Inhibition of mTORC1 by RAD001 (everolimus) potentiates the effects of 1,25-dihydroxyvitamin D(3) to induce growth arrest and differentiation of AML cells in vitro and in vivo.
OBJECTIVE
Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) represents a useful approach for the treatment of acute myelogenous leukemia (AML). We previously showed that Gemini-23-yne-26,27-hexafluoro-D(3) 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)(2)D(3) and rapamycin analog RAD001 (everolimus) in AML cells.


METHODS
Effects of RAD001 and 1,25-(OH)(2)D(3) 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)(2)D(3) 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)(2)D(3) on the promoter of the p21(waf1) gene. U937 murine xenograft model was utilized to explore the effects of RAD001 and 1,25-(OH)(2)D(3) in vivo.


RESULTS
RAD001 potentiated the ability of 1,25(OH)(2)D(3) 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)(2)D(3)-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)(2)D(3)-induced growth inhibition of U937 tumor xenografts in nude mice without adverse effects.


CONCLUSIONS
Concomitant administration of 1,25(OH)(2)D(3) and the mTORC1 inhibitor may be a promising treatment strategy for individuals with AML.
Yang
Jing
J
Department of Hematology and Respiratory Medicine, Kochi University, Nankoku, Japan.
Ikezoe
Takayuki
T
Nishioka
Chie
C
Ni
Lei
L
Koeffler
H Phillip
HP
Yokoyama
Akihito
A
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D039481:CD11b Antigen / Q000378:metabolism; D002117:Calcitriol / Q000819:agonists / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects*; D003114:Colony-Forming Units Assay; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004357:Drug Synergism; D000068338:Everolimus; D005260:Female; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D006657:Histones; D006801:Humans; D007166:Immunosuppressive Agents / Q000819:agonists / Q000494:pharmacology; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D046912:Multiprotein Complexes; D011401:Promoter Regions, Genetic; D011506:Proteins; D018167:Receptors, Calcitriol / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000819:agonists / Q000031:analogs & derivatives* / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D020298:U937 Cells; D014815:Vitamins / Q000819:agonists / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
D039481:CD11b Antigen; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D006657:Histones; C509295:ITGAM protein, human; D007166:Immunosuppressive Agents; D046912:Multiprotein Complexes; D011506:Proteins; D018167:Receptors, Calcitriol; D014157:Transcription Factors; D014815:Vitamins; D000068338:Everolimus; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D002117:Calcitriol; D020123:Sirolimus