Investigation of the effects of mTOR inhibitors rapamycin and everolimus in combination with carboplatin on canine malignant melanoma cells

Abstract

Background Malignant melanoma in dogs is considered to be largely resistant to conventional chemotherapy, although responses to carboplatin have been documented. Invasion and early metastasis are common features of certain melanoma subtypes that contribute to tumour progression despite aggressive local and systemic 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 and 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 further hypothesized that rapamycin or everolimus would impact metabolism by reducing glycolysis in these cells. 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. Results When combined with carboplatin chemotherapy, rapamycin or everolimus treatment was overall synergistic in reducing cell viability. Carboplatin-induced apoptosis was noted at 72 h after treatment compared to the vehicle control. Levels of phosphorylated mTOR were reduced by rapamycin and everolimus in all four cell lines, but activation of the downstream protein p70S6K was not consistently reduced by treatment in two of the cell lines. Both mTOR inhibitors decreased the extracellular acidification rate of canine melanoma cells, indicating reduced cancer cell glycolytic activity. Conclusions Inhibition of mTOR by rapalogs, such as rapamycin and everolimus combined with carboplatin chemotherapy may have activity in canine melanoma. Future mechanistic investigation is warranted, including in vivo assessment of this combination therapy.

Interactive contribution of hyperinsulinemia, hyperglycemia, and mammalian target of rapamycin signaling to valvular interstitial cell differentiation and matrix remodeling

“…Moreover, phosphorylation of P70S6K(Thr 389 ) was not influenced by rapamycin treatment in VIC. This might be due to the relatively low rapamycin concentration chosen for our experiments (10 nM), whereas others also reported cell line dependent responsiveness with this concentration ( 34 ). Murine VIC treated with 100 nM rapamycin showed a decrease in P70S6K phosphorylation ( 35 ), which might be indicative for the need of higher concentrations of rapamycin.…”

Discovery of Clinically Used Octenidine as NRAS Repressor That Effectively Inhibits NRAS-Mutant Melanoma

“…Inspired by the antimelanoma performance of Octenidine, we compared its antimelanoma activity with the most promising clinical and preclinical antimelanoma agents. Different targeted agents were chosen, such as BRAF inhibitor Vemurafenib, MEK inhibitor MEK-162, ERK inhibitor LY3214996, AKT inhibitor MK-2206, and mTOR inhibitor Rapamycin. − Noteworthy, they were all inhibitors of MAPK and PI3K-AKT signaling pathways. On the other hand, the STK19 inhibitor ZT-12-037-01, another antimelanoma agent, could suppress NRAS activation .…”

CD133-Dependent Activation of Phosphoinositide 3-Kinase /AKT/Mammalian Target of Rapamycin Signaling in Melanoma Progression and Drug Resistance

“…Meanwhile, the dual inhibitor of PI3K/mTOR (NVP-BEZ235) has been studied in melanoma [263]. Also, the mTOR analogs including everolimus [264] and temsirolimus [265] have been tested on their inhibitory effect on mTORC1 in melanoma; conversely, mTOR kinase inhibitors were found to target both mTORC1 and mTORC2 [266,267]. In addition, the most common references describing the reliability of the Fyn/CD133/PI3K/mTOR pathway as a therapeutic target in melanoma treatment have been provided in Table 2 and the possible therapeutic targets of the Fyn/CD133/PI3K/Akt/mTOR pathway in melanoma are outlined in Figure 10.…”