EPOX Inhibits Angiogenesis by Degradation of Mcl-1 through ERK Inactivation

Abstract

Purpose: 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. Experimental Design: 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 proteasomedependent 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 angiogenesisrelated pathologies.

Synergistic Interaction between the HDAC Inhibitor, MPT0E028, and Sorafenib in Liver Cancer Cells In Vitro and In Vivo

“…Silencer select siRNA against ERK was purchased from Ambion. Plasmid expressing constitutively active Mek was prepared as described previously (21). Western blot analysis was performed as previously described (13).…”

Endothelial cell survival during angiogenesis requires the pro-survival protein MCL1

“…27 However, Bim deletion was not sufficient to rescue the apoptosis defect caused by the loss of Mcl1 in the limited number of mice studied here. While the small number of T/+ double mutant animals analyzed prevents conclusive interpretation, the data suggest that BIM is not essential for EC apoptosis to proceed following loss of MCL1, and that BIM is not the major antagonist of MCL1 in this system.…”

“…Several stimuli have been reported to promote EC apoptosis through downregulation of MCL1 in vitro. These include hypoxia, 25,26 suppression of ERK 27 and Rho inhibition. 28 Conversely, the S1P receptor agonist FTY720P was reported to delay apoptosis in serum-starved HUVEC by upregulating MCL1.…”

Anti-tumour effects of xanthone derivatives and the possible mechanisms of action

“…It is notable that the cytotoxic effects of the mono-(9) and di- (10,11) acetoxyxanthones were similar to those of the corresponding mono-(2) and di- (3,4) hydroxyxanthones but that the cytotoxic activities of tri- (12) and tetra-(13) acetoxyxanthones were shown to be much weaker than those of the corresponding tri-(7) and tetrahydroxy (8) xanthones, respectively. These data suggest that acetyl groups and hydroxyl groups may affect cytotoxic activity in different ways.…”

“…In recent years, their anti-tumour effects have attracted more and more interest. Some natural and synthetic xanthone derivatives have been described as anti-tumour agents [10][11][12]. Doxorubicin, an anthracycline anti-cancer drug that has been in use for decades, has a nucleus structure similar to that of xanthones.…”