In vitro effects of the activity of novel platinum (II) complex in canine and human cell lines

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

Met interacts with EGFR and Ron in canine osteosarcoma

The receptor tyrosine kinase (RTK) Met is known to be over‐expressed in canine osteosarcoma (OSA). In human cancers, the RTKs Met, epidermal growth factor receptor (EGFR) and Ron are frequently co‐expressed and engage in heterodimerization, altering signal transduction and promoting resistance to targeted therapeutics. We found that EGFR and Ron are expressed in canine OSA cell lines and primary tissues, EGFR and Ron are frequently phosphorylated in OSA tumour samples, and Met is co‐associated with EGFR and Ron in canine OSA cell lines. Transforming growth factor alpha (TGFα) and hepatocyte growth factor (HGF) stimulation induced amplification of ERK1/2 and STAT3 phosphorylation in OSA cells and Met was phosphorylated following TGFα stimulation providing evidence for receptor cross‐talk. Lastly, treatment of OSA cells with combined gefitinib and crizotinib inhibited cell proliferation in an additive manner. Together, these data support the notion that Met, EGFR and Ron interact in OSA cells and as such, may represent viable targets for therapeutic intervention.     

Zhangfei在小鼠动情周期卵巢和子宫中的分布

探讨Zhangfei(ZF)在动情周期小鼠子宫和卵巢中的分布规律.通过阴道涂片方法确定昆明雌性小鼠的动情周期,利用免疫组化SP法检测了ZF在小鼠动情周期子宫和卵巢组织中的定位.ZF在各级卵泡的卵母细胞中均呈阳性分布,且主要定位于胞质中;次级卵泡和三级卵泡的颗粒细胞层中可见ZF的存在;成熟卵泡的颗粒细胞中ZF蛋白的阳性更...     

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.     

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.     

Neurokinin‐1 receptor expression and antagonism by the NK‐1R antagonist maropitant in canine melanoma cell lines and primary tumour tissues

We interrogated the neurokinin‐1 receptor (NK‐1R)/substance P (SP) pathway in canine melanoma tumour tissues and cell lines. NK‐1R messenger RNA (mRNA) and protein expression were observed in the majority of tumour tissues. Immunohistochemical assessment of archived tissue sections revealed NK‐1R immunoreactivity in 11 of 15 tumours, which may have diagnostic, prognostic and therapeutic utility. However, we were unable to identify a preclinical in vitro cell line or in vivo xenograft model that recapitulates NK‐1R mRNA and protein expression documented in primary tumours. While maropitant inhibited proliferation and enhanced apoptosis in cell lines, in the absence of documented NK‐1R expression, this may represent off‐target effects. Furthermore, maropitant failed to suppress tumour growth in a canine mouse xenograft model derived from a cell line expressing mRNA but not protein. While NK‐1R represents a novel target, in the absence of preclinical models, in‐species clinical trials will be necessary to investigate the therapeutic potential for antagonists such as maropitant.     

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.     

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.     

Adipocytes suppress differentiation of muscle cells in a co‐culture system

The development of adipose tissue in skeletal muscle is important for improving meat quality. However, it is still unclear how adipocytes grow in the proximity of muscle fibers. We hypothesized that adipocytes would suppress muscle cell growth so as to grow dominantly within muscle. In this study, we investigated the effect of adipocytes on the differentiation of muscle cells in a co‐culture system. The fusion index of C2C12 myoblasts co‐cultured with 3T3‐L1 adipocytes was significantly lower than that of the control. The expression of myogenin and myosin heavy chain in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes was significantly lower than in the control. Furthermore, the expression of Atrogin‐1 and MuRF‐1 was higher in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes than the control. These results suggest that 3T3‐L1 adipocytes suppress the differentiation of C2C12 myoblasts. In addition, 3T3‐L1 adipocytes induced the expression and secretion of IL‐6 in C2C12 muscle cells. The fusion index and myotube diameter were higher in C2C12 muscle cells co‐cultured with 3T3‐L1 cells in medium containing IL‐6‐neutralizing antibody than the control. Taken together, there is a possibility that adipocyte‐induced IL‐6 expression in muscle cells could be involved in the inhibition of muscle cell differentiation via autocrine.     

1,25-Dihydroxyvitamin D3, recombinant human transforming growth factor-beta 1, and recombinant human bone morphogenetic protein-2 induce in vitro differentiation of canine osteosarcoma cells.

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

Evaluation of the gene for inducible nitric oxide synthase as a radiosensitizer under hypoxic and oxic conditions

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

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.     

