PEG-liposomal doxorubicin as a potential agent for canine metastatic osteosarcoma - in vitro and ex ovo studies.

Introduction: Appendicular osteosarcoma (OSA) is a highly aggressive and metastatic primary bone tumour in dogs. Standard therapy is amputation and adjuvant chemotherapy (e.g. with doxorubicin). Liposomal drug delivery may augment therapeutic efficacy and reduce negative side effects. Polyethylene glycol (PEG)-liposomal doxorubicin treats human metastatic cancers effectively. The study aimed was to evaluate PEG-liposomal doxorubicin's inhibitory effect on canine metastatic proliferation and migration in vitro. It also aimed to appraise the drug's extravasation inhibition in vivo using the human medicine-proven chick embryo chorioallantoic membrane ex ovo model. Material and Methods: The canine D-17 OSA cell line was cultured and inoculated with decreasing concentrations of PEG-liposomal doxorubicin and conventional doxorubicin in a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test of cell viability, proliferation and cytotoxicity. Flow cytometry with Annexin V and Draq 7 staining confirmed the MTT test results, indicating dead, early and late apoptotic, and live cells. The inhibitory effect of the two preparations on cancer cell migration was investigated with a wound-healing assay. Culture plates seeded with cells were prepared. The cell monolayer was scratched and images of cells migrating to the scratch were captured at 0 h, 12 h and 24 h. Also, embryos were removed from three-day-incubated fertilised chicken eggs. On the 12th day, labelled D-17 cells were injected into each embryo. Embryos in one group received 100 µL of phosphate-buffered saline as controls, those in another group 30 µg/mL of PEG-liposomal doxorubicin, and those in the last group 6 µg/mL of conventional doxorubicin. The effectiveness of the intravascular administration of the D-17 cells was confirmed under a microscope. Results: PEG-liposomal doxorubicin inhibited the migration of canine OSA cells more effectively than conventional doxorubicin (P ≤ 0.05). The ex ovo model showed that both drugs had similar impacts on canine metastatic OSA. Conclusion: The liposomal form of the drug may be considered a potentially effective compound in canine metastatic OSA; nevertheless, further in vivo studies are essential to confirm this hypothesis.

Gold Nanoparticles Inhibit Extravasation of Canine Osteosarcoma Cells in the Ex Ovo Chicken Embryo Chorioallantoic Membrane Model.

Canine osteosarcoma (OS) is an aggressive bone tumor with high metastatic potential and poor prognosis, mainly due to metastatic disease. Nanomedicine-based agents can be used to improve both primary and metastatic tumor treatment. Recently, gold nanoparticles were shown to inhibit different stages of the metastatic cascade in various human cancers. Here, we assessed the potential inhibitory effect of the glutathione-stabilized gold nanoparticles (Au-GSH NPs) on canine OS cells extravasation, utilizing the ex ovo chick embryo chorioallantoic membrane (CAM) model. The calculation of cells extravasation rates was performed using wide-field fluorescent microscopy. Transmission electron microscopy and Microwave Plasma Atomic Emission Spectroscopy revealed Au-GSH NPs absorption by OS cells. We demonstrated that Au-GSH NPs are non-toxic and significantly inhibit canine OS cells extravasation rates, regardless of their aggressiveness phenotype. The results indicate that Au-GSH NPs can act as a possible anti metastatic agent for OS treatment. Furthermore, the implemented CAM model may be used as a valuable preclinical platform in veterinary medicine, such as testing anti-metastatic agents.

Procoagulant and anticoagulant plasma indicators in diabetic dogs showing increased antithrombin III levels in canine diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) often leads to dangerous thromboembolic complications in humans. DM is also a relatively common endocrinopathy of dogs. There is scarce information regarding procoagulant and anticoagulant plasma indicators in this disease. The aim of the study was to evaluate the levels of the selected plasma haemostatic parameters in dogs suffering from diabetes. The study group consisted of 20 dogs meeting all the inclusion criteria, with fasting glycaemia exceeding 11.1 mmol/l. The control group consisted of 15 healthy dogs presented for routine examination. An evaluation of the prothrombin time (PT); and fibrinogen, D-dimer and antithrombin III (ATIII) levels was performed. RESULTS: Except for ATIII activity, the haemostatic parameter differences were not statistically significant. High values of ATIII activity were observed in 90% of diabetic dogs. On average, the values amounted to 166.6% and were 31.4% higher than those in the control group. The ATIII activity in the diabetic group was significantly higher than that in the control group (p = 0.0004). CONCLUSIONS: Here, we report elevated levels of ATIII in diabetic dogs. This finding may suggest the protective role of ATIII against potential thrombotic events. However, the exact role of ATIII in dog diabetes remains unclear.

