The effect of deracoxib and piroxicam on viability of canine osteosarcoma cells in vitro

Introduction:  Cyclooxygenase‐2 (COX‐2) inhibitors are being used increasingly in cancer therapy. Although the effects of COX‐2 inhibitors have been evaluated extensively in carcinomas, less is known about their effects in sarcomas. Since the majority of dogs with appendicular osteosarcoma (OSA) are treated for pain with a non‐steroidal anti‐inflammatory drug (some COX‐2 selective) prior to definitive treatment, it is important to determine the effects that commonly used NSAIDS have on tumor cell growth.
Methods:  Established canine osteosarcoma (POS, HMPOS and COS31) and canine fibroblast cell lines were maintained in culture under standard conditions. Cells were incubated with either deracoxib (1 uM to 500 uM) or piroxicam (1 uM to 1000 uM). Cell viability was assessed at 72 hours by cell counts and the MTT assay. The DNA fragmentation analysis was utilized to assess for apoptosis induction.
Results:  Deracoxib concentrations ≥100 uM and piroxicam concentrations ≥500 uM significantly reduced mean cell viability of all three OSA cell lines (lowest cell viability percentages 20% and 32%, respectively). Deracoxib concentrations ≥250 uM and piroxicam concentrations ≥500 uM also reduced viability of fibroblasts; however, the cell viability percent was reduced to only 54% and 68%, respectively, of the control value. Exposure of OSA cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation.
Conclusions:  Deracoxib and piroxicam demonstrated a cytotoxic effect on canine osteosarcoma cells. There was no evidence of apoptosis induction at the concentrations evaluated. Further investigation will need to be performed to determine whether either drug exhibits anti‐tumor effects in vivo .     

Investigation of the effect of pamidronate disodium on the in vitro viability of osteosarcoma cells from dogs

OBJECTIVE: To determine the effect of pamidronate disodium on the in vitro viability of osteosarcoma cells and non-neoplastic cells from dogs. SAMPLE POPULATION: 3 osteosarcoma and 1 fibroblast cell lines derived from dogs. PROCEDURE: Cell counts and cell viability assays were performed in cultures of osteosarcoma cells (POS, HMPOS, and COS31 cell lines) and fibroblasts after 24, 48, and 72 hours of incubation with pamidronate at concentrations of 0.001 to 1000 microM or with no drug (control treatment). Percentage viability was determined in cell samples for each concentration of pamidronate and each incubation time. A DNA fragmentation analysis was performed to assess bisphosphonate-induced apoptosis. RESULTS: Osteosarcoma cell viability decreased significantly in a concentration- and time-dependent manner at pamidronate concentrations ranging from 100 to 1000 microM, most consistently after 48 and 72 hours' exposure. In treated osteosarcoma cells, the lowest percentage cell viability was 34% (detected after 72 hours' exposure to 1000 microM pamidronate). Conversely, 72 hours' exposure to 1000 microM pamidronate did not significantly reduce fibroblast viability (the lowest percentage viability was 76%). After 72 hours of exposure, pamidronate did not cause DNA fragmentation in POS or HMPOS cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that pamidronate may have the potential to inhibit osteosarcoma growth in dogs, possibly through a nonapoptotic mechanism. The clinical relevance of these in vitro findings remains to be determined, but administration of pamidronate may potentially be indicated as an adjuvant treatment in chemotherapeutic protocols used in dogs.     

Evaluation of the effects of histone deacetylase inhibitors on cells from canine cancer cell lines

