Introduction

Rapamycin is an antifungal drug with antitumor activity and acts inhibiting the mTOR complex. Due the rapamycin antitumor potential, the aim of this study was to evaluate the effect of rapamycin on preclinical model of primary mammary gland tumors from female dogs and their respective metastases.

Methods

Two samples from primary tumors and two paired metastases were cultured in vitro, characterized as tumor cell lines and treated with rapamycin. Cells were evaluated for AKT, mTOR, PTEN, and 4EBP1 gene [removed] qPCR) and investigated for rapamycin IC50.Then, the four cell lines were treated with rapamycin IC50 dosage and mRNA and protein were extracted in treated and non-treated cells to perform AKT, mTOR, PTEN, and 4EBP1 gene expression and proteomic analysis by mass spectrometry.

Results

MTT assay demonstrated rapamycin IC50 for the different tumor cells between 2 and 12 M. In the cell lines treated with rapamycin IC50, there was an AKT increased expression after treatment, suggesting a cellular positive feedback to its blockade, which may indicate a mechanism of activation of alternative pathways. The proteomic analysis of cells treated with rapamycin revealed 32 differentially expressed proteins (p<0.05) between the groups (treated versus untreated cells). The three most relevant proteins were phosphoglycerate mutase and L-lactate dehydrogenase that were decreased in the group treated with rapamycin, both are related to cellular metabolic processes, and Myotrophin that was found increased in the same group, which is associated with positive regulation of cell growth.

Conclusion

The results suggested that rapamycin was able to inhibit cell growth of mammary gland tumor cells in vitro, however, concentrations needed to reach the IC50 were high, when compared to other studies. Thus, the in vivo rapamycin dosage necessary to inhibit tumor cells should exceed the maximum recommended dosage in vivo.

Funding Information

This research was founded by São Paulo State Research Foundation (#2015/25400-7).