Enhanced Cytotoxic Effect of Doxorubicin Conjugated to Glutathione-Stabilized Gold Nanoparticles in Canine Osteosarcoma-In Vitro Studies.

Osteosarcoma (OSA) is the most common malignant bone neoplasia in humans and dogs. In dogs, treatment consists of surgery in combination with chemotherapy (mostly carboplatin and/or doxorubicin (Dox)). Chemotherapy is often rendered ineffective by multidrug resistance. Previous studies have revealed that Dox conjugated with 4 nm glutathione-stabilized gold nanoparticles (Au-GSH-Dox) enhanced the anti-tumor activity and cytotoxicity of Dox in Dox-resistant feline fibrosarcoma cell lines exhibiting high P-glycoprotein (P-gp) activity. The present study investigated the influence of Au-GSH-Dox on the canine OSA cell line D17 and its relationship with P-gp activity. A human Dox-sensitive OSA cell line, U2OS, served as the negative control. Au-GSH-Dox, compared to free Dox, presented a greater cytotoxic effect on D17 (IC50 values for Au-GSH-Dox and Dox were 7.9 µg/mL and 15.2 µg/mL, respectively) but not on the U2OS cell line. All concentrations of Au-GSH (ranging from 10 to 1000 µg/mL) were non-toxic in both cell lines. Inhibition of the D17 cell line with 100 µM verapamil resulted in an increase in free Dox but not in intracellular Au-GSH-Dox. The results indicate that Au-GSH-Dox may act as an effective drug in canine OSA by bypassing P-gp.

Distribution of Glutathione-Stabilized Gold Nanoparticles in Feline Fibrosarcomas and Their Role as a Drug Delivery System for Doxorubicin-Preclinical Studies in a Murine Model.

Feline injection site sarcomas (FISS) are malignant skin tumors with high recurrence rates despite the primary treatment of radical surgical resections. Adjunctive radiotherapy or chemotherapy with doxorubicin is mostly ineffective. Cellular and molecular causes of multidrug resistance, specific physio-chemical properties of solid tumors impairing drug transport, and the tumor microenvironment have been indicated for causing standard chemotherapy failure. Gold nanoparticles are promising imaging tools, nanotherapeutics, and drug delivery systems (DDS) for chemotherapeutics, improving drug transport within solid tumors. This study was conducted to assess the distribution of 4-nm glutathione-stabilized gold nanoparticles in FISS and their influence on kidney and liver parameters in nude mice. The role of gold nanoparticles as a doxorubicin DDS in FISS was examined to determine the potential reasons for failure to translate results from in vitro to in vivo studies. Grade III tumors characterized by a large area of necrosis at their core displayed positive immuneexpression of tumor-associated macrophages (TAM) at both the periphery and within the tumor core near the area of necrosis. Gold nanoparticles did not cause necrosis at the injection site and had no negative effect on liver and kidney parameters in nude mice. Gold nanoparticles accumulated in the tumor core and at the periphery and co-internalized with TAM-an important observation and potential therapeutic target warranting further investigation. The large area of necrosis and high immunoexpression of TAM, indicating "pro-tumor macrophages", may be responsible for FISS tumor progression and therapeutic failure. However, further studies are required to test this hypothesis.

Proteomic Differences in Feline Fibrosarcomas Grown Using Doxorubicin-Sensitive and -Resistant Cell Lines in the Chick Embryo Model.

Proteomic analyses are rapid and powerful tools that are used to increase the understanding of cancer pathogenesis, discover cancer biomarkers and predictive markers, and select and monitor novel targets for cancer therapy. Feline injection-site sarcomas (FISS) are aggressive skin tumours with high recurrence rates, despite treatment with surgery, radiotherapy, and chemotherapy. Doxorubicin is a drug of choice for soft tissue sarcomas, including FISS. However, multidrug resistance is one of the major causes of chemotherapy failure. The main aim of the present study was to identify proteins that differentiate doxorubicin-resistant from doxorubicin-sensitive FISS using two-dimensional gel electrophoresis (2DE), followed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using the three-dimensional (3D) preclinical in ovo model, which resembles features of spontaneous fibrosarcomas, three significantly (p ≤ 0.05) differentially expressed proteins were identified in tumours grown from doxorubicin-resistant fibrosarcoma cell lines (FFS1 and FFS3) in comparison to the doxorubicin-sensitive one (FFS5): Annexin A5 (ANXA5), Annexin A3 (ANXA3), and meiosis-specific nuclear structural protein 1 (MNS1). Moreover, nine other proteins were significantly differentially expressed in tumours grown from the high doxorubicin-resistant cell line (FFS1) in comparison to sensitive one (FFS5). This study may be the first proteomic fingerprinting of FISS reported, identifying potential candidates for specific predictive biomarkers and research targets for doxorubicin-resistant FISS.