Osteosarcoma tissues and cell lines from patients with differing serum alkaline phosphatase concentrations display minimal differences in gene expression patterns

Serum alkaline phosphatase (ALP) concentration is a prognostic factor for osteosarcoma in multiple studies, although its biological significance remains incompletely understood. To determine whether gene expression patterns differed in osteosarcoma from patients with differing serum ALP concentrations, microarray analysis was performed on 18 primary osteosarcoma samples and six osteosarcoma cell lines from dogs with normal and increased serum ALP concentration. No differences in gene expression patterns were noted between tumours or cell lines with differing serum ALP concentration using a gene‐specific two‐sample t‐test. Using a more sensitive empirical Bayes procedure, defective in cullin neddylation 1 domain containing 1 (DCUN1D1) was increased in both the tissue and cell lines of the normal ALP group. Using quantitative PCR (qPCR), differences in DCUN1D1 expression between the two groups failed to reach significance. The homogeneity of gene expression patterns of osteosarcoma associated differing serum ALP concentrations are consistent with previous studies suggesting serum ALP concentration is not associated with intrinsic differences of osteosarcoma cells.     

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.     

Radiosensitivity of canine osteosarcoma cells transfected with wild‐type p53 in vitro*

The p53 gene is one of the important tumour suppressor genes that are involved with the cell survival signal pathway. One of the major functions of the p53 protein is to organize cell cycle regulation and induction of apoptosis for cellular genetic stability. It has been documented that more than 50% of all human cancers include a p53 mutation. We evaluated the difference in radiosensitivity between upregulating the expression of canine wild‐type p53 (cp53) in cultured osteosarcoma (D17) cells and naive D17 cells in vitro. We found that upregulating transfected cp53 D17 cells increased their radiation sensitivity in vitro, and there was a significant decrease (P < 0.009) in survival between cp53‐transfected D17 cells and naive D17 cells. In this experiment, a p53 enhancement ratio (p53ER) reached approximately 3.0 at high doses. The transfected cp53 D17 cells were significantly more radiosensitive at all doses evaluated than naive D17 cells, except at 1 Gy where too few data points were available. The p53ER increased rapidly at doses less than 4 Gy, achieving a maximum of about 3.0 for doses of 4 Gy and above. This study shows the enhanced radiosensitivity of the transfected p53 at clinically relevant doses.     

Antineoplastic effect of the cyclooxygenase inhibitor meloxicam on canine osteosarcoma cells

A decisive role in cancer development has been attributed to cyclooxygenase-2 (COX-2) activity, but the significance of COX-2 inhibitors in cancer treatment still needs to be thoroughly investigated. We studied the influence of meloxicam, a non-steroidal antiinflammatory drug with preferential inhibitory effects on COX-2 compared to COX-1, on canine osteosarcoma (D-17) cells. We demonstrated that D-17 cells expressed mRNA and COX-2 protein. Treatment with meloxicam induced a time- and dose-dependent inhibition of cellular growth. To determine if apoptosis plays a role in meloxicam-induced cell death, we performed agarose gel electrophoresis and found a DNA-ladder pattern, typically seen in apoptosis, as well as early apoptotic changes by Annexin V tests. Furthermore, electron microscopy revealed ultrastructural alterations typical of apoptosis. Quantification of apoptotic cells by immunohistochemical staining of caspase 3 confirmed the results. However, further studies with meloxicam are necessary to assess its potential use for treatment of osteosarcomas in dogs.     

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.     

Characterization of Transgenic Pigs That Express Human Decay Accelerating Factor and Cell Membrane‐tethered Human Tissue Factor Pathway Inhibitor

The expression of human complement regulatory proteins (hCRP; hDAF, hCD59, and hMCP) in pig tissues has been suggested as one of strategies to overcome the hyperacute rejection (HAR) in pig‐to‐human transplantation. Expression of human tissue factor pathway inhibitor (hTFPI) in porcine endothelial cells has been suggested as a remedy to overcome microvascular thrombosis. To investigate the effects of these combined transgenes, we established transformed pig cells expressing human decay accelerating factor (hDAF) under the control of enhancer promoter (5′LTR‐PCMVIE), and the fusion protein (hTFPI/hCD4) consisting of the functional domains (K1 and K2) of hTFPI and membrane‐tethering domains (D3 and D4) of hCD4 under the control of PCMVIE. Transgenic pigs were generated with the transformed porcine cells through somatic cell nuclear transfer (SCNT) technology. Analysis of quantitative PCR and real‐time quantitative RT‐PCR showed that four copies of hDAF were integrated and 391 copies of hDAF mRNA expressed in the cells of the transgenic pig. The enhancing activity of 5′LTR was approximately 2 fold compared to CMVIE promoter only. The cell viability test showed that more than 80% of ear cells were viable in the presence of 50% human serum. The chromogenic substrate assay and immunocytochemical staining with tail cells showed that the TFPI activity of fusion protein was observed on the cell membrane. The membrane localization of hDAF and hTFPI proteins was observed by immunocytochemical staining, and the expression of transgenes in heart and liver tissues was also confirmed by immunohistochemistry.