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1.
Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumour size, presence of metastatic disease and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behaviour of osteosarcoma cells differ based on serum ALP concentration. Here, we report on the generation of six canine osteosarcoma cell lines from osteosarcoma‐bearing dogs with differences in serum ALP concentration. To determine whether in vitro behaviour differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP, assays were performed to evaluate proliferation, migration, invasion and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion or chemosensitivity between cell lines associated with normal or increased serum ALP concentration.  相似文献   

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3.
Osteosarcoma is the most common primary malignancy of bone in dogs and is associated with poor long‐term outcomes due to its highly metastatic nature. A better understanding of the signalling pathways and proteins involved with osteosarcoma pathogenesis may aid in improved outcomes through the use of targeted therapies. The Wnt5a protein, a ligand for the non‐canonical Wnt signalling pathway, is implicated in mediating the aggressiveness of cancer cell lines, including those of human osteosarcoma origin. Given the close relationship between human and canine osteosarcoma, the primary goal of this study was to characterize Wnt5a expression in canine osteosarcoma. Second, if Wnt5a expression was present in canine osteosarcoma, the study aimed to determine any potential association with clinical outcome and clinical variables in similarly treated osteosarcoma‐bearing dogs. Wnt5a expression was present in 26 of the 48 (54%) cases of canine osteosarcoma. Wnt5a expression was not associated with progression‐free survival (P = 0.4) or overall survival (P = 0.1).  相似文献   

4.
The anticancer activity of novel platinum derivative, a complex of platinum with tris(2‐carboxyethyl)phosphine (Pt‐TCEP), has been evaluated in canine (D‐17) and human osteosarcoma (U2‐OS) cell lines. Viability of cells after incubation for 24 or 72 hours with increasing concentrations (0.625, 1.25, 2.50, 5, 10 and 20 μM) of Pt‐TCEP was tested in an MTT assay and compared to effect of cisplatin. Longer‐term effect of Pt‐TCEP was evaluated in the colony‐forming unit assay after 24 hours exposure to the Pt‐TCEP (2 and 3 μM) and subsequent incubation for 2 weeks. The influence of the compound on the cell cycle was measured after 24 hours treatment with Pt‐TCEP (3 μM). Its pro‐apoptotic activity was examined after 24 hours treatment with Pt‐TCEP (1.25, 2.50, 5, 10 and 20 μM) using flow cytometry. Expression of main proteins involved in apoptosis was measured after exposure for 24 hours to 3 or 5 μM Pt‐TCEP in Western Blot. The compound much more effectively decreased cell viability than cisplatin in case of both cell lines. IC50 of Pt‐TCEP was 5.93 ± 0.12 in D‐17 and 3.45 ± 0.14 in U2‐OS cell lines after 24 hours, and 1.77 ± 0.14 in D‐17 and 1.53 ± 0.11 in U2‐OS after 72 hours (P < .05). The compound arrested cells in the G2/M phase and inhibited the ability of cells to form colonies. Pt‐TCEP induced caspase‐dependent apoptosis. The expression of the anti‐apoptotic Bcl‐XL protein was decreased after Pt‐TCEP treatment in both cell lines. The results confirmed anti‐cancer activity of Pt‐TCEP against canine and human osteosarcoma cell lines.  相似文献   

5.
Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.  相似文献   

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7.
Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), recombinant human transforming growth factor (rhTGF)-beta 1 and recombinant human bone morphogenetic protein (rhBMP)-2 on differentiation in four different canine osteosarcoma cell lines (POS53B, 53C, 53D and 14A) were examined using markers specifically expressed by phenotypic osteoblasts. 1,25(OH)2D3 increased alkaline phosphatase (ALP) activity in one cell line, osteocalcin production in two lines and type I collagen production in three lines. RhTGF-beta 1 increased ALP activity in one clonal cell, osteocalcin production in one clonal cell and type I collagen production in two clonal cells. RhBMP-2 increased ALP activity in all clonal cells, osteocalcin production in two clonal cells and type I collagen production in three clonal cells. Thus, these agents induced differentiation in osteosarcoma cells at different efficacies. Electron microscopic study revealed that these agents increased cellular activity in all cell lines with no evidence of degeneration of cell organelle by drug cytotoxicity. In some cultures treated with either 1,25(OH)2D3 or rhBMP-2, apoptotic cells were observed. Based on the change in markers, rhBMP-2 and 1,25(OH)2D3 seemed to be more effective than rhTGF-beta 1. These agents are potential inducers of apoptosis.  相似文献   

