 60 nm DDAB:DOPE liposome was formulated using ethanol injection/extrusion. Clinically, downregulation of c-jun may proffer an improved treatment outcome for these tumours originating in bone.

MR diffusion changes correlate with ultra-structurally defined axonal degeneration in murine optic nerve.

Diffusion weighted imaging (DWI) and diffusion tensor imaging (DTI) are widely used to investigate central nervous system (CNS) white matter structure and pathology. Changes in principal diffusivities parallel and perpendicular to nerve fibers or axonal tracts have been associated with axonal pathology and de/dysmyelination respectively. However, the ultra-structural properties and the pathological alterations of white matter responsible for diffusivity changes have not been fully elucidated. We examined the relationship between the directional diffusivities and ultra-structural properties in mouse optic nerve using healthy animals, and mice with optic neuritis (ON) that exhibited marked inflammatory changes and moderately severe axonal pathology. Progressive axonal degeneration in ON resulted in a 23% reduction of parallel diffusivity as detected by diffusion MRI (P<10(-5)), but no change in perpendicular diffusivity. Parallel diffusion changes were highly correlated with the total axolemmal cross-sectional area in the pre-chiasmal portion of the optic nerve (r=0.86, P<0.001). This study provides quantitative evidence that reduced parallel diffusivity in the optic nerve correlates significantly with axolemmal cross-sectional area reductions. MRI-based assessment of axonal degeneration in murine ON is feasible and potentially useful for monitoring of neuro-protective therapies in preclinical trials in animals.

Alzheimer's disease therapeutics: new approaches to an ageing problem.

Abnormal proteinaceous deposits are found in the brain of patients with many different neurodegenerative diseases. In many of these diseases, the production of the deposits is probably associated with disease pathogenesis. In Alzheimer's disease (AD), the amyloid protein (A beta), is produced by the action of enzymes known as secretases, which cleave the beta-amyloid protein precursor. A beta is secreted from cells in the brain, after which it oligomerizes and is deposited in the extracellular compartment of the brain to form amyloid plaques and amyloid angiopathy. Targeting the production of A beta and its aggregation is now a key strategy in the development of novel therapeutic agents for the treatment of AD. This review examines the potential of immunization strategies, cholesterol-lowering drugs, protease inhibitors and nicotinic drugs for the treatment of AD.