Investigation of the Effects of mTOR Inhibitors Rapamycin and Everolimus in Combination with Carboplatin on Canine Malignant Melanoma Cells.
Malignant melanoma in dogs is largely resistant to conventional chemotherapy, although responses to carboplatin have been documented. Invasion and early metastasis are common features of certain melanoma subtypes that are associated with tumour progression despite aggressive therapy. Upregulation of the PI3K/AKT/mTOR pathway has been observed in canine malignant melanoma and may represent a potential target for therapy. Rapamycin (sirolimus) and everolimus are commercially available small molecule inhibitors that target mTOR; therefore, may have anticancer activity in canine melanoma. It was hypothesized that there is synergism between rapamycin or everolimus and platinum chemotherapy, and that combination drug treatment would inhibit target/downstream proteins involved in cell viability/proliferation and increase cell death in canine melanoma cells. It was also hypothesized that rapamycin or everolimus would result in decreased glycolytic pathway. Four canine melanoma cell lines were treated in vitro with rapamycin and everolimus as sole treatment or combined with carboplatin. Cell viability, apoptosis, target modulation, and glycolytic metabolism were evaluated by crystal violet colourimetric assay, Annexin V/PI flow cytometry, western blotting, and Seahorse bioanalyzer, respectively. When combined with carboplatinum chemotherapy, rapamycin or everolimus treatment was overall synergistic in reducing cell viability. Carboplatin-induced apoptosis 72 hours after treatment was noted compared to vehicle control. Levels of p-mTOR were reduced by rapamycin and everolimus in all four cell lines, but its active downstream protein p70S6K was not always affected by the treatment in two cell lines. Both mTOR inhibitors decreased the extracellular acidification rate of canine melanoma. Inhibition of mTOR by rapalogs combined with carboplatin chemotherapy may be beneficial in the treatment of canine melanoma. Future mechanistic investigation is required, including evaluating efficacy of combination therapy in vivo.