EPOX inhibits angiogenesis by degradation of Mcl-1 through ERK inactivation.
OBJECTIVE
Antiangiogenic therapy is considered as an effective strategy for controlling the growth and metastasis of tumors. Among a myriad of biological activities described for xanthone derivatives, the anticancer activity is quite remarkable, but the molecular mechanism is not clearly resolved. In the present study, we investigated the antiangiogenic mechanism of 3,6-di(2,3-epoxypropoxy)xanthone (EPOX), a novel Mcl-1 targeting drug.


METHODS
To evaluate the antiangiogenic activity of EPOX, we did cell viability, cell cycle, tube formation assay in vitro, and Matrigel plug assay in vivo. To evaluate the effect of EPOX on the endothelial signaling pathway, we did immunoblotting, immunoprecipitation, and immunofluorescence analysis. Intracellular glutathione levels were determined with the use of monochlorobimane, a glutathione-specific probe.


RESULTS
EPOX induced endothelial cell apoptosis in association with proteasome-dependent Mcl-1 degradation. Down-regulation of Mcl-1 resulted in an increase in Mcl-1-free Bim, activation of Bax, and then signaling of mitochondria-mediated apoptosis. Additionally, glutathione depletion and extracellular signal-regulated kinase (ERK) inactivation was observed in EPOX-treated cells. Glutathione supplementation reversed the inhibitory effects of EPOX on ERK, which increases the phosphorylation of Mcl-1 at T(163.) Overexpression of mitogen-activated protein/ERK kinase (MEK) partially reversed the effect of EPOX on Mcl-1 dephosphorylation, ubiquitination, and degradation, further implicating ERK in the regulation of Mcl-1 stability.


CONCLUSIONS
This study provides evidence that EPOX induces glutathione depletion, ERK inactivation, and Mcl-1 degradation on endothelial cells, which leads to inhibition of angiogenesis. Our results suggest that EPOX is a novel antiangiogenic agent, making it a promising lead compound for further development in the treatment of angiogenesis-related pathologies.
Sun
Hui-Lung
HL
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan.
Tsai
An-Chi
AC
Pan
Shiow-Lin
SL
Ding
Qingqing
Q
Yamaguchi
Hirohito
H
Lin
Chun-Nan
CN
Hung
Mien-Chie
MC
Teng
Che-Ming
CM
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D004852:Epoxy Compounds / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005978:Glutathione / Q000032:analysis; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014471:Umbilical Veins / Q000378:metabolism; D044004:Xanthones / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
C545138:3,6-di(2,3-epoxypropoxy)xanthone; D020533:Angiogenesis Inhibitors; D051017:Apoptosis Regulatory Proteins; D004852:Epoxy Compounds; C579094:Mcl1 protein, mouse; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D044004:Xanthones; C009689:xanthone; D048049:Extracellular Signal-Regulated MAP Kinases; D005978:Glutathione