OBJECTIVE: To determine whether exposure of canine cancer cells to histone deacetylase (HDAC) inhibitors S(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)benzamide (OSU-HDAC42) or suberoylanilide hydroxamic acid (SAHA) results in increased histone acetylation and decreased cell viability and whether any changes in viability involve induction of apoptosis or alterations in progression of the cell cycle. Sample POPULATION: 9 canine cancer cell lines. PROCEDURES: Cells from 9 canine cancer cell lines were treated with dimethyl sulfoxide vehicle, OSU-HDAC42, or SAHA, then assays of cell viability were performed. Histone acetylation was assessed by use of western blot analysis. Apoptosis was assessed via ELISA to detect fragmentation of cytoplasmic nucleosomal DNA and western blot analysis to detect cleavage of caspase 3. Cell cycle analysis was performed by use of propidium iodide staining and flow cytometry. RESULTS-Concentrations of OSU-HDAC42 and SAHA required to achieve 50% inhibition of cell viability (IC(50)) were reached in cells of 6 and 4 canine cancer cell lines, respectively, and ranged from approximately 0.4 to 1.3 microM for OSU-HDAC42 and 0.6 to 4.8 microM for SAHA. Cells from T-cell lymphoma, mast cell tumor, osteosarcoma, and histiocytic sarcoma lines were most sensitive to HDAC inhibition, with IC(50)s of < 1 microM for OSU-HDAC42 and < 5 microM for SAHA. Induction of apoptosis was indicated via cleavage of caspase 3 and increases in cytoplasmic nucleosomes and the subG(1) cell population. CONCLUSIONS AND CLINICAL RELEVANCE: Micromolar concentrations of HDAC inhibitors OSU-HDAC42 and SAHA induced histone acetylation, cytotoxicity, and apoptosis in canine cancer cells. In general, OSU-HDAC42 was more potent than SAHA.     

Biological activity of dihydroartemisinin in canine osteosarcoma cell lines

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.     

The in vitro effects of piroxicam and meloxicam on canine cell lines

OBJECTIVES: To analyse the direct antiproliferative effects of both piroxicam and meloxicam at a variety of concentrations on a series of canine cancer cell lines and the mechanism of cell death. METHODS: The in vitro effects of piroxicam and meloxicam at various concentrations on canine cell cultures (Madin-Darby canine kidney cells, osteosarcoma, mammary carcinoma, and lymphoma) were assessed with respect to proliferation inhibition and apoptosis induction. Western blot analysis of cyclooxygenase-1 and cyclooxygenase-2 expression was performed on all cell lines. RESULTS: All cell lines used in this study were cyclooxygenase-1 and cyclooxygenase-2 positive apart from Madin-Darby canine kidney cells which were negative for both cyclooxygenase-1 and cyclooxygenase-2. Both meloxicam and piroxicam were able to inhibit proliferation in cell lines in a dose-dependent manner. However, the drug concentration required for a given effect was cell line dependent. CLINICAL SIGNIFICANCE: The results suggest that significant inhibition of proliferation and induction of apoptosis would only occur when drug concentrations were in excess of those that can be achieved in vivo following maximum recommended dose rates. It is possible, however, that local or topical treatment or altered dosing regimens may offer alternative approaches to the use of these drugs as antineoplastic agents.     

The second-generation curcumin analogue RL71 elicits G2/M cell cycle arrest and apoptosis in canine osteosarcoma cells

Canine osteosarcoma is an aggressive cancer, comprising 85% of canine bone neoplasms. Current treatment practices of surgery and chemotherapy increase 1-year survival by only 45%. The curcumin analogue RL71, has demonstrated potent in vitro and in vivo efficacy in several models of human breast cancer through increased apoptosis and cell cycle arrest. Thus, the present study aimed to investigate efficacy of curcumin analogues in two canine osteosarcoma cell lines. Osteosarcoma cell viability was assessed using the sulforhodamine B assay and mechanisms of action were determined by analysing the levels of cell cycle and apoptotic regulatory proteins via Western blotting. Further evidence was obtained using flow cytometry to detect cell cycle distribution and the number of apoptotic cells. RL71 was the most potent curcumin analogue with EC₅₀ values of 0.64 ± 0.04 and 0.38 ± 0.009 μM (n = 3) in D-17 (commercial) and Gracie canine osteosarcoma cells, respectively. RL71 significantly increased the ratio of cleaved-caspase 3 to pro-caspase 3 and the level of apoptotic cells at the 2× and 5× EC₅₀ concentration (p < 0.001, n = 3). Furthermore, at the same concentration, RL71 significantly increased the number of cells in the G2/M phase. In conclusion, RL71 has potent cytotoxic activity in canine osteosarcoma cells triggering G2/M arrest and apoptosis at concentrations achievable in vivo. Future research should further investigate molecular mechanisms for these changes in other canine osteosarcoma cell lines prior to in vivo investigation.     

