Investigation of the effects of deracoxib and piroxicam on the in vitro viability of osteosarcoma cells from dogs

OBJECTIVE: To determine whether exposure of canine osteosarcoma cells to deracoxib or piroxicam results in decreased viability, whether the cytotoxic effects of deracoxib and piroxicam involve induction of apoptosis, and whether deracoxib is a more potent inhibitor of osteosarcoma cell growth than piroxicam. SAMPLE POPULATION: 1 fibroblast and 3 osteosarcoma cell lines. PROCEDURE: Cell counts and viability assays were performed using osteosarcoma cells (POS, highly metastatic POS, and canine osteosarcoma cell 31) and fibroblasts after 72 hours of incubation with deracoxib at concentrations of 0.5 microM to 500 microM or piroxicam at concentrations of 1 microM to 1,000 microM. Percentage viability was determined for each concentration. A DNA fragmentation analysis was performed to assess drug-induced apoptosis. RESULTS: Concentration of deracoxib required for 50% inhibition of cell viability (IC50) was reached in all 3 osteosarcoma cell lines and ranged from 70 to 150 microM, whereas the IC50 for piroxicam was only reached in the POS cell line at 500 microM. Neither deracoxib nor piroxicam induced sufficient toxicity in fibroblasts to reach an IC50. Exposure of osteosarcoma cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation. CONCLUSIONS AND CLINICAL RELEVANCE: Intermediate and high concentrations of deracoxib and high concentrations of piroxicam were cytotoxic to osteosarcoma cells; neither drug inhibited cell viability at typical plasma concentrations in dogs. Deracoxib inhibited viability of cells at concentrations that did not affect fibroblast viability. There was no evidence of apoptosis induction for either drug; however, only 1 cell line was evaluated for apoptosis induction and only for a limited selection of drug concentrations.     

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 .     

Differentiating effect of pamidronate on canine osteo‐sarcoma cell lines in vitro

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^® AQ_uous, 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.     

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.     

Investigating the in vitro interactive effects of ionizing radiation and pamidronate in a canine osteosarcoma cell line

Introduction: Dogs with osteosarcoma suffer intense bone pain. Conventional palliative treatment options include ionizing radiation with or without systemic chemotherapy. Intravenous pamidronate is a first‐line therapeutic agent in people with painful skeletal metastases and is currently being evaluated for the management of osteolytic pain associated with canine osteosarcoma. The theoretical combination of radiation and pamidronate for managing bone pain appears to be a rational treatment option, however, in vitro experiments demonstrating additive or supraadditve cytotoxic effects of these two modalities would further support the clinical institution of this novel combination therapy. The purpose of this study was to evaluate the in vitro cytotoxicity of ionizing radiation and pamidronate in a canine OSA cell line and to characterize the interaction between these two therapeutic modalities when used in combination. Methods: The canine osteosarcoma cell line, HMPOS, was subjected independently to varying doses of ionizing radiation (100 cGy, 300 cGy, 400 cGy, 600 cGy, 800 cGy, or 1000 cGy) or different concentrations of pamidronate (10, 25, 50, 66, 75, 100, 150, 200, and 250 μM). Cells were harvested following 48 hours of incubation and cell viability was assessed by flow cytometric analysis. Dose‐response curves were generated, and a theoretical ED₅₀ for each treatment modality calculated. An ED₅₀ isobologram was created to determine if different combinations of ionizing radiation and pamidronate (isoboles) would demonstrate subadditive, additive, or supraadditve cytotoxic effects. Results: The ED₅₀ of ionizing radiation and pamidronate were 614 cGy and 65.3 μM, respectively. Of the six different isobole combinations evaluated, three produced supraadditve effects, one produced a subadditive effect, and two produced additive effects. Conclusions: Both ionizing radiation and pamidronate independently exert in vitro cytotoxic effects. Positive interactive effects can be generated, but only with specific isobole combinations. The results from this in vitro study support the use of specific combinations of ionizing radiation and pamidronate in order to maximize the cytotoxic properties of both treatment modalities.     