8.
The receptor tyrosine kinase Met is dysregulated in several human cancers including osteosarcoma (OSA) in which overexpression is a negative prognostic indicator and enforced Met expression in normal osteoblasts leads to genomic instability and malignant transformation. Met is also known to be inappropriately expressed in canine OSA tumour samples and cell lines. The purpose of this study was to evaluate the potential utility of an orally bioavailable small molecule Met inhibitor, PF2362376, against canine OSA cell lines as a prelude to future clinical work. PF2362376 inhibited phosphorylation of Met, Gab‐1, Erk and Akt, but not of Src or STAT3. Furthermore, PF2362376 inhibited proliferation of canine OSA cell lines and induced cell death at biologically achievable concentrations. Last, activities associated with Met signalling including migration, invasion, branching morphogenesis and colony formation in soft agar were blocked by PF2362376. These studies support the notion that Met is a relevant target for therapeutic intervention in OSA.  相似文献   

9.
The radiosensitizing effect of inducible nitric oxide synthase (iNOS) was evaluated, in vitro, in a feline vaccine‐associated sarcoma (VAS) cell line and a canine osteosarcoma cell line (D17). The gene encoding the human iNOS was cloned into an expression plasmid under the control of a cytomegalovirus immediate early promoter. Transient transfections were performed in feline VAS cells and D17 cells. Nitric oxide was measured in the supernatant media 48 h later as an indirect measurement of iNOS expression. Cells were irradiated using cobalt‐60 under hypoxic or oxic conditions, and clonogenic assays were used to evaluate the effects of gene transfer on the sensitivity of cells to radiation. The results demonstrated that iNOS had no significant effect on improving the radiosensitivity of cells under oxic conditions. However, under hypoxic conditions, iNOS gene transfer significantly improved radiosensitization in osteosarcoma cells. These results demonstrate the feasibility of improving the outcome of radiotherapy in dogs with large bulky tumours using iNOS gene therapy.  相似文献   

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11.
We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half‐maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152‐induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi‐agent protocols is warranted.  相似文献   

12.
Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti‐inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage‐independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response.  相似文献   

13.
Non‐adherent, 3‐dimensional sphere formation is used as an in vitro surrogate to evaluate cellular potential for tumour initiation and self‐renewal. To determine if a shared molecular program underlies the capacity for sphere formation by cells originating from diverse tumour types, we characterized molecular and functional properties of 10 independent cell lines derived from 3 ontogenetically distinct dog cancers: hemangiosarcoma, osteosarcoma and glial brain tumours. Genome‐wide gene expression profiling identified tumour‐of‐origin‐dependent patterns of adjustment to sphere formation in a uniform culture condition. However, expression of the stem/progenitor markers CD34 and CD117, resistance to cytotoxic drugs and dye efflux (side population assays) showed no association with these gene expression profiles. Instead, primary sphere‐forming capacity was inversely correlated with the ability to reform secondary spheres, regardless of tumour ontogeny. Primary sphere formation seemed to be proportional to the number of pre‐existing cells with sphere‐forming capacity in the cell lines. Cell lines where secondary sphere formation was more proficient than primary sphere formation showed enrichment of genes involved in fatty acid synthesis and immunosuppressive cytokines. In contrast, cell lines where secondary sphere formation was approximately equivalent to or less proficient than primary sphere formation showed upregulation of CD40 and enrichment of genes involved in fatty acid oxidation. Our data suggest that in vitro sphere formation is associated with upregulation of gene clusters involved in metabolic and immunosuppressive functions, which might be necessary for self‐renewal and for tumour initiation and/or tumour propagation in vivo.  相似文献   

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15.
The prevalence of cancer in animals has increased significantly over the years. Mammary tumours are the most common neoplasia in dogs, in which around 50% are presented in the malignant form. Hence, the development and characterization of in vitro models for the study of canine tumours are important for the improvement of cancer diagnosis and treatment. Thus, the aim of this study was to characterize cell lines derived from canine mammary gland neoplasias which could be further used for basic and applied oncology research. Samples of canine mammary carcinomas were taken for cell culture and 2 cell lines were established and characterized in terms of cell morphology, tumourigenicity and global gene expression. Both cell lines presented spindle‐shape morphology and shown common malignant features as in vitro invasion potential and expression of epithelial and mesenchymal proteins. Also, we found gene expression patterns between the 2 cell cultures in comparison to the normal mammary gland tissue. Cells from M25 culture showed a higher invasion and in vivo tumourigenic potential, associated to the overexpression of genes involved in focal adhesion and extracellular matrix communication, such as FN1, ITGA8 and THBS2. The phenotypic characterization of these cells along with their global gene expression profile potentially determine new therapeutic targets for mammary tumours.  相似文献   

