Histiocytic Sarcoma Cell Lines Research Articles

Effect of dasatinib in a xenograft mouse model of canine histiocytic sarcoma and in vitro expression status of its potential target EPHA2.

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic tumor. Previously, the kinase inhibitor dasatinib was shown to have potent growth inhibitory activity against HS cells in vitro, possibly via targeting the EPHA2 receptor. Here, the in vivo effect of dasatinib in HS cells was investigated using a xenograft mouse model. Moreover, the expression status of EPHA2 was examined in six HS cell lines, ranging from insensitive to highly sensitive to dasatinib. In the HS xenograft mouse model, dasatinib significantly suppressed tumor growth, as illustrated by a decrease in mitotic and Ki67 indices and an increase in apoptotic index in tumor tissues. On Western blot analysis, EPHA2 was only weakly detected in all HS cell lines, regardless of sensitivity to dasatinib. Dasatinib likely results in the inhibition of xenograft tumor growth via a mechanism other than targeting EPHA2. The findings of this study suggest that dasatinib is a targeted therapy drug worthy of further exploration for the treatment of canine HS.

Variations in Histiocytic Differentiation of Cell Lines From Canine Cerebral and Articular Histiocytic Sarcomas

Two newly established canine histiocytic sarcoma (HS) cell lines, designated as PWC-HS01 and FCR-HS02, were obtained from brain and articular tumors, respectively. These 2 HS cell lines had phagocytic ability and modal chromosome aberrations. Although morphologic features of both HS cells were similar, immunocytochemical examinations revealed that the PWC-HS01 cell line expressed both dendritic cell (ie, S100, CD208, CD1, and CD4) and macrophage (ie, CD68, CD163, and CD204) markers. In contrast, the FCR-HS02 cell line was immunonegative for CD204 and CD68 but consistently positive for the dendritic cell markers. Moreover, reverse transcription polymerase chain reaction analyses confirmed histiocytic differentiation of both HS cell lines. These results suggest that HS from the central nervous system may have a tendency to be more undifferentiated compared with cases from other organs. In addition, the 2 newly established HS cell lines were also tumorigenic and metastatic in immunodeficient mice, supporting that these cell lines can be used as new tumor models for investigating canine histiocytic diseases.

Persistent Morbillivirus Infection Leads to Altered Cortactin Distribution in Histiocytic Sarcoma Cells with Decreased Cellular Migration Capacity.

Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated in vitro in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread.

Reduced angiogenic gene expression in morbillivirus-triggered oncolysis in a translational model for histiocytic sarcoma.

Histiocytic sarcoma represents a rare malignant tumour with a short survival time, indicating the need of novel treatment strategies including oncolytic virotherapy. The underlying molecular mechanisms of viral oncolysis are largely unknown. As cancer in companion animals shares striking similarities with human counterparts, we chose a permanent canine histiocytic sarcoma cell line (DH82 cells) to identify global transcriptome changes following infection with canine distemper virus (CDV), a paramyxovirus closely related to human measles virus. Microarray analysis identified 3054 differentially expressed probe sets (DEPs), encoding for 892 up‐ and 869 down‐regulated unique canine genes, respectively, in DH82 cells persistently infected with the vaccine strain Onderstepoort of CDV (DH82‐Ond‐pi), compared to non‐infected DH82 cells. Up‐regulated genes were predominantly related to immune processes, as demonstrated by functional enrichment analysis. Moreover, there was substantial enrichment of genes characteristic for classically activated M1 and alternatively activated M2 macrophages in DH82‐Ond‐pi; however, significant polarization into either of both categories was lacking. ‘Angiogenesis’ was the dominant enriched functional term for the down‐regulated genes, highlighting decreased blood vessel generation as a potential mechanism of paramyxovirus‐induced oncolysis in DH82 cells. The anti‐angiogenic effect of infection was verified by immunohistochemistry, which revealed a lower blood vessel density in an in vivo mouse model, xenotransplanted with DH82‐Ond‐pi, compared to mice transplanted with non‐infected DH82 cells. Reduction in angiogenesis appears to be an important oncolytic mechanism of CDV in DH82 cells, suggesting that similar mechanisms might account for human histiocytic sarcoma and maybe other tumours in conjunction with measles virus.

Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines.

Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines.

Abstract B46: Evaluation of JQ1 as a cytotoxic agent in canine sarcoma cell lines

Abstract Spontaneous cancers in dogs present valuable opportunities to evaluate novel treatment approaches. Among the range of cancers seen in dogs, sarcomas are among the most frequent. In dogs as in humans, the treatment of the sarcomas presents clinical challenges. Here we report evaluation of JQ1, a novel bromodomain inhibitor that has been shown to inhibit MYC signaling, as a cytotoxic agent in cell lines derived from two canine sarcomas, osteosarcoma (OS) and histiocytic sarcoma (HS). Osteocytic sarcoma is the most common primary tumor of dogs and humans, with the incidence in dogs being nearly ten times that observed in humans. The survival rate in humans has stagnated in the past two decades, and thus novel approaches are needed. Histiocytic sarcoma is a very rare but highly malignant disorder in humans. In dogs, HS is seen with appreciable frequency in certain breeds, and is especially common in Bernese mountain dogs. Treatment options are limited and the prognosis is poor. In the current study, we evaluated the IC50 of JQ1 in three canine OS cell lines (D17, Abrams and Gracie) along with two human OS cell lines (SAOAS2 and U2OS), and in two canine HS cell lines (DH82 and BD, a novel cell line recently derived in our laboratory). The IC50 of JQ1 varied in the OS cell lines from 0.5 to 12 µM. The IC50 in the canine OS cell lines were 0.5, 12.5 and 0.47 µM, in D17, Abrams and Gracie, respectively. In the human OS cell lines, SAOS2 and U2OS, the IC50 values were 3.5 and 8.37 µM, in the same range as observed in the canine cell lines. In the HS cell lines, the IC50 of JQ1 was 0.05 and 0.19 µM, in DH82 and BD, respectively. These are all within clinically achievable and tolerable plasma concentrations based on mouse studies. Scratch wound healing assays were carried out in D17 and SOAS2 cell lines. JQ1 at 0.4 µM was found to decrease the migration ability of the cells and to delay wound healing. While the gap was closed in untreated cell by 48hrs, the gap was only decreased by about 25% in the JQ1 treated group, indicating that JQ1 inhibited cell migration. These results are very promising for evaluation of JQ1 as a potential therapeutic agent in both of these sarcomas. The effects of JQ1 on the transcriptome of these sarcomas is being further evaluated. In addition, in vivo xenograft studies are underway in nude mice to test efficacy of JQ1 in treating both sets of sarcomas. If the xenograft studies yield promising results, further safety and clinical efficacy studies in dogs can be undertaken. The spontaneous canine sarcomas will allow the early clinical evaluation of novel agents, such as JQ1, and hopefully, decrease the time to translation to the clinic for humans. We gratefully acknowledge that JQ1 used in these studies was a generous gift from Dr. James Bradner and the Dana-Farber Cancer Institute. Citation Format: Ya-Ting Yang, Marilia Takada, Vilma Yuzbasiyan-Gurkan. Evaluation of JQ1 as a cytotoxic agent in canine sarcoma cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B46.

Influence of a survivin suppressor YM155 on the chemoresistance of canine histiocytic sarcoma cells

Histiocytic sarcoma (HS) in dogs exhibits aggressive biological behaviors and currently few effective treatments are available. Survivin could serve as a potential therapeutic target in several cancers. Sepantronium bromide (YM155) is a potential novel survivin-targeting agent and in this study the influence of survivin expression on clinical outcomes and the effects of YM155 on biological activities in HS cells were investigated. Specimens of HS dogs (n = 30) and four canine HS cell lines were used. The correlation between survivin expression and clinical outcome in the HS dogs was retrospectively assessed using quantitative PCR. Following YM155 treatment of cell lines, apoptosis, cell viability, and drug transporter activities were evaluated using annexin V staining, methylthiazole tetrazolium assays, and Hoechst-33342 staining, respectively.Elevated survivin expression in the HS dogs corresponded with reduced disease-free intervals and survival time, and increased chemoresistance, which led to poor clinical outcomes. Furthermore, YM155 treatment suppressed cell-growth and resistance to lomustine in HS cells by inhibiting the activity of ATP-binding cassette transporters. The evidence presented here supports favorable preclinical evaluation and indicates that survivin-targeted therapies might be effective against HS dogs.

