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.     

Effects of transplantation sites on tumour growth,pulmonary metastasis and ezrin expression of canine osteosarcoma cell lines in nude mice

To determine the influence of the transplantation site of canine osteosarcoma (OS) cell lines on tumour growth and pulmonary metastasis, three OS cell lines were transplanted into nude mice via subcutaneous (SC), intratibial (IT) or intravenous (IV) injection. IT‐xenografts exhibited greater potential for developing primary masses and pulmonary metastasis than SC‐xenografts. In IT and IV xenografts, lung micrometastases along with phosphorylated ezrin–radixin–moesin (p‐ERM) overexpression were found in mice xenografted with HMPOS and OOS cells after 1 week and metastasis was found with decreased p‐ERM expression at later time points. The expression of ezrin and p‐ERM in the primary tumours of IT‐xenografted mice was higher than those in SC‐xenografted mice with HMPOS and OOS cells. The results suggest that the orthotopic transplantation site plays an important role in the spontaneous metastasis of canine OS and that ezrin phosphorylation may be involved in the early metastatic mechanism of canine OS cells.     

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.     

Effect of all-trans and 9-cis retinoic acid on growth and metastasis of xenotransplanted canine osteosarcoma cells in athymic mice

OBJECTIVE: To determine effects of all-trans and 9-cis retinoic acid (RA) on tumor growth and metastatic ability of canine osteosarcoma cells transplanted into athymic (nude) mice. ANIMALS: Forty-five 5-week-old female BALB/c nude mice. PROCEDURE: 1 X 10(7) POS osteosarcoma cells were transplanted subcutaneously into the intrascapular region of mice. All-trans RA (3 or 30 microg/kg of body weight in 0.1 ml of sesame oil), 9-cis RA (3 or 30 mg/kg in 0.1 ml of sesame oil), or sesame oil (0.1 ml; control treatment) were administered intragastrically 5 d/wk for 4 weeks beginning 3 days after transplantation (n = 4 mice/group) or after formation of a palpable tumor (5 mice/group). Tumor weight was estimated weekly by measuring tumor length and width, and retinoid toxic effects were evaluated daily. Two weeks after the final treatment, mice were euthanatized, and number of mice with pulmonary metastases was determined. RESULTS: Adverse treatment effects were not detected. Tumor weight was less in mice treated with either dose of 9-cis RA than in control mice, although this difference was not significant. Treatment with 30 mg of 9-cis RA/kg initiated after tumor formation significantly reduced the incidence of pulmonary metastasis, compared with the control group. CONCLUSIONS AND CLINICAL RELEVANCE: 9-cis RA decreased the incidence of pulmonary metastasis in nude mice transplanted with canine osteosarcoma cells and may be a potential adjunct therapy for treatment of osteosarcoma in dogs.     

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.     

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.     

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.     

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.     

Anti-tumor effect of adenoviral vector-mediated p53 gene transfer on the growth of canine osteosarcoma xenografts in nude mice

We evaluated the anti-tumor effect of adenoviral vector-mediated p53 gene therapy on the growth of canine osteosarcoma xenografts formed in nude mice. Nude mice were subcutaneously transplanted with cells of 2 P53 mutant canine osteosarcoma cell lines, POS and CHOS. The osteosarcoma xenografts were injected with either an adenoviral vector that expresses canine wild-type P53 (AxCA-cp53) or LacZ (AxCA-LacZ). Tumor growth was significantly inhibited in the xenografts injected with AxCA-cp53 in comparison to those injected with AxCA-LacZ or PBS during the observation period of 27 days. An increase of the amount of p21(WAF1/CDKN1A) mRNA, and the number of apoptotic cells was shown in the tumors injected with AxCA-cp53 in comparison to those injected with AxCA-LacZ or PBS. The present study revealed that the adenoviral vector-mediated p53 gene transfer had an anti-tumor effect in canine osteosarcoma xenografts formed in nude mice.     

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.     

Establishment and characterization of a cell line, MCO-Y4, derived from canine mammary gland osteosarcoma

A cell line, MCO-Y4, was established from a mammary gland osteosarcoma of a 16-year-old female mongrel dog. Histopathologically the tumor was composed of osteoblastic cells with an osteoid meshwork and chondroid matrix. The mean doubling time of the cells at the 93rd passage was 32.39+/-4.66 hr. Immunohistochemically, the osteoblastic and chondroblastic cells were positive for bone morphogenetic protein (BMP)-2/4 and BMP receptor (BMPR) II. The cultured cells were spindle in shape during the growth and the confluent phases. No tumor matrix was detected in the culture dish by alcian blue staining or von-Kossa silver impregnation. MCO-Y4 cells on the chamber slides showed intense immunoreactivity for BMP-2/4 and BMPR II. Noggin, an antagonist for BMP-2/4, showed the growth inhibition on MCO-Y4 cells. In addition, fibronectin might be potential for stimulating growth of MCO-Y4 cells. When transplanted into severe combined immunodeficiency mice, the cells formed tumors consisting of solid proliferation of osteoblastic and fibroblastic cells with woven-bone trabeculae. These tumor cells were intensely positive for BMP-2/4 and BMPR II. Our results suggested that the cell line might be useful for studying the role of BMPs in canine osteosarcoma and the mechanism of ossification.     

