Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity.
Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors.
Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Hajime
H
Kashima
Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie 514-8507, Japan.
Fumiyasu
F
Momose
Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Hiroshi
H
Umehara
Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Nao
N
Miyoshi
Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Naohisa
N
Ogo
Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Daisuke
D
Muraoka
Mie University Center for Comprehensive Cancer Immunotherapy, Mie 514-8507, Japan.
Hiroshi
H
Shiku
Mie University Center for Comprehensive Cancer Immunotherapy, Mie 514-8507, Japan.
Naozumi
N
Harada
Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Akira
A
Asai
D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification* / Q000494:pharmacology; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D015251:Epirubicin / Q000302:isolation & purification* / Q000494:pharmacology; D005260:Female; D051858:Forkhead Transcription Factors / Q000037:antagonists & inhibitors*; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects* / Q000276:immunology
D000970:Antineoplastic Agents; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D015251:Epirubicin; D008156:Luciferases