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines.

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.

Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells)

DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcoma (HS) and are commonly used in various fields of research upon infection and cancer, respectively. Despite its frequent use, data on cell surface antigen expression of this cell line are fragmentary and in part inconsistent. We therefore aimed at a detailed morphological and antigenic characterization of DH82 cells with respect to passage-dependent differences. Cellular morphology of early (≤13) and late (≥66) passages of DH82 cells was evaluated via scanning electron microscopy. Moreover, cells were labelled with 10 monoclonal antibodies directed against CD11c, CD14, CD18, CD44, CD45, CD80, CD86, MHC-I, MHC-II, and ICAM-1 for flow cytometric analysis. Early passage cells were characterized by round cell bodies with abundant small cytoplasmic projections whereas later passages exhibited a spindle-shaped morphology with large processes. The percentage of CD11c-, CD14-, CD18-, CD45-, and CD80 positive cells significantly decreased in late passages whereas the expression of CD44, CD86, MHC-I, MHC-II and ICAM-1 remained unchanged. DH82 cells represent a remarkably heterogeneous cell line with divergent antigenic and morphologic properties. The present findings have important implications for future studies, which should consider distinct characteristics with regard to the used passage.

Cytotoxicity evaluation of curcumin treatment in DH82 canine histiocytic sarcoma cell line

Background The identification of targets and new drugs to fight cancer is one the greatest challenges for Biotechnology researchers. Not only humans, but also animals are affected by this disease. It is estimated for example that one in every 3-4 dogs will develop some type of cancer during its lifetime, twice as much as the human being [1,2]. DNA methylation is an important epigenetic mechanism which control gene expression during cell proliferation and differentiation. Deregulation of this mechanism is one of the events which can contribute to the development of cancer [3]. The identification of epigenetic drugs, which are molecules that can revert aberrant DNA methylation, is one of the main areas of cancer research nowadays. In this regard, natural products are a rich source of possibilities to increase the arsenal of epigenetic drugs [1]. This study aims to analyze the cytotoxicity effect of curcumin treatments in DH82 canine histiocytic sarcoma cell line. This polyphenol derived from Curcuma longa has emerged as a potent multimodal cancer-preventing agent which modulates multiple cell signaling pathways and also been described as an inhibitor of DNA methylation[4]. The knowledge of the biological effects of these substances can pinpoint targets for the development of epigenetic drugs, with a positive impact for the treatment of canine and human cancer.

Abstract 2042: Deregulated TS promotes tumor progression in diverse hematopoietic and mesenchymal cell lineages

