In vitro effects of Yunnan Baiyao on canine hemangiosarcoma cell lines

Yunnan Baiyao is a Chinese herbal medicine that has been utilized for its anti‐inflammatory, haemostatic, wound healing and pain relieving properties in people. It has been utilized in the veterinary profession to control bleeding in dogs with hemangiosarcoma (HSA) and has been anecdotally reported to prolong survival times in dogs with this neoplasm. This study evaluated the in vitro activity of Yunnan Baiyao against three canine HSA cell lines after treatment with increasing concentrations of Yunnan Baiyao (50, 100, 200, 400, 600 and 800 µg mL^?1) at 24, 48 and 72 h. Mean half maximum inhibitory concentration (IC₅₀) at 72 h for DEN, Fitz, SB was 369.9, 275.9 and 325.3 µg mL^?1, respectively. Caspase‐3/7 activity increased in correlation with the IC₅₀ in each cell line which was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, APO‐BRDU Kit; BD Biosciences, San Jose, CA, USA) assay. VEGF in cell supernatant was also quantified. Overall, the study found that Yunnan Baiyao causes dose and time dependent HSA cell death through initiation of caspase‐mediated apoptosis, which supports future studies involving Yunnan Baiyao.     

Anticancer effects of resveratrol in canine hemangiosarcoma cell lines

Hemangiosarcoma (HSA) is a highly malignant tumour with aggressive biological behaviour. HSAs are more common in dogs than other domestic animals. The median survival time of dogs with HSA remains short, even with chemotherapy and surgery. Therefore, there is a critical need to improve the adjuvant chemotherapeutic regimens to improve clinical outcomes in dogs with HSA. Resveratrol has been shown to possess strong anti‐proliferative and/or pro‐apoptotic properties in human cancer cell lines. Nevertheless, the potential anticancer effects of resveratrol have not been reported in canine HSAs. The objective of this study is to determine the growth inhibitory effects of resveratrol in HSA cells when used alone or in combination with doxorubicin, a commonly used chemotherapeutic agent. Frog and DD‐1 canine HSA cell lines were treated with varying concentrations of resveratrol with and without doxorubicin. Cell viability was measured by the MTT assay. The expression of apoptotic proteins, activation of p38 mitogen‐activated protein kinase (MAPK), AMP‐activated protein kinase (AMPK) and extracellular signal‐regulated kinase 1/2 (ERK1/2) were assessed by western blotting. Similar to human cancer cell lines, resveratrol markedly inhibited the growth and induced apoptosis in both HSA cell lines. Mechanistically, resveratrol activated p38 MAPK, but did not affect the AMPK or the ERK1/2 pathways. Additional experiments showed that resveratrol augmented the growth‐inhibitory and apoptotic effects of doxorubicin in both HSA cell lines. These findings suggest that resveratrol has pro‐apoptotic effects in canine HSA cells; therefore, its use as a potential adjunct therapy in canine HSA patients warrants further investigation.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

In vitro effects of the tyrosine kinase inhibitor, masitinib mesylate, on canine hemangiosarcoma cell lines

This study evaluated the in vitro activity of masitinib mesylate against canine hemangiosarcoma (HSA) cell lines after treatment with increasing concentrations of masitinib mesylate (0.01-100?μM) for 24, 48 and 72 h. Results indicated that masitinib mesylate caused a dose- and time-dependent decrease in HSA cell proliferation. The 50% inhibitory concentration (IC(50) ) at 72 h for three HSA cell lines (DEN, Fitz and SB) was found to be 8.56, 9.41 and 10.65?μM, respectively. Further investigation demonstrated that masitinib mesylate induced apoptosis in all HSA cell lines, including activation of caspase-3/7. Measurement of VEGF levels in cell supernatant found a statistically significant increased VEGF in close proximity to the IC(50) of each cell line followed by a decline back towards baseline. These findings indicate that masitinib mesylate causes dose-dependent HSA cell death in vitro and supports future clinical trials of masitinib for canine HSA.     

A randomized trial investigating the efficacy and safety of water soluble micellar paclitaxel (Paccal Vet) for treatment of nonresectable grade 2 or 3 mast cell tumors in dogs

Background

Effective treatments for dogs with advanced stage mast cell tumors (MCT) remain a pressing need. A micellar formulation of paclitaxel (paclitaxel [micellar]) has shown promise in early‐phase studies.

Hypothesis/Objectives

The objective was to demonstrate greater activity for paclitaxel (micellar) compared with lomustine. The null hypothesis was μ_p = μ_L (ie, proportion of responders for the paclitaxel [micellar] and lomustine groups, respectively).

Animals

Two hundred and fifty‐two dogs with advanced stage nonresectable grade 2 or 3 MCT.

Methods

Prospective multicenter randomized double‐blind positive‐controlled clinical trial. The primary endpoint was confirmed overall response rate (CORR) at 14 weeks. A secondary endpoint, biologic observed response rate (BORR), also was calculated. Safety was assessed by the characterization and grading of adverse events (AE).

Results

Overall CORR (7% versus 1%; P = .048) and BORR (23% versus 10%; P = .012) were greater for paclitaxel (micellar) compared with lomustine. Paclitaxel (micellar)‐treated dogs were 6.5 times more likely to have a confirmed response and 3.1 times more likely to experience a biologic observed response. The majority of AE with paclitaxel (micellar) were transient and clinically manageable. Twenty‐seven dogs (33%) receiving lomustine were discontinued because of hepatopathy compared with 3 dogs (2%) receiving paclitaxel (micellar) (P < .0001; odds ratio 26.7).

Conclusions and Clinical Importance

Paclitaxel (micellar)'s activity and safety profile are superior to lomustine. The addition of an active and novel taxane to the veterinary armamentarium could fill a substantial need and, as its mechanism of action and AE profile do not overlap with currently available TKI, its availability could lead to effective combination protocols.     

Tumour necrosis factor‐related apoptosis‐inducing ligand induces apoptosis in canine hemangiosarcoma cells in vitro

Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is an apoptosis‐inducing cytokine that shows potential therapeutic value for human neoplasms, and is effective in some canine tumours; however, its potential for killing canine hemangiosarcoma (HSA) cells is unknown. Thus, we evaluated the proapoptotic effect of TRAIL in nine canine HSA cell lines. Cells (JuA1, JuB2, JuB2‐1, JuB4, Re11, Re12, Re21, Ud2 and Ud6) were cultured with three recombinant human TRAILs (rhTRAILs): TRAIL‐TEC derived from Escherichia coli, TRAIL‐TL derived from mammalian cells and isoleucine zipper recombinant human TRAIL (izTRAIL) containing an isoleucine‐zippered structure that facilitates trimerization. TRAIL‐TEC did not decrease the cell viability in any of the cell lines tested, whereas the other two rhTRAILs effectively decreased the viability of all cell lines as assessed by the WST‐1 assay. In canine HSA cells, izTRAIL induced apoptosis more effectively than TRAIL‐TL. In JuB4, Re12, and Ud6 cells, izTRAIL increased the activation of caspase‐3 and caspase‐8 and caused poly (ADP‐ribose) polymerase degradation. Moreover, izTRAIL treatment increased the proportion of Annexin V+/ Propidium iodide (PI)? apoptotic cells and nuclear fragmentation in izTRAIL‐sensitive cells. These results show that rhTRAIL can induce apoptosis in canine HSA cells, but the sensitivity of TRAIL was different depending on the cell lines. Therefore, TRAIL could be an effective therapeutic agent against canine HSA, but the specific mechanism of resistance should be determined to clarify under what conditions this treatment would be most effective.     

Epidermal growth factor enhances the malignant phenotype in canine mammary carcinoma cell lines

Canine mammary gland tumours (CMTs) are the most common malignancies in female dogs. The receptor tyrosine kinase EGFR (erbb1), a receptor for epidermal growth factor (EGF) and related factors, mediates multiple oncogenic functions in human epithelial neoplasms. While previous studies have demonstrated EGFR expression in canine tumours, its function has not been studied in canine cancer. The purpose of this study was to determine the in vitro effects of EGF and vandetanib (ZD6474), a small molecule inhibitor of VEGFR-2, EGFR and RET tyrosine kinases, on proliferation, invasion, survival and chemosensitivity in CMT cells. In low serum, EGF enhanced proliferation and chemotaxis, attenuated apoptosis, and stimulated vascular endothelial growth factor (VEGF) production. Vandetanib dose-dependently inhibited EGFR phosphorylation as well as PI3K/Akt activation, and inhibited all EGF-induced phenotypic effects. In conclusion, EGF stimulates multiple features promoting the malignant phenotype in CMT. Thus, CMT may be an important translational model for the investigation of novel EGFR-directed therapies.     

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.     

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.     

Evaluation of a multi‐agent chemotherapy protocol combining lomustine,procarbazine and prednisolone (LPP) for the treatment of relapsed canine non‐Hodgkin high‐grade lymphomas

The standard of care treatment for canine lymphoma is multi‐agent chemotherapy containing prednisolone, cyclophosphamide, vincristine and an anthracycline such as doxorubicin (CHOP) or epirubicin (CEOP). Lomustine, vincristine, procarbazine, and prednisone (LOPP) has been evaluated as a rescue, with encouraging results; however, resistance to vincristine is likely in patients relapsing on CHOP/CEOP, and this agent may enhance LOPP toxicity without improving efficacy. The aim of this study was to evaluate responses to a modified‐LOPP protocol that does not include vincristine (LPP) and is administered on a 21‐day cycle. Medical records of dogs with high‐grade multicentric lymphoma from 2012 to 2017 were reviewed. Dogs with relapsed lymphoma that received LPP as a rescue protocol were enrolled. Response, time from initiation to discontinuation (TTD) and toxicity of LPP were assessed. Forty‐one dogs were included. Twenty‐five dogs (61%) responded to LPP including 12 complete responses (CR) and 13 partial responses (PR). Responders had a significantly longer TTD (P < .001) compared to non‐responders with 84 days for CR and 58 days for PR. Neutropenia was documented in 20 dogs (57%): 12 grade I to II, 8 grade III to IV. Thrombocytopenia was infrequent (20%): 5 grade I to II, 2 grade III to IV. Twelve dogs developed gastrointestinal toxicity (30%): 10 grade I to II and 2 grade III. Nineteen dogs had elevated ALT (59%): 9 grade I to II, 10 grade III to IV. Treatment was discontinued due to toxicity in 8 dogs (19%). The LPP protocol shows acceptable efficacy and toxicity‐profile and minimizes in‐hospital procedures.     

A combination chemotherapy protocol with MOPP and CCNU consolidation (Tufts VELCAP-SC) for the treatment of canine lymphoma

A chemotherapy protocol using a consolidation phase of alkylating agents was used for treating 94 dogs with lymphoma. Fifty‐seven percent of dogs were in stage V, 63% were ill and 38% had T‐cell lymphoma. The complete remission (CR) rate was 70% and is comparable to results achieved with previously published chemotherapy protocols. Anorexia predicted the remission; of the 40 dogs without anorexia, 35 (88%) achieved CR whereas of 52 dogs with anorexia, 30 (58%) achieved CR. Median first CR duration was 168 days and 1‐ and 2‐year CR rates were 17.4 and 15.5%, respectively. Platelet count affected length of first CR, with a 53.2% reduced chance of coming out of remission with each log increase in platelet count. Median survival time was 302 days. One and 2‐year survival rates were 44 and 13%, respectively. Anorexia and no dose reduction of any drug were independent negative variables. Of 93 dogs with toxicity data, 65 dogs (70%) required a dose reduction. Cyclophosphamide was most commonly reduced with reductions in 31 (38%) of 82 dogs. A dose reduction was significantly more likely in dogs with B‐cell lymphoma than in those with T‐cell lymphoma.     

Antitumour effects of Liporaxel (oral paclitaxel) for canine melanoma in a mouse xenograft model

Paclitaxel, a member of the taxane family, exhibits antitumour effects by targeting the microtubules in cancer cells. Recently, oral paclitaxel has been developed to overcome the side effects of intravenous paclitaxel administration in human patients. The objective of this study was to investigate the antitumour effects of oral paclitaxel in vitro and in vivo. Three weeks after inoculation, oral paclitaxel (25 and 50 mg/kg) or saline was administered every week for three consecutive weeks. To explore the underlying mechanism, tumour angiogenesis was examined by immunohistochemistry with an anti‐CD31 antibody. Tumour cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick‐End Labeling assay, and cell cycle arrest was confirmed by western blot analysis. Oral paclitaxel treatment of canine melanoma cells exerted mediated antiproliferative effects and mediated cell cycle arrest in vitro. In animal experiments, after oral paclitaxel administration, the average tumour size decreased to approximately 30% of that in the control. Histologically, oral paclitaxel showed anti‐angiogenic effects and induced the apoptosis in tumour tissues. Oral paclitaxel also downregulated the intratumoural expression of cyclin D1 and inhibited cell proliferation. The study findings support potential application of oral paclitaxel as a novel chemotherapeutic strategy to treat canine melanoma. This is the first study to investigate the potential of oral paclitaxel as a therapeutic drug against canine tumours.     

