Reovirus changes the expression of anti-apoptotic and proapoptotic proteins with the c-kit downregulation in canine mast cell tumor cell lines.

Although reovirus has reached phase II and III clinical trials in human cancers, the exact mechanism of reovirus oncolysis is still not completely understood. Previously, we have shown that canine mast cell tumor (MCT) cell lines were highly susceptible to reovirus, as compared with other kinds of canine cancer cell lines. In this study, we showed that reovirus infection not only led to the dephosphorylation but also downregulation of c-kit in four canine MCT cell lines, where c-kit activation is required for proliferation. Consistent with c-kit dysregulation, downstream signaling of c-kit, the level of Ras-GTP and phosphorylation of all the downstream effectors of Ras (Raf, MEK, and ERK) and Akt decreased in all the cell lines after reovirus infection, except for Akt in one of cell lines. Pro-apoptotic and anti-apoptotic proteins such as Bim, Bad and Mcl-1 were also altered by reovirus infection in these cell lines. In short, reovirus infection degraded c-kit in all the canine MCT cell lines, leading to the downregulation of downstream signaling of c-kit, which may relate to the cell death induced by reovirus.

Combination Therapy with Reovirus and ATM Inhibitor Enhances Cell Death and Virus Replication in Canine Melanoma.

Oncolytic virotherapy using reovirus is a promising new anti-cancer treatment with potential for use in humans and dogs. Because reovirus monotherapy shows limited efficacy in human and canine cancer patients, the clinical development of a combination therapy is necessary. To identify candidate components of such a combination, we screened a 285-compound drug library for those that enhanced reovirus cytotoxicity in a canine melanoma cell line. Here, we show that exposure to an inhibitor of the ataxia telangiectasia mutated protein (ATM) enhances the oncolytic potential of reovirus in five of six tested canine melanoma cell lines. Specifically, the ATM inhibitor potentiated reovirus replication in cancer cells along with promoting the lysosomal activity, resulting in an increased proportion of caspase-dependent apoptosis and cell cycle arrest at G2/M compared to those observed with reovirus alone. Overall, our study suggests that the combination of reovirus and the ATM inhibitor may be an attractive option in cancer therapy.

Oncolytic reovirus synergizes with chemotherapeutic agents to promote cell death in canine mammary gland tumor.

The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC50) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy.

Les effets oncolytiques des reovirus dans divers cancers ont été prouvés lors de plusieurs essais cliniques en médecine humaine. La virothérapie oncolytique pour les cancers canins utilisant des reovirus est présentement en développement dans notre laboratoire. L'objectif de cette étude était d'examiner les effets synergiques anticancéreux d'une combinaison de reovirus et de faibles doses d'agents chimio-thérapeutiques variés sur les tumeurs des glandes mammaires (TGM) chez les chiens. La première partie de l'étude a démontré l'efficacité du reovirus sur des TGM in vitro et in vivo. Les reovirus utilisés seuls ont produit une quantité significative de mort cellulaire dans des lignées cellulaires canines de TGM via l'apoptose dépendante de la caspase. Une injection unique de reovirus interféra avec la croissance de TGM canines chez des souris ayant reçu une xénogreffe, mais était insuffisante pour induire une régression complète de la tumeur. La deuxième partie de cette étude a mis en évidence les effets anti-tumoraux des reovirus en combinaison avec de faibles doses de paclitaxel, de carboplatin, de gemcibatine, ou de toceranib. Une activité synergique augmentée fut observée dans la lignée cellulaire TGM traitée de manière concomitante avec du reovirus et tous les agents chimio-thérapeutique sauf le toceranib. De plus, en combinant le reovirus avec du paclitaxel ou de la gemcibatine à la moitié de la dose de la moitié de la concentration inhibitrice maximale (IC50) on augmenta la cytotoxicité en activant la caspase 3. Nos données suggèrent que la combinaison de reovirus et de faibles doses d'agents chimio-thérapeutiques fournie une option intéressante pour le traitement de cancer canin.(Traduit par Docteur Serge Messier).

The oncolytic effects of reovirus in canine solid tumor cell lines.