Abstract Our laboratory recently showed that elevated TS plays a direct causal role in tumorigenesis of NIH3T3 cells in vitro and in the development of endocrine pancreatic mouse tumors in vivo. This observation changed the paradigm of TS as a passive biomarker and has significance for cancer treatment as it refocuses attention on the importance of TS as a tumor-promoting signal. To address whether TS plays an early role in tumorigenesis that is restricted to specific tumor types or if TS plays a late role associated with enhanced progression of established tumors of diverse histologic origin, we have now cross-bred human TS (hTS) transgenic mice with Ink4a/Arf null mice, a common somatic mutation frequently found in adult human cancers. Median survival of hTS/Ink4a/Arf+/- (n=15) mice was reduced 24% (313 vs. 411 days, p <0.05). Both hTS/Ink4a/Arf+/- and hTS/Ink4a/Arf-/- mice (n=16) showed an increase in lymphoma, histiocytic sarcoma (HS) and soft tissue sarcoma (SS). For example, the incidence of HS in hTS/Ink4a/Arf-/- male mice was 31% versus 12% in Ink4a/Arf-/- minus (p<0.01), and tumor cells had spread to multiple tissues (as many as 9 different organs) as compared to hTS minus mice where tumors were localized in lymph nodes and spleen. Strikingly, the tumor burden was markedly increased in hTS/Ink4a/Arf-/- mice: average spleen weight 1.11 vs. 0.49 g, p<0.001, lymph nodes, 1.06 g vs. 0.42 g, p<0.001. We also detected an increase in size and extent of tumor mass of SS (average weight 5.8 g). We detected increased aneuploidy in hTS/Ink4a/Arf-/- HS cell lines as compared to Ink4a/Arf-/- HS cell lines: chromosomal loss 32.1% vs. 16.7%, p<0.05, gain 24.5% vs. 0%, p<0.001 and translocations 9.4% vs. 0%, p<0.05 by spectral karyotyping analysis. Treatment of TS positive HS tumor cells with lenti-TS shRNAs downregulated TS expression and resulted in tumor growth inhibition in vitro as well as in mouse orthotopically transplanted xenograft. In addition, combined TS shRNAs with gemcitabine or pemetrexed treatment showed synergistic inhibition of HS tumor cell growth. These data demonstrate that deregulated TS enhanced genome instability and promote tumor progression in diverse hematopoietic and mesenchymal tumor cells. Citation Format: Min Chen, Akbar Nawab, Frederic J. Kaye, Maria Zajac-Kaye. Deregulated TS promotes tumor progression in diverse hematopoietic and mesenchymal cell lineages. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2042. doi:10.1158/1538-7445.AM2014-2042

Abstract 3931: A novel canine histiocytic sarcoma cell line provides a potential path to effective treatments with relevance for translational and comparative studies in humans

Abstract Histiocytic sarcoma in people is an uncommon and highly aggressive hematopoietic malignancy that accounts for less than 1% of all non-Hodgkin's lymphomas. Regardless of treatment, patients invariably carry a poor prognosis and a high mortality rate. The low number of cases limits investigations for more efficacious forms of treatment for this disorder. To date, there is no human histiocytic sarcoma cell line available for research purposes. Therefore, the establishment of a representative cell line would be highly beneficial. In the present study, we established a cell line from a tumor in a Bernese mountain dog that was morphologically characterized as a malignant histiocytic sarcoma. The diagnosis was further supported by the positive cellular expression of CD18 and vimentin by immunohistochemistry, and CD11c and MHC II by flow cytometry techniques. Together, the expression of these markers indicated a dendritic cell origin, in accordance to the cell type observed in the majority of histiocytic sarcoma cases in dogs. The established cell line has been maintained in tissue culture for a minimum of 50 passages over 12 months in RPMI 1640 medium supplemented with 15% heat-inactivated fetal bovine serum. Results from a pHrodo™ E. coli Bioparticles® assay indicated that the cells had phagocytic behavior. Furthermore, the cell line was successfully transplantable as a xenograft in immunodeficient mice when injected subcutaneously. A palpable tumor was observed within two weeks of injection and the tumor retrieved from the mice was histopathologically similar to the primary tumor. Finally, we performed cytotoxic assays in vitro with classical chemotherapeutic agents and novel therapeutic drugs, including tyrosine kinase inhibitors. The results from these assays demonstrated that dasatinib, a receptor tyrosine kinase pan-inhibitor, was able to significantly inhibit the growth of the cells in vitro within a clinically achievable, and tolerable plasma concentration. Interestingly, the antiproliferative response to dasatinib was augmented when combined to doxorubicin, a classical chemotherapeutic agent, indicating the potential benefits of drug combination for the treatment of this malignancy. In this study we successfully established a canine histiocytic sarcoma cell line that can be used as model for translational studies for similar disorders in humans. In addition, studies that require a cell line derived from dendritic cells can also benefit from this new established cell line. Citation Format: Marilia Takada, Maciej Parys, Emmalena Gregory-Bryson, Vilma Yuzbasiyan-Gurkan. A novel canine histiocytic sarcoma cell line provides a potential path to effective treatments with relevance for translational and comparative studies in humans. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3931. doi:10.1158/1538-7445.AM2014-3931

