Inhibition of mTORC1 by RAD001 (everolimus) potentiates the effects of 1,25-dihydroxyvitamin D3 to induce growth arrest and differentiation of AML cells in vitro and in vivo

Yang, Jing; Ikezoe, Takayuki; Nishioka, Chie; Ni, Lei; Koeffler, H. Phillip; Yokoyama, Akihito

doi:10.1016/j.exphem.2010.03.020

« Previous Next »Experimental Hematology
Volume 38, Issue 8 , Pages 666-676, August 2010

Inhibition of mTORC1 by RAD001 (everolimus) potentiates the effects of 1,25-dihydroxyvitamin D₃ to induce growth arrest and differentiation of AML cells in vitro and in vivo

Jing Yang
- Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan
Takayuki Ikezoe
- Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan
- Offprint requests to: Takayuki Ikezoe, M.D., Department of Hematology and Respiratory Medicine, Kochi University, Nankoku, Kochi 783-8505, Japan
Chie Nishioka
- Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan
Lei Ni
- Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan
H. Phillip Koeffler
- Division of Hematology and Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, Calif., USA
Akihito Yokoyama
- Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan

Received 25 December 2009; received in revised form 8 March 2010; accepted 30 March 2010. published online 12 April 2010.

Objective

Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) represents a useful approach for the treatment of acute myelogenous leukemia (AML). We previously showed that Gemini-23-yne-26,27-hexafluoro-D₃ inhibited the proliferation of MCF-7 breast cancer cells in association with inhibition of the mammalian target of rapamycin (mTOR) signaling. This study explored the drug interaction of 1,25(OH)₂D₃ and rapamycin analog RAD001 (everolimus) in AML cells.

Materials and Methods

Effects of RAD001 and 1,25-(OH)₂D₃ on the proliferation and differentiation of U937 cells were assessed by colony-forming assay and quantification of CD11b cell surface antigens and their endocytic capability, respectively. Effects of RAD001 and 1,25-(OH)₂D₃ on Akt/mTOR complex-1 (mTORC1) signaling and cell-cycle−related molecules were explored by Western blot analysis. The reporter gene and chromatin immunoprecipitation assays were employed to examine the effects of RAD001 and 1,25-(OH)₂D₃ on the promoter of the p21^waf1 gene. U937 murine xenograft model was utilized to explore the effects of RAD001 and 1,25-(OH)₂D₃ in vivo.

Results

RAD001 potentiated the ability of 1,25(OH)₂D₃ to induce growth arrest and differentiation of AML cells in parallel with downregulation of the levels of p-S6K and p-4E-BP1, substrates of mTORC1. In addition, RAD001 significantly enhanced 1,25(OH)₂D₃-mediated transcriptional activity of p21^waf1 in association with increased levels of the acetylated forms of histone H3 and vitamin D receptor bound to the p21^waf1 promoter in U937 cells. Moreover, RAD001 (3 mg/kg, every another day) significantly enhanced 1,25(OH)₂D₃-induced growth inhibition of U937 tumor xenografts in nude mice without adverse effects.