Simultaneous inhibition of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways augment the sensitivity to actinomycin D in Ewing sarcoma.
OBJECTIVE
Ewing sarcoma cells, of which over 85% retain chimeric fusion gene EWS/Fli-1, are by and large more resistant to chemotherapeutics compared to nonneoplastic cells. The purpose of this study is to determine the role of EWS/Fli-1 fusion and its downstream targets regarding the cells' resistance against actinomycin D (ActD), which is one of the most commonly used antitumor agents in combination chemotherapy of Ewing sarcomas.


METHODS
Cytotoxicity was measured by WST-8 assay. Caspase-dependent and -independent cell death was examined by fluorescence microscope. Protein expression was analyzed by western blotting. Caspase activity was determined by Caspase-Glo assay.


RESULTS
ActD-induced caspase-dependent apoptotic cell death to Ewing sarcoma TC-135 cells in a dose- and time- dependent manner. Knockdown of EWS/Fli-1 fusion by siRNA resulted in enhancement of ActD-induced apoptosis. ActD treatment activated both mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways although in a distinctive manner. Combined administration of U0126 (MEK inhibitor) and LY294002 (PI3K inhibitor) significantly enhanced ActD-induced apoptosis in vitro and suppressed xenograft tumor growth in vivo.


CONCLUSIONS
The present study demonstrated for the first time that combination of U0126 and LY294002 can augment the cytotoxicity of ActD against Ewing sarcoma cells in vitro and in vivo. Our results indicate that further study on combination of conventional chemotherapies with MEK and PI3K inhibitors may be considered for innovative treatments of Ewing sarcoma patients.
Yamamoto
Takatoshi
T
Department of Orthopedic Surgery, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan.
Ohno
Takatoshi
T
Wakahara
Kazuhiko
K
Nagano
Akihito
A
Kawai
Gou
G
Saitou
Mitsuru
M
Takigami
Iori
I
Matsuhashi
Aya
A
Yamada
Kazunari
K
Shimizu
Katsuji
K
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis; D001859:Bone Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D002070:Butadienes / Q000627:therapeutic use; D016923:Cell Death; D045744:Cell Line, Tumor; D002867:Chromones / Q000627:therapeutic use; D003609:Dactinomycin / Q000627:therapeutic use*; D019008:Drug Resistance, Neoplasm; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009025:Morpholines / Q000627:therapeutic use; D009570:Nitriles / Q000627:therapeutic use; D015514:Oncogene Proteins, Fusion / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051789:Proto-Oncogene Protein c-fli-1; D034741:RNA, Small Interfering / Q000378:metabolism; D034802:RNA-Binding Protein EWS; D012512:Sarcoma, Ewing / Q000188:drug therapy* / Q000201:enzymology; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002070:Butadienes; D002867:Chromones; D003609:Dactinomycin; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D009570:Nitriles; D015514:Oncogene Proteins, Fusion; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051789:Proto-Oncogene Protein c-fli-1; D034741:RNA, Small Interfering; D034802:RNA-Binding Protein EWS; D014157:Transcription Factors; C106031:EWS-FLI fusion protein; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one