Applying Advanced Imaging Techniques to a Murine Model of Orthotopic Osteosarcoma.

INTRODUCTION: Reliable animal models are required to evaluate novel treatments for osteosarcoma. In this study, the aim was to implement advanced imaging techniques in a murine model of orthotopic osteosarcoma to improve disease modeling and the assessment of primary and metastatic disease. MATERIALS AND METHODS: Intra-tibial injection of luciferase-tagged OPGR80 murine osteosarcoma cells was performed in Balb/c nude mice. Treatment agent [pigment epithelium-derived factor (PEDF)] was delivered to the peritoneal cavity. Primary tumors and metastases were evaluated by in vivo bioluminescent assays, micro-computed tomography, [(18)F]-Fluoride-PET and [(18)F]-FDG-PET. RESULTS: [(18)F]-Fluoride-PET was more sensitive than [(18)F]-FDG-PET for detecting early disease. Both [(18)F]-Fluoride-PET and [(18)F]-FDG-PET showed progressive disease in the model, with fourfold and twofold increases in standardized uptake value (p < 0.05) by the study endpoint, respectively. In vivo bioluminescent assay showed that systemically delivered PEDF inhibited growth of primary osteosarcoma. DISCUSSION: Application of [(18)F]-Fluoride-PET and [(18)F]-FDG-PET to an established murine model of orthotopic osteosarcoma has improved the assessment of disease. The use of targeted imaging should prove beneficial for the evaluation of new approaches to osteosarcoma therapy.

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.

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.

Anti-chondrosarcoma effects of PEDF mediated via molecules important to apoptosis, cell cycling, adhesion and invasion.

Chondrosarcoma develops as a result of overgrowth of chondrocytes and overproduction of cartilage matrix. It is currently surgically treated, although non-invasive methods are being sought. In this report, pigment epithelium-derived factor (PEDF) induced apoptosis in the chondrosarcoma cell line - JJ012, with upregulation of Bax, Fas, caspase-3 and -6 and downregulation of Bcl-2. Cell cycling was also decreased with decreased expression of p38, p-Akt, p-Erk and JNK1 and increased expression of p73 and E2F1. Furthermore, PEDF increased adhesion of cells to collagen-I, with decreased expression of p-Fak, RhoA and cdc42. Invasion of cells through collagen-I was also reduced by PEDF, with decreased expression of uPAR, MMP-14 and increased expression of PAI-1. These findings seminally indicate that PEDF may have potential as an anti-cancer agent against chondrosarcoma.

Dz13 induces a cytotoxic stress response with upregulation of E2F1 in tumor cells metastasizing to or from bone.

The oligonucleotide Dz13 is a DNA enzyme (deoxyribozyme) that cleaves c-Jun mRNA. It has efficacious effects against tumors directly, is active against tumor-induced angiogenesis, inhibits neointima formation after arterial injury, and controls inflammatory responses. The off-target effects of Dz13 may in fact be driving some of these potentially therapeutic effects, though no mechanisms have been clearly defined in target cells. To this end, we here show that when a panel of human tumor cells that naturally propagate in bone are challenged with Dz13, the tumor suppressor E2F1 is upregulated regardless of cellular p53 status. The piddosomal components, p53-induced protein with a death domain and caspase-2, were translocated to the nucleus when deoxyribozymes were incubated with cells, but RIP associated Ich-1/CED homologous protein with death domain levels increased throughout the cell with either Dz13 or its scrambled control oligonucleotide. In response to Dz13-mediated cytotoxicity, cells upregulated levels of ERK, Akt, and p38. Summarily, these results suggest a cytotoxic stress (resembling DNA damage) response of tumor cells to Dz13, which induces apoptosis via the activation of inhibitor of caspase-activated deoxyribonuclease and protein kinase C delta. In vivo, in tumor-in-bone orthotopic and clinically relevant models for prostate and breast cancer metastasis, and a novel spontaneously metastasizing model for osteosarcoma (OS), Dz13 decreased growth in bone, and also metastasis for OS. This new model for OS was assessed to be clinically relevant in its expression of typical bone markers, osteopontin and osteocalcin. These results provide an off-target mechanism for Dz13 function, but this may be useful therapeutically against tumors.

Direct anti-metastatic efficacy by the DNA enzyme Dz13 and downregulated MMP-2, MMP-9 and MT1-MMP in tumours.

The DNA enzyme Dz13, targeted against the oncogene c-Jun, is capable of inhibiting various model tumours in mice albeit in ectopic models of neoplasia. In previous studies using orthotopic models of disease, the inhibitory effects of Dz13 on secondary growth was a direct result of growth inhibition at the primary lesion site. Thus, the direct and genuine effects on metastasis were not gauged. In this study, Dz13 was able to inhibit both locoregional and distal metastasis of tumour cells in mice, in studies where the primary tumours were unaffected due to the late and clinically-mimicking nature of treatment commencement. In addition, the effect of Dz13 against tumours has now been extended to encompass breast and prostate cancer. Dz13 upregulated the matrix metalloproteinase (MMP)-2 and MMP-9, and decreased expression of MT1-MMP (MMP-14) in cultured tumour cells. However, in sections of ectopic tumours treated with Dz13, both MMP-2 and MMP-9 were downregulated. Thus, not only is Dz13 able to inhibit tumour growth at the primary site, but also able to decrease the ability of neoplastic cells to metastasize. These findings further highlight the growing potential of Dz13 as an antineoplastic agent.

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.

Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery.

