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Evaluation of the mammalian target of rapamycin pathway and the effect of rapamycin on target expression and cellular proliferation in osteosarcoma cells from dogs

Ira K. Gordon DVM1, Fang Ye MS2, and Michael S. Kent DVM, MAS3
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  • 1 Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616.
  • | 2 Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616.
  • | 3 Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616.

Abstract

Objective—To investigate activation of the mammalian target of rapamycin (mTOR) pathway and the antitumor effect of rapamycin in canine osteosarcoma cells.

Sample Population—3 established primary canine osteosarcoma cell lines generated from naturally developing tumors.

Procedures—Expression of total and phosphorylated mTOR and p70S6 kinase was assessed by use of western blot analysis in canine osteosarcoma cells with and without the addition of rapamycin. A clonogenic assay was performed to determine the surviving fraction of osteosarcoma cells at various concentrations of rapamycin.

Results—Total and phosphorylated mTOR and p70S6 kinase expression was evident in all 3 cell lines evaluated, which was indicative of activation of this pathway. Treatment with rapamycin resulted in a time-dependent decrease in phosphorylated mTOR expression and a lack of detectable phosphorylated p70S6 kinase. No detectable change in expression of total mTOR and total p70S6 kinase was identified after rapamycin treatment. The clonogenic assay revealed a significant dose-dependent decrease in the surviving fraction for all 3 cell lines when treated with rapamycin.

Conclusions and Clinical Relevance—These data indicated that mTOR and its downstream product are present and active in canine osteosarcoma cells. The pathway can be inhibited by rapamycin, and treatment of cells with rapamycin decreased the surviving tumor cell fraction. These data support the molecular basis for further investigation into the use of mTOR inhibitors as an antineoplastic approach for dogs with osteosarcoma.

Abstract

Objective—To investigate activation of the mammalian target of rapamycin (mTOR) pathway and the antitumor effect of rapamycin in canine osteosarcoma cells.

Sample Population—3 established primary canine osteosarcoma cell lines generated from naturally developing tumors.

Procedures—Expression of total and phosphorylated mTOR and p70S6 kinase was assessed by use of western blot analysis in canine osteosarcoma cells with and without the addition of rapamycin. A clonogenic assay was performed to determine the surviving fraction of osteosarcoma cells at various concentrations of rapamycin.

Results—Total and phosphorylated mTOR and p70S6 kinase expression was evident in all 3 cell lines evaluated, which was indicative of activation of this pathway. Treatment with rapamycin resulted in a time-dependent decrease in phosphorylated mTOR expression and a lack of detectable phosphorylated p70S6 kinase. No detectable change in expression of total mTOR and total p70S6 kinase was identified after rapamycin treatment. The clonogenic assay revealed a significant dose-dependent decrease in the surviving fraction for all 3 cell lines when treated with rapamycin.

Conclusions and Clinical Relevance—These data indicated that mTOR and its downstream product are present and active in canine osteosarcoma cells. The pathway can be inhibited by rapamycin, and treatment of cells with rapamycin decreased the surviving tumor cell fraction. These data support the molecular basis for further investigation into the use of mTOR inhibitors as an antineoplastic approach for dogs with osteosarcoma.

Contributor Notes

Presented in part at the 26th Annual Conference of the Veterinary Cancer Society, Callaway Gardens, Ga, October 2006.

The authors thank John Doval for assistance with preparation of the figures.

Address correspondence to Dr. Kent.