Cytotoxic Effects of Loperamide Hydrochloride on Canine Cancer Cells

Loperamide is a peripheral opiate agonist that can cause apoptosis and G2/M arrest in human cancer cell lines and may sensitize cells to chemotherapy. The objectives of this study were to investigate the effects of loperamide on viability, apoptosis and cell cycle kinetics in canine cancer cells and to establish whether the drug sensitizes cells to doxorubicin. Cell viability was assessed using Alamar Blue. Cell death and cell cycle were studied using flow cytometry with 7-Aminoactinomycin-D (7-AAD) and propidium iodide (PI), respectively. Loperamide decreased cell viability in a dose-dependent fashion and was most effective against canine osteosarcoma cells. In all cell lines, it induced a dose and time dependent apoptosis and resulted in accumulation in G0/G1. When co-incubated with doxorubicin, loperamide induced a synergistic cell kill in canine carcinoma cells. Investigation is warranted into the role of loperamide in the treatment of canine cancer.     

In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type-1

OBJECTIVE: To establish the in vitro efficacy of 4 novel drugs (ie, ganciclovir, cidofovir, penciclovir, and foscarnet) against feline herpesvirus type-1 (FHV-1) and compare their antiviral efficacy with that of acyclovir and idoxuridine. SAMPLE POPULATION: Cultured Crandell-Reese feline kidney (CRFK) cells and FHV-1 strain 727 PROCEDURE: For each drug, antiviral effect was estimated by use of conventional plaque-reduction assays, and inhibitory concentration 50 (IC50; drug concentration at which plaque numbers were reduced by 50% relative to the number of plaques for nontreated control wells) was calculated. To determine whether observed antiviral effects were related to alterations in the number or viability of CRFK cells, cytotoxicity assays were performed at 1, 2, and 10 times the median IC50 for each antiviral drug. RESULTS: Median IC50 for each drug was as follows: ganciclovir, 5.2 microM; cidofovir, 11.0 microM; penciclovir, 13.9 microM; foscarnet, 232.9 microM; idoxuridine, 4.3 microM; and acyclovir, 57.9 microM. Obvious changes in morphologic characteristics, confluence, or viability of CRFK cells were not observed at concentrations up to and including 2 times the IC50 for each drug. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro efficacy of idoxuridine and ganciclovir against FHV-1 was approximately equivalent and about twice that of cidofovir and penciclovir. Foscarnet appeared to be comparatively ineffective. Given the reasonable clinical efficacy of idoxuridine in cats infected with FHV-1, clinical trials of ganciclovir, cidofovir, and penciclovir or their prodrug forms appear to be warranted.     

Anti‐Tumor Effect of Adenovirus‐Mediated P53 Gene Therapy on the Growth of Canine Osteosarcoma Transplanted into Nude Mice

Introduction:  It has been reported that 40–50% of canine osteosarcoma cases have p53 mutations. The p53 tumor supressor gene plays a central role in cell cycle regulation and induction of apoptosis. We previously showed that adenoviral vector expressing canine P53 (AxCA‐cp53) inhibited growth of cultured canine osteosarcoma cell lines. Here, we evaluated anti‐tumor effect of adenovirus‐mediated p53 gene therapy on the growth of canine osteosarcomas transplanted into nude mice.
Methods:  Nine nude mice were subcutaneously injected with cells of a canine osteosarcoma cell line (POS) having p53 gene mutation. The transplanted tumors formed into nude mice were injected with AxCA‐cp53, AxCA‐LacZ (adenovirus vector expressing LacZ) or PBS (3 mice each) 7 times during 15 days. Tumor sizes were measured every 3 days for 27 days after injection with the adenovirus vectors. Expression efficiency of the adenovirus‐mediated gene transfer was examined by X‐gal staining and P53 immunostaining. Effects of the P53 expression on cell cycle control were examined by RT‐PCR for expression of p21 gene downstream of P53.
Results:  Significant differences in the tumor size was observed between the transplanted osteosarcoma tissues injected with AxCA‐cp53 and those injected with AxCA‐LacZ or PBS. Expressions of LacZ and P53 were confirmed at the injection sites of the tumors. Moreover, p21 mRNA expression was shown to be induced in the AxCA‐cp53‐injected tumors, indicating the funciton of P53 to induce cell cycle arrest.
Conclusions:  Adenoviral vector expressing canine P53 inhibited the growth of canine ostersarcoma transplanted into nude mice.     

