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.     

Retinoids induce growth inhibition and apoptosis in mast cell tumor cell lines

Retinoids are well recognized as promising antitumor agents in humans. However, there have only been a few reports about the effect of retinoids in canine cancers. To investigate the antitumor effect of retinoids on mast cell tumors (MCT), inhibitory effect on cell growth and induction of apoptosis were examined in vitro. Although sensitivity of these cells differed among the cells, the growth of three MCT cell lines (CoMS, CM-MC and VI-MC) were inhibited dose dependently when they were treated with retinoids. FACS analysis of PI-stained nuclei revealed an apoptotic fraction in CM-MC cells about 30% when treated with retinoids, while those of control cells were less than 5%. Caspase-3 activation was observed after retinoid treatment in CM-MC cells. This was confirmed by inhibiting the retinoid-induced apoptosis using the pan-caspase inhibitor, ZVAD-FMK. Both retinoid receptors, RARs and RXRs, were detected by immunoprecipitation followed by western blot analysis in all the three MCT cells. These data suggests that retinoids inhibit the growth of MCTs partly through apoptosis, and this growth inhibition by retinoids may be mediated by RARs and RXRs. We conclude that retinoid may be a potential adjunctive chemotherapeutic agent for the treatment of canine MCT.     

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 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.     

Relationship between retinoic acid receptor alpha gene expression and growth-inhibitory effect of all-trans retinoic acid on canine tumor cells

Retinoids show antitumor effects on human acute promyelocytic leukemia and other tumors via retinoid receptors. In dogs, the role of retinoid receptors in inhibiting tumor development remains unclear. To evaluate the correlation between the degree of expression of retinoic acid receptor alpha (RARalpha) mRNA and the antiproliferative effects of all-trans retinoic acid (ATRA) treatments, expression analysis of RARalpha mRNA and cell growth inhibition assay were performed on 17 established canine tumor cell lines, including 6 mammary gland tumor (MGT) cell lines, 3 osteosarcoma cell lines, 5 melanoma cell lines, and 3 mast cell tumor (MCT) cell lines. Among the cell lines investigated, all 3 MCT cell lines showed high expression of RARalpha, and the most effective cell growth inhibition was observed in ATRA-treated MCT cell lines. However, remarkable antiproliferative effects of ATRA treatments were not observed on other tumor cell lines with moderate or low RARalpha mRNA expression. As a result of the relationship between RARalpha mRNA expression and ATRA treatment with regression analysis, statistically significant correlation was suggested. Furthermore, real-time quantitative polymerase chain reaction analysis of RARalpha was performed on MCT tissue samples of dogs with spontaneous disease, and 5 of 9 tissues showed high expression. These results suggest that ATRA may be an effective antitumor agent for MCT in dogs, and that prior measurement of expression of RARalpha mRNA may be a good indicator of the effectiveness of ATRA treatment.     

Results of hyperamplification of centrosomes in naturally developing tumors of dogs.

OBJECTIVE: To evaluate results of centrosome hyperamplification in naturally developing tumors of dogs. SAMPLE POPULATION: Tumor specimens from 9 dogs with tumors (rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myxosarcoma, and mammary gland tumor) and 2 canine osteosarcoma cell lines. PROCEDURE: 3 antibodies for centrosome proteins (ie, anti-gamma-tubulin, anti-BRCA1, and anti-pericentrin) were used for immunohistochemical analysis. Double immunostaining for centrosomes was used to confirm the specificity of these antibodies for centrosomes. Mutational analysis of the canine p53 gene was carried out by polymerase chain reaction-single-strand conformation polymorphism analysis, and expression of canine MDM2 protein was evaluated by use of immunohistochemical analysis, using anti-MDM2 antibody. RESULTS: Immunohistochemical analysis of dog osteosarcoma cell lines with apparent aneuploidy revealed frequent hyperamplification of centrosomes in the osteosarcoma cell lines. Similar hyperamplified centrosomes were detected in the tumor tissues from all of the 9 tumors. The frequency of cells with hyperamplified centrosomes (3 to 20/cell) in each tumor tissue ranged from 9.50 to 48.1%, whereas centrosome hyperamplification was not observed in normal lymph nodes from these dogs. In 8 of the 9 tumors, mutation of p53 gene or overexpression of MDM2, or both, was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Various types of naturally developing tumors in dogs often have hyperamplification of centrosomes associated with chromosome instability. Hyperamplification of centrosomes is a novel tumor marker for use in cytologic and histologic examinations of clinical specimens obtained from dogs.     

In vitro retinoid-induced growth inhibition and morphologic differentiation of canine osteosarcoma cells

OBJECTIVE: To determine differentiation and growth inhibition effects of retinoids on canine osteosarcoma cells. SAMPLE POPULATION: 3 osteosarcoma cell lines established from osteosarcomas in dogs. PROCEDURE: Osteosarcoma cells were incubated with various concentrations of all-trans-retinoic acid and 9-cis-retinoic acid or control medium, counted daily for 10 days, and evaluated for morphologic changes. Synthesis of DNA was measured by use of a cell proliferation ELISA. To analyze effect of retinoids on colony formation on plastic dishes, cells were cultured for 14 days, fixed, and stained; number of colonies was counted. RESULTS: In a dose-dependent manner, both retinoids induced morphologic differentiation and growth inhibition in the 3 osteosarcoma cell lines and inhibited each cell's ability to form anchorage-dependent colonies. CONCLUSION AND CLINICAL RELEVANCE: Retinoids induced differentiation of osteosarcoma cells of dogs, resulting in altered expression of their malignant phenotype. Induction of differentiation by retinoids may have potential as an adjunctive treatment for osteosarcoma in dogs.     