Abstract 4383: Effects of inhibition of upregulated microRNAs in canine histiocytic sarcoma

Abstract Histiocytic diseases encompass a spectrum of proliferative diseases in humans and dogs. While these orphan malignancies are rare in the human, histiocytic diseases are relatively frequent in certain breeds of dog. In the dog, these diseases range from benign histiocytoma to malignant histiocytic sarcoma (HS). Disseminated HS has a poor prognosis and lacks effective treatment options in both humans and dogs. The overall goal of this study is to unravel the mechanisms of tumorigenesis in histiocytic diseases and identify better treatments. Our studies have focused on microRNAs (miRNAs), which are master regulators of gene expression and may have a significant role in tumorigenesis in histiocytic diseases. MiRNA profiling of canine histiocytic diseases was conducted on cases of reactive histiocytosis, HS, and hemophagocytic HS. These samples were compared to normal canine histiocytes derived from peripheral blood or peritoneal fluid. Several miRNAs were identified to be upregulated in the disease samples and were selected for validation by qRT-PCR. From these results, two miRNA targets were selected for further evaluation. We are probing the significance of the upregulation of these miRNAs by inhibiting their function in canine HS cell lines using chemical miRNA inhibitors and sponge constructs designed to competitively bind the upregulated miRNAs, thus preventing them from acting on their targets. Our results to date indicate that inhibiting these miRNAs in the canine HS cell line, DH82, decreases growth rate by as much as 39%. Studies into other aspects of tumorigenicity potentially affected by the inhibition of these miRNAs are underway. Our preliminary data suggests these miRNAs themselves may be effective therapeutic targets for histiocytic diseases. Additionally, further analysis of the pathways regulated by these miRNAs may identify critical gene targets in HS tumorigenesis and thus increase our understanding of these malignancies and discover novel and effective therapies. Comparative studies have the potential to provide opportunities for advancement in these orphan malignancies, and in this case, improve clinical outcome in dogs and humans. Citation Format: Emmalena J. Gregory-Bryson, Maciej Parys, Matti Kiupel, Vilma Yuzbasiyan-Gurkan. Effects of inhibition of upregulated microRNAs in canine histiocytic sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4383. doi:10.1158/1538-7445.AM2014-4383

Molecular cloning and gene expression of canine apoptosis inhibitor of macrophage.

Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM.

Erythrophagocytosis Enhances Heme-Dependent Cytotoxicity of Antimalarial Drugs in Canine Histiocytic Sarcoma Cell Line DH82

ABSTRACTAntimalarial drugs, dihydroartemisinin (DHA) and artesunate (ATS), exhibit iron-dependent cytotoxicity in tumor cells. We hypothesized that erythrophagocytic uptake of heme-iron enhances the cytotoxicity of DHA and ATS. Erythrophagocytic (EP) treatment of the canine histiocytic sarcoma cell line DH82 markedly increased the cytotoxicity of DHA and ATS compared to controls. Succinyl acetone, an inhibitor of intracellular heme synthesis, decreased the cytotoxicity of DHA and ATS in normal cells, but this change was not observed in EP cells. These results suggest that exogenous heme derived from erythrocytes can enhance the cytotoxicity of DHA and ATS. Furthermore, our study suggests that heme could be a novel component of tumor treatment in veterinary medicine.

Identification of dasatinib as an in vitro potent growth inhibitor of canine histiocytic sarcoma cells

Canine histiocytic sarcoma (HS) is an aggressive and fatal neoplasm that has a high recurrence rate and metastatic nature. In the present report, compounds were screened for their growth inhibitory activity in two HS cell lines using a chemical library known to target specific signalling pathways. Among 171 compounds screened, dasatinib, which targets several types of kinases, clearly inhibited cell growth in one of the two HS lines. The growth inhibitory properties of dasatinib were then examined using six HS cell lines and MDCK cells. Dasatinib demonstrated potent growth inhibitory activity against four HS cell lines with calculated IC50 values of 5.4–54.5nM, while the IC50 values in the other cell lines were in the micromolar range. In conclusion, a kinase enzyme targeted by dasatinib appears to be crucial for growth in some subsets of HS and the on-target activity of dasatinib could underlie the marked growth inhibition in HS cells.