The effects of baicalein on canine osteosarcoma cell proliferation and death

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.     

Retinoid receptors and the induction of apoptosis in canine osteosarcoma cells

Retinoids, all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA), induced morphological changes and apoptosis-like cell death characterized by cell shrinkage, chromatin condensation and nuclear disintegration in three canine osteosarcoma cells, OOS, HOS and POS, at a concentration of 10(-5) M. Both retinoid receptors, RARs and RXRs, were identified in these cells. 9-cis-RA bound to both the RXRs and the RARs, whereas ATRA bound to only the RARs in these cells. Those results indicate that the induction of apoptosis in canine osteosarcoma cells may be mediated by the specific control of RARs and RXRs.     

Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.
Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC₅₀) of vincristine or doxorubicin.
Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC₅₀ of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.
Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.     

Effects of vitamin D and retinoids on the differentiation and growth in vitro of canine osteosarcoma and its clonal cell lines

Although canine osteosarcoma is one of the most malignant, aggressive and lethal neoplasms originating from undifferentiated bone cells, it may retain some capacity for normal differentiation. The purpose of this study was to ascertain if the residual capacity for differentiation could be used to suppress its malignant properties. We tested the efficacy of vitamin D and retinoids in inducing differentiation and inhibiting growth of the POS canine osteosarcoma and four of its clonal cell lines, POS 14A (fibroblast type), POS 53B (chondroblast type), POS 53C (undifferentiated type) and POS 53D (osteoblastic type). Treatment with 10(-10)to 10(-8)M concentrations of calcitriol, OCT, cholecalciferol, all-trans retinoic acid (ATRA) and 9-cis retinoic acid for 48-120 hours changed the morphology of POS, POS 53B, POS 53C and POS 53D cells to cells that were elongated and spindle-shaped. Increased number of cytoplasmic organelles and pronounced nuclear activities were induced by concentrations of 10(-8)M and 10(-7)M for 120 hours. All drugs at concentrations of 10(-10)to 10(-8)M for 72 hours inhibited POS growth dose-dependently. OCT significantly reduced the cell number in all cell lines when used at concentrations between 10(-9)and 10(-8)M for 72 hours and exerted significant anti-proliferative effects for eight days culture. This study demonstrated that changed morphology and inhibition of growth was induced by treatment of the cells with these vitamins, that the loss of control of differentiation in the neoplasia was not irreversible and that these drugs may be useful in the clinic.     

Differentiation induction of canine osteosarcoma cell lines by retinoids

The effect of two retinoids, all- trans and 9- cis retinoic acid, on the differentiation of three canine osteosarcoma cells (OOS, HOS, and POS) was examined using markers specifically expressed by phenotypic osteoblasts. Both retinoids induced morphologic differentiation in all the canine osteosarcoma cells. Retinoids enhanced cell flattening and spreading, as well as reduction in cell overlapping. Alkaline phosphatase (ALP) activity and ALP staining was enhanced in OOS, and HOS cells, but decreased in POS cells. These results may suggest that OOS and HOS cells have immature osteoblastic properties and POS cells have mature osteoblastic properties. Retinoids decreased osteocalcin production in all the osteosarcoma cells. They induced an increase in production of type I collagen in HOS and POS cells, but a decrease in OOS cells. These results indicate that retinoids induce differentiation of canine osteosarcoma cells, resulting in an altered expression of their malignant phenotype.     