16.
OBJECTIVE: To evaluate the biological activity of dihydroartemisinin on canine osteosarcoma cell lines in vitro. SAMPLE POPULATION: 4 canine osteosarcoma cell lines. PROCEDURES: Cell viability assays were performed on canine osteosarcoma cell lines OSCA2, OSCA16, OSCA50, and D17 after 24, 48, and 72 hours of treatment with dihydroartemisinin at concentrations of 0.1 to 100 microM. Apoptosis was assessed by use of an ELISA for free nuclosomal DNA fragmentation and by western blot analysis for cleavage of caspase 3. Cell cycle analysis was performed by use of staining with propidium iodide and flow cytometry. Detection of reactive oxygen species (ROS) was conducted in the D17 cell line by use of 6-carboxy-2',7'-dihydrofluorescein diacetate and flow cytometry. RESULTS: The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was achieved in all 4 canine osteosarcoma cell lines and ranged from 8.7 to 43.6 microM. Induction of apoptosis was evident as an increase in nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the population in the sub G0/G1 phase of the cell cycle detected by flow cytometry. Exposure to dihydroartemisinin also resulted in a decrease in the G0/G1 population. Iron-dependent generation of ROS was detected in dihydroartemisinin-treated D17 cells; ROS generation increased in a dose-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Incubation with dihydroartemisinin resulted in biological activity against canine osteosarcoma cell lines, which included induction of apoptosis and arrest of the cell cycle. Clinical trials of dihydroartemisinin in dogs with osteosarcoma should be conducted.  相似文献   

17.
Background: Spirocerca lupi is a nematode of Canidae that matures within the esophageal wall to form fibroblastic nodules with potential for malignant transformation. Diagnosis is based on histopathologic examination, but false‐negative results may be obtained from samples collected by endoscopy. Serum alkaline phosphatase (ALP) activity, frequently increased in hepatobiliary disease, is also increased in a variety of neoplastic conditions in dogs, including appendicular osteosarcoma, and has also been reported to be increased in dogs with spirocercosis. Objective: The aim of this study was to evaluate serum ALP activity as a marker for malignant transformation of esophageal nodules in S. lupi‐infected dogs. Methods: In this retrospective study, medical records of dogs diagnosed with spirocercosis from 1991 to 2008 were reviewed, and serum ALP activity determined at presentation was compared between dogs with nonneoplastic and neoplastic nodules. Owing to use of multiple analyzers, ratios of ALP activity to the upper reference interval for ALP were calculated and compared. Results: Median ALP activity ratios were 0.65 (0.07–4.00) and 0.86 (0.10–3.40) for dogs with nonneoplastic (n=88) and neoplastic (n=32) nodules, respectively, with no significant difference (P=.18) and substantial overlap between groups. Tumors included osteosarcoma (15 dogs), fibrosarcoma (15 dogs), and anaplastic sarcoma (2 dogs); there was no difference in ALP activity between the dogs with osteosarcoma and fibrosarcoma. Conclusion: ALP is a poor marker of malignant transformation in canine spirocercosis.  相似文献   

18.
Immunotherapies targeting checkpoint molecule programmed cell death 1 (PD‐1) protein were shown to be effective for treatment of non‐Hodgkin lymphoma in people, but little is known about the expression of PD‐1 or its ligand PD‐L1 by canine lymphoma. Therefore, flow cytometry was used to analyse expression of PD‐1 and PD‐L1 in canine lymphoma, using fine‐needle aspirates of lymph nodes from 34 dogs with B cell lymphoma (BCL), 6 dogs with T cell lymphoma (TCL) and 11 dogs that had relapsed. Furthermore, fine‐needle aspirates were obtained from 17 healthy dogs for comparison. Lastly, the impact of chemotherapy resistance on expression of PD‐1 and PD‐L1 was assessed in vitro. These studies revealed increased expression of PD‐L1 by malignant B cells compared to normal B cells. In the case of TCL, tumour cells and normal T cells both showed low to negative expression of PD‐1 and PD‐L1. In addition, tumour infiltrating lymphocytes from both BCL and TCL had increased expression of both PD‐1 and PD‐L1 expression compared to B and T cells from lymph nodes of healthy animals. In vitro, chemotherapy‐resistant BCL and TCL cell lines exhibited increases in both PD‐1 and PD‐L1 expression, compared to non‐chemotherapy selected tumour cells. These findings indicate that canine lymphomas exhibit upregulated checkpoint molecule expression, though the impact of checkpoint molecule expression on tumour biological behaviour remains unclear.  相似文献   