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.     

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.     

Exposure to L-Ascorbic Acid or α-Tocopherol Facilitates the Development of Porcine Denuded Oocytes from Metaphase I to Metaphase II and Prevents Cumulus Cells from Fragmentation

It is known that alpha-tocopherol (vitamin E) and L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidants. The objective of the present study was to investigate the effects of alpha-tocopherol and L-ascorbic acid on porcine oocyte meiotic maturation, viability and the functions of cumulus cells. In two independent experiments, porcine oocytes with or free from cumulus cells were exposed to different levels of alpha-tocopherol (0, 10, 100 and 200 microM) or L-ascorbic acid (0, 50, 250 and 750 microM). Cumulus expansion, cumulus cell DNA fragmentation, meiotic maturation and degeneration of oocytes were assessed 48 h after in vitro culture. The results showed that: (1) neither alpha-tocopherol nor L-ascorbic acid influenced cumulus expansion but both prevented cumulus cell DNA fragmentation. (2) Alpha-tocopherol lowered the percentage of denuded oocytes (DOs) arrested at germinal vesicle stage (GV). Among the oocytes undergoing germinal vesicle breakdown (GVBD) proportion, fewer DOs treated by alpha-tocopherol were at metaphase I (MI) and more at metaphase II (MII). L-ascorbic acid caused lower percentage of DOs arrested at GV stage and higher percentage of DOs undergoing GVBD, especially at MII. The influences of alpha-tocopherol and L-ascorbic acid were not obvious in cumulus-enclosed oocytes (CEOs). (3) Both vitamins compromised the viability of CEOs and DOs. These results indicate that exposure to alpha-tocopherol or L-ascorbic acid promotes the development of porcine DOs from MI to MII and prevents cumulus cell DNA fragmentation at certain levels, especially 10 microM alpha-tocopherol or 250 microM L-ascorbic acid.     

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.     

Intrinsic radiosensitivity and repair of sublethal radiation-induced damage in canine osteosarcoma cell lines

OBJECTIVE: To characterize the radiosensitivity and capacity for sublethal damage repair (SLDR) of radiation-induced injury in 4 canine osteosarcoma cell lines. SAMPLE POPULATION: 4 canine osteosarcoma cell lines (HMPOS, POS, COS 31, and D17). PROCEDURES: A clonogenic colony-forming assay was used to evaluate the cell lines' intrinsic radiosensitivities and SLDR capacities. Dose-response curves for the cell lines were generated by fitting the surviving fractions after radiation doses of 0 (control cells), 1, 2, 3, 6, and 9 Gy to a linear quadratic model. To evaluate SLDR, cell lines were exposed to 2 doses of 3 Gy (split-dose experiments) at an interval of 0 (single 6-Gy dose), 2, 4, 6, or 24 hours, after which the surviving fractions were assessed. RESULTS: Mean surviving fraction did not differ significantly among the 4 cell lines at the radiation doses tested. Mean surviving fraction at 2 Gy was high (0.62), and the alpha/beta ratios (predictor of tissue sensitivity to radiation therapy) for the cell lines were low (mean ratio, 3.47). The split-dose experiments revealed a 2.8- to 3.9-fold increase in cell survival when the radiation doses were applied at an interval of 24 hours, compared with cell survival after radiation doses were applied consecutively (0-hour interval). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that these canine osteosarcoma cell lines are fairly radioresistant; alpha/beta ratios were similar to those of nonneoplastic, late-responding tissues. Future clinical investigations should involve increasing the fraction size in a manner that maximizes tumor killing without adverse effects on the nonneoplastic surrounding tissues.     

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.     

Influence of vitamin D and retinoids on the induction of functional differentiation in vitro of canine osteosarcoma clonal cells

The efficacy of 22-oxacalcitriol (OCT), calcitriol, cholecalciferol, all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) to differentiate in vitro four clonal cells of the canine osteosarcoma cell line POS into cells having properties of a functionally mature osteoblast bone cell were investigated. The induction of intracellular alkaline phosphatase (ALP) activity, osteocalcin (GLA-OC) and type I collagen (PIP) production after 72 h treatment were used as markers of differentiation. At a concentration of 10(-8)M, OCT and calcitriol significantly induced all markers, and ATRA only the ALP of osteoblast, chondroblast and undifferentiated clonal cells. At the same concentration, 9-cis RA and cholecalciferol induced ALP of chondroblast and osteoblast cells, respectively; ATRA, 9-cis RA and cholecalciferol induced PIP of chondroblast and undifferentiated cells. None of the drugs significantly differentiated fibroblast cells. The ability of these agents to differentiate osteosarcoma cells into cells that exhibit properties of functionally mature osteoblastic bone cells may promote normal osteogenesis and reverse the loss of control of their differentiation.     

Inhibitory effects of 22-oxa-calcitriol and all- trans retinoic acid on the growth of a canine osteosarcoma derived cell-line in vivo and its pulmonary metastasis in vivo

Pulmonary metastasis is a major cause of death and a major obstacle to the successful treatment of canine osteosarcoma. However, the residual capacity of the neoplasia for differentiation and its susceptibility to undergo apoptosis may be used to suppress its growth and metastatic properties. The highly metastasizing POS (HMPOS) canine osteosarcoma cell line which preferentially metastasize to the lungs was used to test the possible efficacy of 22-oxa-calcitriol (OCT) and all- trans retinoic acid (ATRA) to inhibit growth and pulmonary metastasis of the subcutaneously grown osteosarcoma in nude mice. Treatments in vitro, morphologically elongated and increased alkaline phosphatase activity and staining of cells. Tumour growth in vivo was inhibited significantly and the combination treatment of OCT and ATRA (OCT + ATRA) exerted a synergistic and stronger suppression at concentration of 1.0 microg kg(-1)body weight when given subcutaneously three times a week for 5 weeks. The subcutaneous tumours of the control mice consisted of osteoblast-like cells and isolated chondroblast-like cells, but formed several areas of osteoid and increased amount of collagen tissue in all treated mice. Pinpoint macrometastatic nodules developed only in all control mice. Micrometastatic nodule developed only in two of six mice treated with ATRA. However, nodule size and number, and lung wet weight were all reduced significantly. Metastasis were not seen in the mice treated with OCT or OCT + ATRA. This study demonstrated that inhibition of growth and pulmonary metastasis was induced by subcutaneous treatment with these drugs and suggest that both its differentiating and apoptotic inducing activities may be responsible for the antitumour effects. These drugs may be useful in the clinic as an adjunct for the treatment of canine osteosarcoma.     

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.     

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.     

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.     

A comparison of two pamidronate dosing protocols on bone turnover in normal dogs and dogs with osteosarcoma

Introduction: Dogs with appendicular osteosarcoma (OSA) excrete higher concentrations of urine cross‐linked N‐telopeptide of type I collagen (NTx) than normal dogs. NTx is a specific biochemical marker of osteoclastic activity. Pamidronate is a bone‐modulating agent that exerts potent inhibitory effects on osteoclasts. The use of pamidronate is currently being evaluated for the management of osteolytic bone pain in dogs with appendicular osteosarcoma. Despite pamidronate's increasing usage in veterinary oncology, optimal dosing has yet to be determined. Commonly utilized dosages range from 1–2 mg/kg, given intravenously (IV) as a 2‐hour constant rate infusion every 28 days. The purpose of this prospective study was to compare the biological activity of two pamidronate doses (1 mg/kg vs. 2 mg/kg) in the suppression of urine NTx excretion in normal dogs and dogs with appendicular osteosarcoma. Methods: Seventeen OSA dogs receiving single‐agent pamidronate as palliative therapy were evaluated. Group 1A (n = 10) received a dose of 1 mg/kg and group 2A (n = 7) received a dose of 2 mg/kg IV. Urine NTx level were measured at day 0 and 28 using a commercial ELISA (Ostex International). Urine NTx level were also measured in 6 normal dogs: Group 1B (n = 3) received a dose of 1 mg/kg and group 2B (n = 3) received a dose of 2 mg/kg. In normal dogs, urine NTx levels were recorded weekly for six consecutive weeks. Results: In dogs with osteosarcoma, greater reductions in urine NTx excretion from baseline values were demonstrated at 2 mg/kg versus 1 mg/kg (57% and 23%, respectively). Likewise, in normal dogs, urine NTx excretion was suppressed to a greater extent with a dosage of 2 mg/kg versus 1 mg/kg (69% and 23%, respectively). Conclusion: Pamidronate possesses biologic activity in both normal dogs and in dogs with osteosarcoma, as assessed by reductions in urine NTx excretion. Based upon reductions in urine NTx excretion, a dosage of 2 mg/kg appears more effective than 1 mg/kg.     