Oncolytic virotherapy is a new strategy for cancer treatment for humans and dogs. Reovirus has been proven to be a potent oncolytic virus in human medicine. Our laboratory has previously reported that canine mast cell tumor and canine lymphoma were susceptible to reovirus. In this study, canine solid tumor cell lines (mammary gland tumor, osteosarcoma and malignant melanoma) were tested to determine their susceptibility towards reovirus. We demonstrated that reovirus induces more than 50% cell death in three canine mammary gland tumors and one canine malignant melanoma cell line. The reovirus-induced cell death occurred via the activation of caspase 3. Ras activation has been shown to be one of the important mechanisms of reovirus-susceptibility in human cancers. However, Ras activation was not related to the reovirus-susceptibility in canine solid tumor cell lines, which was similar to reports in canine mast cell tumor and canine lymphoma. The results of this study highly suggest that canine mammary gland tumor and canine malignant melanoma are also potential candidates for reovirus therapy in veterinary oncology.

Expression of O(6)-methylguanine-DNA methyltransferase causes lomustine resistance in canine lymphoma cells.

The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT) causes resistance to nitrosoureas in various human cancers. In this study, we analyzed the correlation between canine lymphomas and MGMT in vitro. Two of five canine lymphoma cell lines required higher concentrations of lomustine to inhibit cell growth by 50%, but their sensitivity to the drug increased when they were cultured with an MGMT inhibitor. Fluorometric oligonucleotide assay and real-time polymerase chain reaction of these cell lines revealed MGMT activity and high MGMT mRNA expression, respectively. We analyzed the methylation status of the CpG islands of the canine MGMT gene by the bisulfite-sequencing method. Unlike human cells, the canine lymphoma cell lines did not show significant correlation between methylation status and MGMT suppression levels. Our results suggest that in canine lymphoma MGMT activity may influence sensitivity to nitrosoureas; thus, inhibition of MGMT activity would benefit nitrosourea-resistant patients. Additional studies are necessary to elucidate the mechanism of regulation of MGMT expression.

La protéine de réparation O6-méthylguanine-DNA méthyltransferase (MGMT) cause de la résistance aux produits nitroso-urée dans divers cancers humains. Dans la présente étude nous avons analysé in vitro la corrélation entre les lymphomes canins et le MGMT. Deux des cinq lignées cellulaires de lymphome canin ont nécessité des concentrations plus élevées de lomustine pour inhiber de 50 % la croissance cellulaire, mais leur sensibilité au médicament augmenta lorsqu'elles furent mises en culture avec un inhibiteur de MGMT. Une épreuve fluorométrique des oligonucléotides et une épreuve d'amplification en chaîne par la polymérase en temps réel sur ces lignées cellulaires ont révélé, respectivement, une activité MGMT et une expression élevée d'ARNm de MGMT. Nous avons analysé le statut de méthylation des ilots CpG du gène MGMT canin par la méthode de séquençage au bisulfite. Contrairement aux cellules humaines, les lignées cellulaires canines de lymphome n'ont pas montré de corrélation significative entre le statut de méthylation et les niveaux de suppression de MGMT. Nos résultats suggèrent que lors de lymphome canin l'activité de MGMT peut influencer la sensibilité aux produits nitroso-urée; ainsi, l'inhibition de l'activité MGMT bénéficierait les patients résistants au nitroso-urée. Des études additionnelles sont nécessaires pour élucider le mécanisme de régulation de l'expression de MGMT.(Traduit par Docteur Serge Messier).

Seroepidemiology of reovirus in healthy dogs in six prefectures in Japan.

Reovirus infection is common in mammals. However, seroepidemiological data of reovirus neutralizing antibodies are limited in dogs. In this study, sera of 65 healthy dogs from six prefectures across Japan were tested for neutralizing antibodies against reovirus serotype 1 strain Lang (T1L), serotype 2 strain Amy (T2A) and serotype 3 strain Dearing (T3D) using plaque reduction neutralization test (PRNT). Seropositivity against reovirus T1L, T2A and T3D was 53.85%, 33.85% and 46.15%, respectively. Distribution of reovirus seropositive samples displayed no distinguishable geographical pattern. However, reovirus seropositivity increased with age and in dogs housed outdoor. Co-infection of multiple reovirus serotypes in dogs was also detected. These data will provide valuable insights towards the usage of reovirus in oncolytic virotherapy in canine cancers.

Oncolytic reovirus in canine mast cell tumor.

The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility.