Salinomycin decreases feline sarcoma and carcinoma cell viability when combined with doxorubicin.

BACKGROUND: Cancer is a significant health threat in cats. Chemoresistance is prevalent in solid tumors. The ionophore salinomycin has anti-cancer properties and may work synergistically with chemotherapeutics. The purpose of our study was to determine if salinomycin could decrease cancer cell viability when combined with doxorubicin in feline sarcoma and carcinoma cells. RESULTS: We established two new feline injection-site sarcoma cell lines, B4 and C10, and confirmed their tumorigenic potential in athymic nude mice. B4 was more resistant to doxorubicin than C10. Dose-dependent effects were not observed until 92 µM in B4 cells (p = 0.0006) vs. 9.2 µM (p = 0.0004) in C10 cells. Dose-dependent effects of salinomycin were observed at 15 µM in B4 cells (p = 0.025) and at 10 µM in C10 cells (p = 0.020). Doxorubicin plus 5 µM salinomycin decreased viability of B4 cells compared to either agent alone, but only at supra-pharmacological doxorubicin concentrations. However, doxorubicin plus 5 µM salinomycin decreased viability of C10 cells compared to either agent alone at doxorubicin concentrations that can be achieved in vivo (1.84 and 4.6 µM, p < 0.004). In SCCF1 cells, dose-dependent effects of doxorubicin and salinomycin were observed at 9.2 (p = 0.036) and 2.5 (p = 0.0049) µM, respectively. When doxorubicin was combined with either 1, 2.5, or 5 µM of salinomycin in SCCF1 cells, dose-dependent effects of doxorubicin were observed at 9.2 (p = 0.0021), 4.6 (p = 0.0042), and 1.84 (p = 0.0021) µM, respectively. Combination index calculations for doxorubicin plus 2.5 and 5 µM salinomycin in SCCF1 cells were 0.4 and 0.6, respectively. CONCLUSIONS: We have developed two new feline sarcoma cell lines that can be used to study chemoresistance. We observed that salinomycin may potentiate (C10 cells) or work synergistically (SCCF1 cells) with doxorubicin in certain feline cancer cells. Further research is indicated to understand the mechanism of action of salinomycin in feline cancer cells as well as potential tolerability and toxicity in normal feline tissues.

Biological indicators of chemoresistance: an ex vivo analysis of γH2AX and p53 expression in feline injection-site sarcomas.

BACKGROUND: The response of soft tissue sarcomas to cytotoxic chemotherapy is inconsistent. Biomarkers of chemoresistance or chemosensitivity are needed in order to identify appropriate patients for treatment. Given that many chemotherapeutics kill cells through direct DNA interactions, we hypothesized that upregulation of DNA damage response mechanisms would confer resistance to cytotoxic chemotherapy in sarcomas. To study this, we used spontaneously-occurring feline injection-site sarcomas (FISS). METHODS: γH2AX and p53 expression were determined in biopsy samples of FISS. γH2AX expression was determined via immunohistochemistry whereas p53 expression was determined via qRT-PCR. Cell lines derived from these sarcoma biopsies were then treated with carboplatin (N = 11) or doxorubicin (N = 5) and allowed to grow as colonies. Colony forming-ability of cells exposed to chemotherapy was compared to matched, untreated cells and expressed as percent survival relative to controls. ImageJ was used for quantification. A mixed model analysis was performed to determine if an association existed between relative survival of the treated cells and γH2AX or p53 expression in the original tumors. Cell lines were validated via vimentin expression or growth as subcutaneous sarcomas in nude mice. RESULTS: An association was detected between γH2AX expression and relative survival in cells exposed to carboplatin (P = 0.0250). In the 11 FISS tumors evaluated, γH2AX expression ranged from 2.2 to 18.8% (mean, 13.3%). Cells from tumors with γH2AX expression higher than the sample population mean had fourfold greater relative survival after carboplatin exposure than cells from tumors with γH2AX expression less than the mean. There was no association between relative survival after carboplatin exposure and p53 expression (P = 0.1608), and there was no association between relative survival after doxorubicin exposure and either γH2AX (P = 0.6124) or p53 (P = 0.8645) expression. Four cell lines were validated via growth as sarcomas in nude mice. Vimentin expression was confirmed in the other 7 cell lines. CONCLUSIONS: γH2AX expression, but not wild type p53, may potentially serve as a biomarker of resistance to platinum therapeutics in soft tissue sarcomas. To further investigate this finding, prospective, in vivo studies are indicated in animal models.