A dose‐finding study with a novel water‐soluble formulation of paclitaxel for the treatment of malignant high‐grade solid tumours in dogs

A new formulation of water‐soluble paclitaxel (Paccal® Vet) has been developed for canine cancer patients, without the need for pre‐medication (traditionally required in non‐water‐soluble paclitaxel formulations). The objective of the study was to determine a clinically safe and efficacious dose of Paccal Vet and to estimate progression‐free and overall survival and to evaluate single‐dose pharmacokinetics in tumour‐bearing dogs. A positive risk:benefit ratio was established for Paccal Vet administered at 150 mg m^–2 intravenous (IV) for three or more treatment cycles. Preliminary efficacy was demonstrated by best objective response rate (86%), median time to response (14 days) and median progression‐free survival (131 days). Paccal Vet was associated with expected adverse events (AE) (e.g. myelosuppression), however the majority were transient, clinically silent and manageable. This is the first clinical report of a water‐soluble formulation of paclitaxel suggesting successful administration and being safely used without pre‐medication in dogs.     

In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models

Canine osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs, and it has a high metastatic rate and poor prognosis. Toceranib phosphate (TOC; Palladia, Zoetis) is a veterinary tyrosine kinase inhibitor that selectively inhibits VEGFR‐2, PDGFRs and c‐Kit, but its efficacy is not yet fully understood in the treatment of canine OSA. Here, we evaluated the functional effects of TOC on six OSA cell lines by transwell, wound healing and colony formation assays. Subsequently, two cell lines (Wall and Penny) were selected and were inoculated in mice by intrafemoral injection to develop an orthotopic xenograft model of canine OSA. For each cell line, 30 mice were xenografted; half of them were used as controls, and the other half were treated with TOC at 40 mg/kg body weight for 20 days. TOC inhibited cell growth of all cell lines, but reduced invasion and migration was only observed in Penny and Wall cell lines. In mice engrafted with Penny cells and subjected to TOC treatment, decreased tumour growth was observed, and PDGFRs and c‐Kit mRNA were downregulated. Immunohistochemical analyses demonstrated a significant reduction of Ki67 staining in treated mice when compared to controls. The results obtained here demonstrate that TOC is able to slightly inhibit cell growth in vitro, while its effect is evident only in a Penny cell xenograft model, in which TOC significantly reduced tumour size and the Ki67 index without modifying apoptosis markers.     

Retrospective analysis of factors affecting clinical outcome following CHOP‐based chemotherapy in dogs with primary nodal diffuse large B‐cell lymphoma

Numerous factors are known to affect the prognosis of dogs with chemotherapy‐treated lymphomas. However, prognostic factors for dogs with specific subtypes of lymphoma are less clearly defined. The objective of this study was to identify prognostic factors for dogs receiving CHOP‐based chemotherapy for primary nodal diffuse large B‐cell lymphoma (DLBCL). Medical records of dogs treated for DLBCL at the Purdue Veterinary Teaching Hospital (PUVTH) from 2006 to 2016 were reviewed. Factors potentially related to prognosis were analysed using multivariable statistical methods. Ninety‐eight dogs were included in the study. Best overall response to chemotherapy was complete remission in 80 dogs (81.6%) and partial remission in 18 dogs (18.4%). Median progression‐free survival (PFS) for the entire population was 252 days (range 19‐1068). Factors significantly associated with achieving partial (rather than complete) remission following CHOP included presence of thrombocytopenia at diagnosis (OR 6.88; 95% CI 1.98‐23.93; P = .002), baseline serum globulin concentration (OR 2.63; 95% CI 1.03‐6.75; P = .044), and age at diagnosis (OR 1.36; 95% CI 1.08‐1.71; P = .009). Factors significantly associated with PFS in the lowest quartile (≤93 days) included presence of thrombocytopenia at diagnosis (OR 8.72; 95% CI 1.54‐49.33; P = .014), age at diagnosis (OR 1.47; 95% CI 1.12‐1.94; P = .005), and baseline neutrophil count (OR 1.18; 95% CI 1.02‐1.37; P = .025). Presence of thrombocytopenia, greater age, higher neutrophil count, and higher serum globulin concentration all may be associated with a particularly poor outcome in dogs receiving CHOP‐based chemotherapy for DLBCL.     

In vitro anti‐tubulin effects of mebendazole and fenbendazole on canine glioma cells

Benzimidazole anthelmintics have reported anti‐neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC₅₀) (±SD) obtained from performing the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay after treating J3T, G06‐A, and SDT‐3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550 ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC₅₀ showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.