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 four canine melanoma cell lines

Four canine melanoma cell lines were established from the subcutaneous, oral gingival and mucosal melanoma tissues at the primary and metastatic sites. These cell lines were designated as CMeC-1, CMeC-2, KMeC and LMeC. The cells were spindles in shape, similar to that of primary tumor cells. The doubling times of these cells ranged from 34.1 +/- 5.61 to 57.9 +/- 3.28 hr and their chromosome number ranged from 46 to 80. When transplanted into nude mice, CMeC-1 and LMeC produced tumors, whereas CMeC-2 and KMeC did not. The morphology of the tissue formed by xenotransplantation of these cells was similar to their primary tumors.     

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.     

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.     

Effect of thalidomide on growth and metastasis of canine osteosarcoma cells after xenotransplantation in athymic mice

OBJECTIVE: To determine whether thalidomide inhibits the growth of primary and pulmonary metastatic canine osteosarcoma in mice after xenotransplantation. ANIMALS: Athymic nude mice. PROCEDURE: Canine osteosarcoma cells were injected SC in 50 mice. Mice were randomly placed into the following groups: control group (n = 13; DMSO [drug vehicle] alone [0.1 mL/d, IP]); low-dose group (12; thalidomide [100 mg/kg, IP]), mid-dose group (13; thalidomide [200 mg/kg, IP]); and high-dose group (12; thalidomide [400 mg/kg, IP]). Starting on day 8, treatments were administered daily and tumor measurements were performed for 20 days. On day 28, mice were euthanatized and primary tumors were weighed. Lungs were examined histologically to determine the number of mice with metastasis and tumor emboli. Mean area of the pulmonary micrometastatic foci was determined for mice from each group. RESULTS: Primary tumor size and weight were not significantly different among groups. The number of mice in the mid-dose (200 mg/kg) and high-dose (400 mg/kg) groups with micrometastasis was significantly less than the number of control group mice; however, the number of mice with tumor emboli was not affected by thalidomide treatment. Size of micrometastasis lesions was not affected by thalidomide treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Mean area of micrometastases was not affected by treatment; however, growth of micrometastases had not yet reached an angiogenesis-dependent size. Although thalidomide did not affect growth of primary tumors in mice after xenotransplantation of canine osteosarcoma cells, our findings indicate that thalidomide may interfere with the ability of embolic tumor cells to complete the metastatic process within the lungs.     

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.     

Comparison of examination of thoracic radiographs and thoracic computed tomography in dogs with appendicular osteosarcoma

Appendicular osteosarcoma (OSA) is a highly metastatic tumour in dogs. The aim of the study was to compare thoracic radiographs with thoracic computed tomography (CT) in the staging of canine appendicular OSA. In all, 39 canine patients histologically diagnosed with OSA were reviewed in the retrospective study. All dogs underwent radiographic examination as well as CT examination of the thoracic cavity. Pulmonary nodules were detected radiographically in two cases (5%), whereas the CT imaging showed that pulmonary nodules were evident in 11 cases (28%, P = 0.024). There was an improved detection of small pulmonary nodules in the lung parenchyma with CT (P = 0.021). The number of nodules in CT examination had a significant negative influence on survival time (P = 0.005). However, whether nodules were present in CT or not did not influence overall survival (P = 0.368). CT examination was superior to thoracic radiography in the screening and detection of pulmonary nodules in dogs with OSA.     

Distribution and activity levels of matrix metalloproteinase 2 and 9 in canine and feline osteosarcoma

Overexpression of matrix metalloproteinases (MMPs) has been associated with increased tumor aggressiveness and metastasis dissemination. We investigated whether the contrasting metastatic behavior of feline and canine osteosarcoma is related to levels and activities of MMP2 and MMP9. Zymography and immunohistochemistry were used to determine expression levels of MMP2 and MMP9 in canine and feline osteosarcoma. Using immunohistochemistry, increased MMP9 levels were identified in most canine osteosarcomas, whereas cat samples more often displayed moderate levels. High levels of pro-MMP9, pro-MMP2, and active MMP2 were detected by gelatin zymography in both species, with significantly higher values for active MMP2 in canine osteosarcoma. These findings indicate that MMP2 is probably involved in canine and feline osteosarcoma and their expression and activity could be associated with the different metastatic behavior of canine and feline osteosarcoma.