Metabolic Abnormalities Detected in Phase II Evaluation of Doxycycline in Dogs with Multicentric B-Cell Lymphoma.

Doxycycline has antiproliferative effects in human lymphoma cells and in murine xenografts. We hypothesized that doxycycline would decrease canine lymphoma cell viability and prospectively evaluated its clinical tolerability in client-owned dogs with spontaneous, nodal, multicentric, substage a, B-cell lymphoma, not previously treated with chemotherapy. Treatment duration ranged from 1 to 8 weeks (median and mean, 3 weeks). Dogs were treated with either 10 (n = 6) or 7.5 (n = 7) mg/kg by mouth twice daily. One dog had a stable disease for 6 weeks. No complete or partial tumor responses were observed. Five dogs developed grade 3 and/or 4 metabolic abnormalities suggestive of hepatopathy with elevations in bilirubin, ALT, ALP, and/or AST. To evaluate the absorption of oral doxycycline in our study population, serum concentrations in 10 treated dogs were determined using liquid chromatography tandem mass spectrometry. Serum levels were variable and ranged from 3.6 to 16.6 µg/ml (median, 7.6 µg/ml; mean, 8.8 µg/ml). To evaluate the effect of doxycycline on canine lymphoma cell viability in vitro, trypan blue exclusion assay was performed on canine B-cell lymphoma cell lines (17-71 and CLBL) and primary B-cell lymphoma cells from the nodal tissue of four dogs. A doxycycline concentration of 6 µg/ml decreased canine lymphoma cell viability by 80%, compared to matched, untreated, control cells (mixed model analysis, p < 0.0001; Wilcoxon signed rank test, p = 0.0313). Although the short-term administration of oral doxycycline is not associated with the remission of canine lymphoma, combination therapy may be worthwhile if future research determines that doxycycline can alter cell survival pathways in canine lymphoma cells. Due to the potential for metabolic abnormalities, close monitoring is recommended with the use of this drug in tumor-bearing dogs. Additional research is needed to assess the tolerability of chronic doxycycline therapy.

Prevention of laryngospasm with rocuronium in cats: a dose-finding study.

OBJECTIVE: To identify the dose of rocuronium that will prevent a laryngeal response to water spraying of the glottis in anesthetized cats. STUDY DESIGN: Randomized crossover study. ANIMALS: Eight healthy, adult, short-haired cats, aged 1-4 years, weighing 3.2-6.0 kg. METHODS: Each cat was anesthetized four times and administered one of four doses of rocuronium (0.1, 0.2, 0.3 and 0.6 mg kg(-1) ) in random order. The larynx was observed with a video-endoscope inserted through a laryngeal mask airway. Video-clips of the laryngeal response to a sterile water spray (0.2 mL) were obtained at baseline (without rocuronium) and at maximal effect of each treatment. Glottal closure score (0-2), duration of glottal closure, and number of adductive arytenoid movements were obtained from video-clips of laryngeal responses (reproduced in slow motion) at baseline and after treatment. Two observers blinded to treatment allocation scored the vigor of the laryngeal response on a visual analog scale (VAS). The duration of apnea (up to 5 minutes) was recorded for each treatment. RESULTS: Compared with baseline, rocuronium 0.3 mg kg(-1) and 0.6 mg kg(-1) significantly decreased all glottal scores obtained from the videos (all p < 0.03). Both observers gave lower VAS scores after 0.3 mg kg(-1) (both p = 0.015). Apnea lasting ≥ 5 minutes occurred in none, one, three and seven of eight cats administered doses of rocuronium 0.1, 0.2, 0.3 and 0.6 mg kg(-1) , respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Rocuronium 0.3 mg kg(-1) and 0.6 mg kg(-1) consistently decreased the completeness and duration of the laryngeal response to water spray, and reduced the number of arytenoid adductive movements in response to that stimulus. However, a laryngeal response was never completely prevented. Rocuronium 0.3 mg kg(-1) may be useful for facilitating tracheal intubation. Positive pressure ventilation must be available for cats administered rocuronium.