Temsirolimus, The Mtor Inhibitor, Induces Autophagy In Adenoid Cystic Carcinoma

Purpose:Adenoid cystic carcinoma (ACC), a highly aggressive neoplasm mostly occurring in the salivary gland, accounts for approximately15-25%of all the carcinomas at this location. This neoplasm often presents a prolonged clinical course. After curative surgery, radiotherapy, and chemotherapy, the disease-specific survival at10years for patients with ACC remains to be29to40%. Most deaths from salivary ACC are caused by local recurrence and distant metastasis, those accepted conventional therapies. Therefore, more effective agents with minimal side-effect are urgently needed to control the growth and deterioration of ACC.Autophagy is a process whereby cell components, for example proteins and organelles, are captured within autophagosomes, and are delivered to the lysosomes for degradation. Autophagy facilitates cellular survival by maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation; it also functions in many physiopathologic processes (i.e., differentiation and development, antiaging, innate and adaptive immunity) and cancer. According to the recent report, mild or slow autophagy may enhance cell survival while more severe or rapid autophagy would take part in inducing cell death. Temsirolimus, approved for the treatment of advanced renal cell carcinoma, is a prodrug as a Mammalian target of rapamycin (mTOR) inhibitor, and its primary active metabolite is rapamycin. It also shows preclinical potential in hematological malignancies and pancreatic cancer.In our study, we investigated the role of temsirolimus in ACC as autophagy revulsant, and detected that temsirolimus not only significantly inhibited cells growth in vitro, up-regulated the expression of gene regulating autophagy in ACC cells, but also obviously restrained the xenografts development in vivo, relating with the inhibition of mTOR pathway.Materials and Methods:In vitro study, ACC-M cells were treated with temsirolimus in appropriate concentration, the cytotoxic activity of temsirolimus was determined by MTT assay, and the concentration of drug that produced cytotoxicity against50%of the cultured cells (half maximal inhibitory concentration, IC50) was calculated using the linear regression method; the transmission electron microscopy (TEM) was performed to observe autophagosomes in ACC-M cells; autophagy was reflected by the expression of LC3and Beclinl, which were detected by western blot analysis. In addition, the xenograft model was designed to study the induced-autophagy effect and molecular mechanism of temsirolimus in vivo.Results:The results of MTT assay showed that temsirolimus caused a decrease in the cell viability of ACC-M cells, the inhibition effect assumed an obvious dose-response relationship, IC50of temsirolimus treated ACC-M cells approached20μmol/l; compared with the control group, numerous autophagosomes were observed by TEM in temsirolimus treatment groups; what’s more, the result of western blot analysis showed that the expression of LC3and Beclinl was significantly up-regulated in experiment groups. More importantly, the growth of the ACC xenograft was significantly inhibited by temsirolimus,(Fig.4A and Fig.4D), and the body weight of nude mice showed no significant difference between two groups, either pretreatment or posttreatment. The xenograft model provided further evidence of temsirolimus-induced autophagy in vivo, by inhibiting mTOR activation as well as up-regulation the expression of Beclinl.Conclusion:This study showed that temsirolimus caused a decrease in the cell viability of ACC-M cells; and temsirolimus induced high expression of autophagy related proteins in ACC-M cells; what’s more, temsirolimus could induce autophagy and inhibit xenograft growth in vivo. These results suggest that temsirolimus could act as an mTOR inhibitor to induce autophagy in adenoid cystic carcinoma both in vitro and in vivo.