The growth of the canine glioblastoma cell line D‐GBM and the canine histiocytic sarcoma cell line DH82 is inhibited by the resveratrol oligomers hopeaphenol and r2‐viniferin

Vineatrol(®) 30 is a grapevine-shoot extract, which contains resveratrol as well as considerable amounts of so-called resveratrol oligomers such as hopeaphenol and r2-viniferin. In this study, we analysed whether the two above-mentioned resveratrol oligomers were able to inhibit the growth of the canine glioblastoma cell line D-GBM and the canine histiocytic sarcoma cell line DH82, compared their potency to inhibit tumour cell growth with that of resveratrol and determined whether the induction of apoptosis via caspase 9 and 3/7 activation underlies the tumour cell growth-inhibiting effect of hopeaphenol and r2-viniferin. Vineatrol(®) 30, resveratrol, hopeaphenol and r2-viniferin inhibited the growth of D-GBM and DH82 cells in a concentration-dependent manner, whereby hopeaphenol and r2-viniferin were more potent than resveratrol itself in inhibiting the growth of the canine tumour cell lines. Moreover, the anti-proliferative effect of both resveratrol oligomers in D-GBM cells is based on their capacity to induce caspase 9 and 3/7 activation.

Evaluation of dysregulation of the receptor tyrosine kinases Kit, Flt3, and Met in histiocytic sarcomas of dogs

To evaluate canine histiocytic sarcoma cell lines and tumor samples for dysregulation of the Kit/stem-cell factor (SCF), Flt3/Flt3 ligand (Flt3L), and Met/hepatocyte growth factor (HGF) receptor tyrosine kinase signaling pathways, as these are known to contribute to the differentiation and survival of normal dendritic cells as well as malignant transformation of dendritic cells in mouse models. 4 histiocytic sarcoma tumor cell lines and 35 formalin-fixed histiocytic sarcoma specimens obtained from dogs. Histiocytic sarcoma cell lines were evaluated for expression of Kit/SCF, Flt3/Flt3L, and Met/HGF by use of reverse transcriptase-PCR procedures. Histiocytic sarcoma cell lines and tumor samples were evaluated for mutations in Kit, Flt3, and Met by use of PCR analysis of genomic DNA, followed by both sequencing and fluorescent PAGE for deletions or internal tandem duplications. The ability of the multi-targeted split-kinase inhibitor SU11654 to block proliferation and induce apoptosis of histiocytic sarcoma cell lines was also evaluated. No mutations in Kit, Flt3, and Met were identified in any of the cell lines or tumor samples evaluated. Furthermore, SU11654 did not induce cell-cycle arrest or apoptosis of histiocytic sarcoma lines, even at supratherapeutic doses. These data suggest that dysregulation of Kit/SCF, Flt3/Flt3L, and Met/HGF signaling pathways is unlikely to occur in histiocytic sarcomas of dogs and that inhibitors of the Kit, Flt3, and Met pathways are unlikely to provide clinical benefit to dogs with histiocytic sarcomas.

Establishment and Biological Characterization of Canine Histiocytic Sarcoma Cell Lines

Seven novel cell lines from canine histiocytic sarcoma (HS), three of which were disseminated cutaneous HS and four of which were synovial HS, were established. All of the established cell lines had the same morphological (by light and electron microscopic findings), cytochemical (alpha-naphthyl butyrate esterase-positive), and immunohistochemical (vimentin- and lysozyme-positive, and cyto-keratin-negative) characteristics as the original HS tumor cells. All of the established cell lines injected into nude mice subcutaneously produced solid tumors. Because the established cell lines also showed phagocytic and processing activities, the HS tumor cells appear to originate from the mononuclear phagocytic system cells, despite their differences in locations or organs.