title: New anti-cancer chemicals Ertredin and its derivatives, regulate oxidative phosphorylation and glycolysis and suppress sphere formation in vitro and tumor growth in EGFRvIII-transformed cells.
abstract: EGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2-7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation.
  
  
  
  We constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis, quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis.
  
  
  
  In the course of screening 30,000 substances, a reagent, "Ertredin" was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed both oxidative phosphorylation and glycolysis under 3D conditions, and promoted cell apoptosis.
  
  
  
  We developed a high throughput screening method based on anchorage-independent sphere formation induced by EGFRvIII-dependent transformation. In the course of screening, we identified Ertredin, which inhibited anchorage-independent 3D growth and tumor formation in nude mice. Functional analysis suggests that Ertredin suppresses both mitochondrial oxidative phosphorylation and cytosolic glycolysis in addition to promoting EGFRvIII degradation, and stimulates apoptosis in sphere-forming, EGFRvIII-overexpressing cells.
authors.affiliation: Laboratory of Oncology, Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, 141-0021, Tokyo, Japan. atsumi@bikaken.or.jp.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Atsumi
authors.affiliation: Laboratory of Oncology, Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, 141-0021, Tokyo, Japan.
authors.forename: Chisato
authors.initials: C
authors.lastname: Nosaka
authors.affiliation: Numazu Branch, Institute of Microbial Chemistry, Miyamoto, Numazu-shi, Shizuoka, Japan.
authors.forename: Hayamitsu
authors.initials: H
authors.lastname: Adachi
authors.affiliation: Laboratory of Synthetic Organic Chemistry, Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kimura
authors.affiliation: Laboratory of Synthetic Organic Chemistry, Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: Laboratory of Synthetic Organic Chemistry, Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Takada
authors.affiliation: Laboratory of Synthetic Organic Chemistry, Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo, Japan.
authors.forename: Takumi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Numazu Branch, Institute of Microbial Chemistry, Miyamoto, Numazu-shi, Shizuoka, Japan.
authors.forename: Shun-Ichi
authors.initials: S
authors.lastname: Ohba
authors.affiliation: Numazu Branch, Institute of Microbial Chemistry, Miyamoto, Numazu-shi, Shizuoka, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Inoue
authors.affiliation: Laboratory of Oncology, Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, 141-0021, Tokyo, Japan.|Numazu Branch, Institute of Microbial Chemistry, Miyamoto, Numazu-shi, Shizuoka, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Kawada
authors.affiliation: Laboratory of Oncology, Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, 141-0021, Tokyo, Japan.|Numazu Branch, Institute of Microbial Chemistry, Miyamoto, Numazu-shi, Shizuoka, Japan.|Laboratory of Synthetic Organic Chemistry, Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo, Japan.
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Shibasaki
authors.affiliation: Institute of Physiology and Medicine, Jobu University, Takasaki-shi, Gunma, Japan.
authors.forename: Masabumi
authors.initials: M
authors.lastname: Shibuya
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006019:Glycolysis / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D011810:Quinoxalines / Q000737:chemistry / Q000494:pharmacology*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018874:Spheroids, Cellular / Q000187:drug effects* / Q000378:metabolism; D014184:Transplantation, Homologous; D047368:Tumor Burden / Q000187:drug effects* / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D011810:Quinoxalines; C410723:epidermal growth factor receptor VIII; C000623342:ertredin; D066246:ErbB Receptors
keywords: 3D; Anchorage-independent; Apoptosis; EGFRvIII; Ertredin; Glycolysis; Oxidative phosphorylation; Sphere