Proteins and peptides are increasingly recognized as potential leads for the development of new therapeutics for a variety of human ailments. Due to their relatively specific mode of action, proteins and peptides can be administered at relatively low doses for therapeutic effects. As natural biological products, these low doses reduce the risk otherwise caused by other small molecular drugs or larger charged molecules. Unfortunately, their therapeutic potential and clinical application is frequently hampered by various obstacles to their successful delivery. This review discusses the recent developments in the fields of liposome, microparticle and nanoparticle pertinent to protein and peptide delivery covering those systems tested and/or validated in vivo.

DNAzyme delivery systems: getting past first base.

DNAzyme technology has evolved into a discipline with the potential for presenting drug agents against cancer and atherosclerosis. However, current approaches still rely on sub-optimal drug delivery systems (DDSs) for DNAzymes. Certain DDSs have shown potential, such as chitosan and polyethylenimine (PEI), although more emphasis needs to be placed on actual efficacy and safety, in addition to establishing the pharmacokinetics of the molecule being tested. Unfortunately, the plethora of DDSs reported for antisense delivery--the trailblazer for target gene knockdown agents--have yet to yield even one entity capable of being used clinically, and clinicians have resorted to administering continuous systemic free oligonucleotides with promising, albeit lukewarm results. The challenge ahead for DNAzymes to be considered genuine drug candidates alongside siRNA and antisense simply lies in the better implementation of DDSs.

Cancer, chitosan nanoparticles and catalytic nucleic acids.

OBJECTIVES: The aim of this review was to examine gene therapy involving DNAzyme and siRNA encapsulation into chitosan nanoparticles, discussing the current and future status of this drug delivery system in enhancing drug delivery and cancer therapy. KEY FINDINGS: Cancer is a disease state in which the cells in our body undergo mutations at the genetic level and are transformed, acquiring the ability to replicate limitlessly. Conventional cancer treatment involves the use of surgery and cytotoxic chemotherapy and/or radiotherapy, which have the potential of harming normal, otherwise healthy, non-neoplastic cells. Newer forms of therapy such as immunotherapy and gene therapy have shown initial promise, but still require better ways to limit exposure to cancerous lesions in the body. As a result drug delivery systems have been developed in attempts to deliver therapeutics specifically to the target lesion site. One recent drug delivery system has revolved around the use of chitosan nanoparticle technology, where therapeutics are encapsulated into nanoparticles and targeted to tumours. SUMMARY: Though few, attempts at encapsulating therapeutics such as deoxyribozymes and small or short interfering RNA have been optimistic and encouraging.

Review: doxorubicin delivery systems based on chitosan for cancer therapy.

OBJECTIVES: This review sheds insight into an increasingly popular polymer that has been widely explored as a potential drug delivery system. The abundant, biodegradable and biocompatible polysaccharide chitosan, with many other favourable properties, has been favoured as a drug delivery system for the purposes of encapsulating and delivery of doxorubicin with reduced side-effects. KEY FINDINGS: Doxorubicin is frequently used as a frontline chemotherapeutic agent against a variety of cancers. It has largely been able to demonstrate anti-tumour effects, though there are major shortfalls of doxorubicin, which include serious side-effects such as cardiomyopathy and myelosuppression, and also an ever-present danger of extravasation during drug administration. In view of this, drug delivery systems are currently being explored as alternative methods of drug delivery in a bid to more effectively direct doxorubicin to the specific lesion site and reduce its systemic side-effects. Liposomes and dendrimers have been tested as potential carriers for doxorubicin; however they are not the focus of this review. SUMMARY: Recent advancements in doxorubicin and chitosan technology have shown some preliminary though promising results for cancer therapy.

Osteosarcoma: Conventional treatment vs. gene therapy.

Osteosarcoma (OS) remains the most common primary malignant bone cancer affecting children and adolescents. Majority of patients are subjected to surgery and chemotherapy with limited alternatives to consider. However, with increasing knowledge of the molecular pathogenesis of OS, advancements in OS therapy research fields have developed potential agents for a more targeted and localised approach to treatment. Thus, this review attempts to examine some common treatment strategies (surgery, radiotherapy and chemotherapy) employed clinically as well as recent developments in newer experimental therapy methods (gene therapy) which might potentially benefit patients.

Acute pediatric rhabdomyolysis: causes and rates of renal failure.

OBJECTIVES: The goals were to (1) compare the causes, clinical presentation, and prevalence of acute renal failure in pediatric rhabdomyolysis with the published data for adults; (2) determine predictors of acute renal failure in pediatric patients with rhabdomyolysis; and (3) explore the relationship of acute renal failure with treatment modalities such as fluid and bicarbonate administration. METHODS: We performed a retrospective chart review to identify patients with creatinine kinase levels of > 1000 IU/L who were treated in the emergency department of a tertiary pediatric hospital between 1993 and 2003, and we constructed regression models. RESULTS: Two hundred ten patients were studied. One hundred ninety-one patients met study eligibility (128 male and 63 female), with a median age of 11 years. The most common documented symptoms were muscle pain (45%), fever (40%), and symptoms of viral infection (39%). The most common causes of pediatric rhabdomyolysis were viral myositis (38%), trauma (26%), and connective tissue disease (5%). Six of 37 patients with creatinine kinase levels of > or = 6000 IU/L had previously undiagnosed dermatomyositis or hereditary metabolic disease, compared with 10 of 154 patients with creatinine kinase levels of 1000 to 5999 IU/L. Nine of 191 patients developed acute renal failure. None of 99 patients with initial urinary heme dipstick results of < 2+ developed acute renal failure, compared with 9 of 44 patients with urinary heme dipstick results of > or = 2+. Higher initial creatinine kinase levels and higher fluid administration rates were associated with higher maximal creatinine levels. CONCLUSIONS: The cause of acute pediatric rhabdomyolysis is different from that of adult rhabdomyolysis. The risk of acute renal failure in children is much less than the risk reported for adults.