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.     

Induction of functional differentiation and growth inhibition in vitro of canine osteosarcoma by 22-oxacalcitriol, calcitriol and all-trans retinoic acid

The effects of 22-oxacalcitriol (OCT), calcitriol and all-trans retinoic acid (ATRA) on the induction of functional differentiation and growth inhibition of the canine osteosarcoma cell line POS were investigated in vitro via bone differentiation markers and proliferation assays, respectively. The intracellular alkaline phosphatase (ALP) activity and the gamma-carboxyglutamic acid osteocalcin (GLA-OC) and procollagen type I C peptide (PIP) production were used as markers of differentiation. Treatment with 10(-8) M concentrations of all drugs for 72 h significantly inhibited growth (P < 0.0001) and increased ALP activity and GLA-OC and PIP production in POS. OCT, calcitriol and ATRA significantly increased the: ALP activity from 1.58 +/- 0.14 mumol/min/mg protein (mean +/- SD; control) to 2.50 +/- 0.09 (P < 0.0001), 2.30 +/- 0.14 (P < 0.0001) and 2.00 +/- 0.14 (P = 0.0008), respectively; GLA-OC production from 0.71 +/- 0.01 ng/ml (control) to 2.87 +/- 0.01 (P < 0.0001), 2.87 +/- 0.11 (P < 0.0001) and 1.36 +/- 0.06 (P < 0.0001), respectively; and PIP production from 433.91 +/- 23.29 ng/ml (control) to 536.54 +/- 15.46 (P = 0.0002), 497.06 +/- 1.99 (P = 0.0028) and 481.66 +/- 0.01 (P = 0.0104), respectively. This study demonstrated that treatment with these drugs induced a phenotypic maturation of POS cells into cells that exhibit the properties of functionally mature bone cells with parallel growth inhibition. The effects of these drugs on functional differentiation may be useful to complement the progression of a normal osteogenic differentiation process in the sarcoma.     

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.     

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.     

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.     

Evaluation of the mammalian target of rapamycin pathway and the effect of rapamycin on target expression and cellular proliferation in osteosarcoma cells from dogs

OBJECTIVE: To investigate activation of the mammalian target of rapamycin (mTOR) pathway and the antitumor effect of rapamycin in canine osteosarcoma cells. SAMPLE POPULATION: 3 established primary canine osteosarcoma cell lines generated from naturally developing tumors. PROCEDURES: Expression of total and phosphorylated mTOR and p70S6 kinase was assessed by use of western blot analysis in canine osteosarcoma cells with and without the addition of rapamycin. A clonogenic assay was performed to determine the surviving fraction of osteosarcoma cells at various concentrations of rapamycin. RESULTS: Total and phosphorylated mTOR and p70S6 kinase expression was evident in all 3 cell lines evaluated, which was indicative of activation of this pathway. Treatment with rapamycin resulted in a time-dependent decrease in phosphorylated mTOR expression and a lack of detectable phosphorylated p70S6 kinase. No detectable change in expression of total mTOR and total p70S6 kinase was identified after rapamycin treatment. The clonogenic assay revealed a significant dose-dependent decrease in the surviving fraction for all 3 cell lines when treated with rapamycin. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicated that mTOR and its downstream product are present and active in canine osteosarcoma cells. The pathway can be inhibited by rapamycin, and treatment of cells with rapamycin decreased the surviving tumor cell fraction. These data support the molecular basis for further investigation into the use of mTOR inhibitors as an antineoplastic approach for dogs with osteosarcoma.     

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.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> chemosensitivity of canine mast cell tumors grades II and III to all-<Emphasis Type="Italic">trans</Emphasis>-retinoic acid (ATRA).

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect the skin and soft tissue of dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. While retinoids are well recognized as promising antitumor agents, there have been only a few reports about retinoids’ effect on canine cancers. The aim of this study was to investigate the chemosensitivity of MCT grades II and III to all-trans retinoic acid (ATRA). Immediately after surgical resection, MCT were prepared for primary culture. Samples of MCTs were also fixed in formalin for histopathology and grading according to the classification of Patnaik et al. (Veterinary Pathology 21(5):469–474, 1984). The best results were obtained when neoplastic mast cells were co-cultivated with fibroblasts. Cultured mast cells were, then, treated with concentrations of 10⁻⁴ to 10⁻⁷ M of ATRA, in order to evaluate their chemosensitivity to this retinoid. MTT assay was performed to estimate cell growth and death. The highest level of mast cell chemosensivity was obtained at the dose of 10⁻⁴ M (p < 0,002). MCT of grades II or III were equally susceptible to the treatment with ATRA. Cell death was observed on the first 24 h until 48 h. According to these results, ATRA may be a potential chemotherapeutic agent for the treatment of canine MCT.     

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.