The Use of Liposomes and Nanoparticles as Drug Delivery Systems to Improve Cancer Treatment in Dogs and Cats.

BACKGROUND: Cancer remains a leading cause of death in companion animals. In human medicine, liposomes and nanoparticles have been extensively investigated as drug delivery systems (DDS) for anticancer agents due to their ability to target cancerous cells and reduce the negative side effects of free cytostatic drugs. In this review, the authors discuss the results of clinical trials using liposomes and polymer-based nanoparticles as DDS to improve cancer treatment in dogs and cats, indicating which ones seem worth further evaluation. The authors then overview ongoing animal cancer clinical trials, evaluating nano-DDS registered on the American Veterinary Medical Association Animal Health Studies Database. Finally, the authors indicate the nano-drugs that require further in vivo evaluation based on the encouraging results obtained from in vitro studies. CONCLUSIONS: Liposomes have been the most investigated nano-DDS in veterinary medicine. The lack of cardiotoxicity of the commercially available liposomal doxorubicin (Doxil/Caelyx) suggests it should be used in dogs with cardiac disorders, rather than using free doxorubicin. Cisplatin-incorporated hyaluronic acid nanoparticles, nanocrystals of cisplatin, and paclitaxel are the most promising nano-drugs for potent applications in treating various canine cancers (e.g. oral melanoma, oral sarcoma, and anal gland adenocarcinoma) and their translation into the treatment of human diseases.

Current knowledge on feline injection-site sarcoma treatment.

Feline injection-site sarcomas (FISS) are malignant skin tumours of mesenchymal origin, the treatment of which is a challenge for veterinary surgeons. The role of surgery, radiotherapy and chemotherapy in FISS treatment has been studied, and a correlation between "clean" surgical margins and disease-free survival has been shown. In addition, clean surgical margins are one of the most important factors for achieving a low recurrence rate. The most effective method of FISS treatment includes combining radical surgery with pre- or postoperative radiotherapy. Chemotherapy may be used as a palliative method of treatment or may be considered an adjunctive therapy for surgery and radiotherapy. In cats with FISS without metastasis, the use of immunostimulant treatment with Oncept IL-2, intended as a complementary immunotherapy in association with surgery and brachytherapy, may also be considered to reduce the risk of relapse and increase the time to relapse. Additionally, this review focuses on recent advances in FISS treatment, including the use of novel compounds, such as doxorubicin conjugated to glutathione-stabilized gold nanoparticles, liposomal doxorubicin or tyrosine kinase inhibitors.

3D chick embryo chorioallantoic membrane model as an in vivo model to study morphological and histopathological features of feline fibrosarcomas.

BACKGROUND: The chick embryo chorioallantoic membrane (CAM) model is well described in human medicine as a cost-effective, easy to perform preclinical oncological model for observing pro- and antiangiogenic response, tumor biology and metastasis. The main objective of this article was to present the modification of the CAM assay in order to evaluate tumor growth from two feline fibrosarcoma cell lines (FFS1, FFS3) and describe their morphological and histopathological features. RESULTS: The authors described morphological and histopathological features of two feline fibrosarcoma cell lines (FFS1 and FFS3) grown on the CAM. Tumors from the FFS1 cell line showed high malignancy (grade III), while tumors from the FFS3 cell line were grade II. Proliferation markers (Ki-67 and PCNA) were determined and the positive correlation between PCNA and tumor grade (r = 0.8247; p < 0.001) was demonstrated, as opposed to Ki-67. CONCLUSIONS: The results obtained indicate that PCNA may be helpful to evaluate the tumor grade, better than Ki-67, for feline fibrosarcomas. However, further investigations of proliferation marker, in bigger number of feline spontaneous fibrosarcomas and feline fibrosarcomas grown on the CAM from different cell lines, are needed to confirm these observations.

Experimental tumor growth of canine osteosarcoma cell line on chick embryo chorioallantoic membrane (in vivo studies).

The chick embryo chorioallantoic membrane (CAM) model is extensively used in human medicine in preclinical oncological studies. The CAM model has several advantages: low cost, simple experimental approach, time saving and following "3R principles". Research has shown that the human osteosarcoma cell lines U2OS, MMNG-HOS, and SAOS can form tumors on the CAM. In veterinary medicine, this has been described only for feline fibrosarcomas, feline mammary carcinomas and canine osteosarcomas. However, in case of canine osteosarcomas, it has been shown that only non-adherent osteosarcoma stem cells isolated from KTOSA5 and CSKOS cell lines have the ability to form microtumors on the CAM after an incubation period of 5 days, in contrast to adherent KTOSA5 and CSKOS cells. In the presented study, we have proven that the commercial adherent canine osteosarcoma cell line (D-17) can form vascularized tumors on the CAM after the incubation period of 10 days.