Molecular characterisation of canine nonsteroidal anti-inflammatory drug-activated gene (NAG-1)

Nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1), a divergent member of the transforming growth factor beta superfamily, was previously identified as a gene induced by several anti-tumorigenic compounds, including NSAIDs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands in humans. In this study, canine NAG-1 was characterised from a canine genomic database. Gene induction by some NSAIDs and PPARgamma ligands was demonstrated in canine osteosarcoma cell lines. Phylogenetic analysis indicates that canine NAG-1 is more homologous with the corresponding mouse and rat genes than with human NAG-1. Expression of canine NAG-1 was increased by treatment with piroxicam and SC-560 (NSAIDs) and the PPARgamma ligand rosiglitazone. This study demonstrates that canine NAG-1 is up-regulated by some anti-tumorigenic compounds in osteosarcoma cell lines and may provide an important target of chemotherapy in canine cancer.     

Investigating the molecular mechanism of pamidronate‐induced in vitro cytotoxicity in canine osteosarcoma cells

Introduction: Pamidronate is used to treat metastatic bone lesions in human cancer patients. By directly inhibiting the mevalonate pathway, pamidronate disrupts GTPase‐binding protein prenylation, resulting in osteoclast apoptosis. Pamidronate has been demonstrated to exert dose‐ and time‐dependent cytotoxic effects in several canine malignant osteoblastic cell lines. However, the exact cytotoxic mechanism remains speculative. The purpose of this study was to investigate and characterize the molecular mechanism of pamidronate‐induced cytotoxicity in canine malignant osteoblasts. Methods: The involvement of farnesyl or geranylgeranyl pyrophosphate synthase inhibition was evaluated by performing rescue experiments with cells incubated for 48 hours with cytotoxic concentrations of pamidronate (50 and 100 μM), with or without farnesol (FOH) and geranylgeraniol (GGOH). Cell lysates were fluorometrically‐assessed for caspase‐3‐like activity (R&D Systems) following incubation with varying concentrations of pamidronate (control, 50 and 100 μM) for 48 hours. The expression of a prenylated GTPase protein, Rap1 A, was qualitatively evaluated with immunoblotting using a polyclonal goat antibody (Santa Cruz Biotechnology) in cells incubated for 48 hours with varying concentrations of pamidronate (0, 1, 10, 50, and 100 μM). Results: In cells treated with lower cytotoxic concentrations of pamidronate (50 μM) for 48 hours, the addition of GGOH, but not FOH, was able to diminish the degree cytotoxicity, p ≤ 0.05. However, cells incubated with higher cytotoxic concentrations of pamidronate (100 μM), neither GGOH nor FOH were able to reduce the level of pamidronate‐induced cytotoxicity. Caspase‐3‐like activity directly correlated with the degree of pamidronate‐induced cytotoxicity. Cells treated with pamidronate at 50 μM and 100 μM increased caspase‐3‐like activity by 5.3 and 7.1‐fold, respectively over untreated controls. The detection of Rap1A by immunoblotting requires further optimization. Conclusions: Pamidronate‐induced cytotoxicity in canine osteosarcoma cells may share a similar mechanism as has been demonstrated for osteoclasts. Inhibition of the mevalonate pathway appears to contribute to the observed cytotoxicity at lower doses of pamidronate. In addition, caspase‐3‐like activity contributes to the apoptotic process induced by pamidronate in malignant canine osteoblasts.     