Natural cytotoxicity in the dog: description of two new allogeneic tumour targets

Three canine tumour cells were studied for their susceptibility to cytotoxicity by allogeneic canine natural killer (NK) cells: a lymphoma line, 3132 of B cell origin, and two adherent cell lines emanating from the same non-lymphoid tumour isolate, one (A72F) with a fibroblast morphology and one (A72E) with an epithelioid appearance. Both 3132 and A72E have preliminary evidence of retrovirus infection. Unstimulated canine peripheral blood mononuclear cells, used as the source of NK cells, were able to mediate significant lysis of 3132 and A72F cells at effector cell:target cell ratios of under 50:1, although an 18 h incubation was necessary for maximum cytotoxicity. NK activity against the 3132 tumour cells proved to be variable both within a group of dogs as well as on different occasions utilising the same individual donor. The epithelioid form of the A72 tumour cell line, A72E, had gained a marked resistance to NK lysis, although like the 3132 cells, there is preliminary evidence of persistent retrovirus infection in this cell line. Interestingly the A72F cells were as successful as homologous 3132 cells in the cold target inhibition of labelled 3132 cytotoxicity, while A72E did not. This latter result could indicate that not only do A72F and 3132 share NK determinants recognised by the same NK receptor, but the A72E line has lost this important recognition determinant.     

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild‐type p53

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.     

Double-Blind Placebo-Controlled Trial of Adjuvant Pamidronate with Palliative Radiotherapy and Intravenous Doxorubicin for Canine Appendicular Osteosarcoma Bone Pain

Background: Canine osteosarcoma (OSA) causes focal malignant osteolysis leading to severe pain. Despite the documented efficacy of radiotherapy or IV aminobisphosphonates for managing cancer bone pain, their potential combined therapeutic value has not been reported in OSA-bearing dogs.
Hypothesis: Pamidronate combined with standardized palliative therapy will improve pain control and bone biologic effects in OSA-bearing dogs.
Animals: Fifty dogs with appendicular OSA treated with standardized palliative therapy and either pamidronate or sterile saline.
Methods: Randomized, prospective, double-blinded, placebo-controlled study. Treatment responses for dogs receiving standardized palliative therapy with (n = 26) or without (n = 24) adjuvant pamidronate were serially evaluated for changes in subjective pain scores, urine N-telopeptide (NTx) excretion, primary tumor relative bone mineral density ( r BMD), and computerized pressure platform gait analysis.
Results: Median duration of subjective pain relief for dogs treated with adjuvant pamidronate or placebo was 76 and 75 days, respectively ( P = .39). Forty percent (20/50; pamidronate [11/26] and placebo [9/24]) of dogs experienced durable analgesia, defined by pain alleviation ≥112 days. For patients achieving durable pain control, dogs treated with pamidronate achieved greater reductions in NTx excretion and larger increases in r BMD compared with placebo controls. Changes in peak vertical force assessed by computerized pressure platform gait analysis correlated with pain alleviation in OSA-bearing dogs.
Conclusions and Clinical Importance: Combining pamidronate with standardized palliative therapy is safe, but does not clearly improve pain alleviation. However, in dogs achieving durable pain control, adjuvant pamidronate appears to decrease focal bone resorption in the local tumor microenvironment.