Doxorubicin Conjugated to Glutathione Stabilized Gold Nanoparticles (Au-GSH-Dox) as an Effective Therapeutic Agent for Feline Injection-Site Sarcomas-Chick Embryo Chorioallantoic Membrane Study.

Feline injection-site sarcomas are malignant skin tumours with a high local recurrence rate, ranging from 14% to 28%. The treatment of feline injection-site sarcomas includes radical surgery, radiotherapy and/or chemotherapy. In our previous study it has been demonstrated that doxorubicin conjugated to glutathione-stabilized gold nanoparticles (Au-GSH-Dox) has higher cytotoxic effects than free doxorubicin for feline fibrosarcoma cell lines with high glycoprotein P activity (FFS1, FFS3). The aim of the present study was to assess the effectiveness of intratumoural injection of Au-GSH-Dox on the growth of tumours from the FFS1 and FFS3 cell lines on chick embryo chorioallantoic membrane. This model has been utilized both in human and veterinary medicine for preclinical oncological studies. The influence of intratumoural injections of Au-GSH-Dox, glutathione-stabilized gold nanoparticles and doxorubicin alone on the Ki-67 proliferation marker was also checked. We demonstrated that the volume ratio of tumours from the FFS1 and FFS3 cell lines was significantly (p < 0.01) decreased after a single intratumoural injection of Au-GSH-Dox, which confirms the positive results of in vitro studies and indicates that Au-GSH-Dox may be a potent new therapeutic agent for feline injection-site sarcomas.

Enhancing anti-tumor efficacy of Doxorubicin by non-covalent conjugation to gold nanoparticles - in vitro studies on feline fibrosarcoma cell lines.

BACKGROUND: Feline injection-site sarcomas are malignant skin tumors of mesenchymal origin, the treatment of which is a challenge for veterinary practitioners. Methods of treatment include radical surgery, radiotherapy and chemotherapy. The most commonly used cytostatic drugs are cyclophosphamide, doxorubicin and vincristine. However, the use of cytostatics as adjunctive treatment is limited due to their adverse side-effects, low biodistribution after intravenous administration and multidrug resistance. Colloid gold nanoparticles are promising drug delivery systems to overcome multidrug resistance, which is a main cause of ineffective chemotherapy treatment. The use of colloid gold nanoparticles as building blocks for drug delivery systems is preferred due to ease of surface functionalization with various molecules, chemical stability and their low toxicity. METHODS: Stability and structure of the glutathione-stabilized gold nanoparticles non-covalently modified with doxorubicin (Au-GSH-Dox) was confirmed using XPS, TEM, FT-IR, SAXRD and SAXS analyses. MTT assay, Annexin V and Propidium Iodide Apoptosis assay and Rhodamine 123 and Verapamil assay were performed on 4 feline fibrosarcoma cell lines (FFS1WAW, FFS1, FFS3, FFS5). Statistical analyses were performed using Graph Pad Prism 5.0 (USA). RESULTS: A novel approach, glutathione-stabilized gold nanoparticles (4.3 +/- 1.1 nm in diameter) non-covalently modified with doxorubicin (Au-GSH-Dox) was designed and synthesized. A higher cytotoxic effect (p<0.01) of Au-GSH-Dox than that of free doxorubicin has been observed in 3 (FFS1, FFS3, FFS1WAW) out of 4 feline fibrosarcoma cell lines. The effect has been correlated to the activity of glycoprotein P (main efflux pump responsible for multidrug resistance). CONCLUSIONS: The results indicate that Au-GSH-Dox may be a potent new therapeutic agent to increase the efficacy of the drug by overcoming the resistance to doxorubicin in feline fibrosarcoma cell lines. Moreover, as doxorubicin is non-covalently attached to glutathione coated nanoparticles the synthesized system is potentially suitable to a wealth of different drug molecules.

Functioning unilateral adrenocortical carcinoma in a dog.

An 11-year-old, 24-kg, intact female Siberian husky dog in anestrus had a 2-month history of polyuria and polydipsia. The dog had signs of mineralocorticoid excess such as hypertension and hypokalemia refractory to potassium supplementation. Abdominal ultrasound revealed an irregular mass in the left adrenal gland. The ACTH stimulation test for aldosterone concentration did not reveal hyperaldosteronism. Unilateral adrenalectomy was performed and histopathology identified adrenal cortical carcinoma. All clinical signs of mineralocorticoid excess ceased after surgery.