Effects of Mycobacterial Cell Wall‐DNA Complex (MCC), Alendronate and Pamidronate on Canine Osteosarcoma Cell Lines

Introduction:  MCC, a cell wall composition prepared from Mycobacterium phlei ., inhibits the proliferation and induces apoptosis in a wide range of tumor cells. Bisphosphonates have been reported to inhibit the proliferation of canine osteosarcoma cell lines. In this study, we have determined the activity of MCC alone and in combination with the bisphosphonates alendronate and pamidronate on canine osteosarcoma cell lines.
Methods:  Canine osteosarcoma cell lines D17 and D22 were incubated with different concentrations of MCC (0.01–100 μg/ml) and suboptimal concentrations of alendronate and pamidronate for 72 hours. Cellular proliferation was measured by MTT reduction. Nuclear DNA condensation was determined using with Hoescht 33258 staining, and apoptosis by flow cytometry using active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies.
Results:  MCC inhibited the proliferation of both canine osteosarcoma D17 and D22 cell lines in a concentration‐dependent manner. The IC₅₀ for D17 cells was 3.9 μg/ml and for D22 cells 44.4 μg/ml. Cells incubated with 100 μg/ml MCC were positive for Hoescht staining, active caspase‐3 and cleaved PARP, indicative of cell death by apoptosis. The addition of alendronate or pamidronate was found to potentiate the apoptosis‐inducing activity of MCC. Maximal activity was observed when 5 μM alendronante or 10 μM pamidronate were used in combination with 100 μg/ml MCC.
Conclusion:  MCC inhibits the proliferation and induces apoptosis in canine osteosarcoma cell lines in vitro . This anticancer activity can be potentiated by the use of alendronate and pamidronate. These data support the development of MCC as a therapeutic agent for the treatment of canine osteosarcoma.     

Establishment and characterization of the growth and pulmonary metastasis of a highly lung metastasizing cell line from canine osteosarcoma in nude mice.

Highly lung metastasizing model of canine osteosarcoma in nude mice was established from five subcutaneous implantation cycles of lung tumor deposits. The selection of cells with increased metastatic properties from the parent POS canine osteosarcoma cell line recovered medium sized and polygonal Highly Metastasizing POS cells (HMPOS). The doubling time of HMPOS and POS in culture averaged 30 +/- 1.2 hr and 32 +/- 1.3 hr respectively, and their cell growth patterns in vitro were comparable to their in vivo growth patterns. HMPOS cells produced more tumor deposits (> 20 nodules, > 1 -mm in diameter) of various sizes with replacement of lung tissues at 12 weeks after implantation. POS cells produced fewer and smaller lung deposits (< 10 nodules, 1-mm in diameter). Tumor size and number of metastatic tumor deposits showed a regular association. HMPOS cells developed an osteoblastic type of cellular differentiation subcutaneously and in the lungs. HMPOS micrometastasis along the alveolar walls and blood vessels at 4 weeks averaged 6-7 small tumor locus. Each micrometastatic locus contained an average of 5-7 tumor cells, and developed a pleomorphic osteoblastic type of cellular differentiation. An average of 4 macrometastatic nodules could be seen at 6 weeks, composed of an average of 23 tumor cells, 10 nodules at 8 weeks, 12 nodules at 10 weeks and 20 nodules at 12 weeks. These model provides an opportunity for the evaluation of new treatments against canine lung metastatic osteosarcoma in a nude mice model.     

The effects of sulforaphane on canine osteosarcoma proliferation and invasion

Recent evidence in in vitro and in vivo models suggests that sulforaphane (SFN), found in raw cruciferous vegetables, may have utility in chemoprevention, as an antineoplastic agent and as a free radical scavenger. The effects of SFN alone or with doxorubicin on cell viability were examined, as well as cell cycle kinetics, invasion capabilities and apoptosis in three canine osteosarcoma cell line (D17, OS 2.4 and HMPOS). Results showed that SFN could not induce cell death at potentially physiological concentrations (<50 μM), but significantly diminished cell invasion and downregulation of focal adhesion kinase (FAK) signaling. Modest cell cycle changes were observed in each cell line. When doxorubicin was used in conjunction with SFN, there was a protective effect to doxorubicin‐induced cytotoxicity in D17 and OS 2.4 cells. Further studies examining SFN as a supplement are warranted, particularly in light of pro‐proliferative and cytoprotective properties in canine osteosarcoma.