19.
Introduction: Pamidronate has been traditionally used to manage pathologic osteoclastic disorders. In addition to its effects on osteoclasts, pamidronate has also been demonstrated to promote phenotypic maturation and inhibition of proliferation in osteoblasts. Canine osteosarcoma (OSA) consists of malignant, undifferentiated osteoblasts. The objective of this study was to determine if micromolar concentrations of pamidronate could induce malignant osteoblastic differentiation as evaluated by an increase in alkaline phosphatase (ALP) activity and/or osteocalcin (OC) production, two specific markers of normal osteoblastic activity. Methods: Two canine OSA cell lines (HMPOS and COS 31) were used for all experiments. Cells were incubated for 48 or 72 hours with various pamidronate concentrations (0, 0.1, 1, 5, 10, and 20 μM). After incubation, the supernatants were sampled and the relative amounts of viable cells were determined with a cell proliferation assay (Cell Titer 96® AQuous, Promega). An ALP detection kit (Starbright®, Sigma®) was used to measure the ALP activity and an ELISA (Osteocalcin EIA kit, Biomedical Technologies) was used to determine the concentration of osteocalcin in the supernatants. The ALP and osteocalcin values were corrected for the amount of viable cells. Results: Pamidronate induced a dose‐dependent reduction in the number of viable COS 31 and HMPOS cells at both 48 and 72 hours. A dose‐dependent elevation in ALP activity from baseline was observed. At 20 μM, a 2.3‐fold increase was observed for HMPOS at 72 hours, while a 1.43‐fold increase was observed for COS 31 at 72 hours. Very low level (less than 2 ng/ml) of osteocalcin pre‐ and post‐pamidronate treatment was detected for both COS 31 and HMPOS. Conclusion: The data suggests that pamidronate increases alkaline phosphatase activity in canine OSA cells in a dose‐dependent manner. However, cytotoxic assays are needed in order to accurately characterize any concurrent decrease in the number of viable cells. The potential differentiating effect of pamidronate on malignant osteoblasts provides an additional argument for its use in the palliative treatment of OSA.  相似文献   

20.
Since William Coley utilized bacterial immunotherapy to treat sarcomas in the late 19th century, an association between infection and improved survival has been reported for human and canine osteosarcoma patients. One of the reasons for this improved survival is likely a reactivation of the host immune system towards an inflammatory anti‐tumour response, and one of the key players is the macrophage. Yet, despite their importance, the response of macrophages to infectious agents in the context of osteosarcoma has not been thoroughly evaluated. The aim of this study was to evaluate how in vitro exposure to a bacterial agent (Staphylococcus aureus) influenced canine and human macrophage differentiation in the presence of osteosarcoma. Our hypothesis was that S. aureus would, in the presence of osteosarcoma, induce a macrophage phenotype with significantly increased inflammatory signatures. Consistent with our hypothesis, human macrophages co‐cultured with osteosarcoma and S. aureus exhibited increased IFN‐γ, TNF‐α and IL‐12p70 cytokine secretion, decreased TGF‐β cytokine secretion and increased mRNA expression of TNF‐α when compared with macrophages co‐cultured with osteosarcoma and to macrophages cultured alone. Canine macrophages similarly exhibited increased IFN‐γ and TNF‐α cytokine secretion, decreased TGF‐β cytokine secretion, increased mRNA expression of TNF‐α and increased surface receptor expression of CD80 when co‐cultured with osteosarcoma and S. aureus. Collectively, the findings of this study suggest that infection upregulates the inflammatory immune response to counteract osteosarcoma‐induced immune suppression. This work informs a potential therapeutic strategy to optimize inflammatory stimuli for triggering an anti‐osteosarcoma macrophage response.  相似文献   

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