Cell experiment:

To evaluate cytotoxic effects of ENMD-1198 on tumor cells, HUH-7 and HepG2 cells are seeded into 96-well plates (1×103/well; 12 wells per condition) and exposed to various concentrations of ENMD-1198 for 24 and 48 hours at 37℃. Methylthiazole tetrazolium (MTT) assay is used to assess cell proliferation.

Animal experiment:

In brief, 2 holes are drilled through the bone cortex of the upper right tibia with a 25-gauge needle (25G 5/8) and bone marrow is flushed out. Subsequently, 250,000 MDA-BO2 cells/10 μL PBS is injected into the right tibiae of 6-week-old nude mice. Three days after intraosseous inoculation of MDA-BO2 cells, the animals are randomly divided in groups (n = 10). In a first experiment, 4 groups of mice (n = 10) receive either ENMD-1198 (200 mg/kg/d, per oral gavage) or CTX (30 mg/kg/d, through the drinking water) or a combination of ENMD-1198 and CTX or vehicle control. Treatment starts at day 7 after inoculation and is continued throughout the experiment. After 6 weeks, the animals are sacrificed by cervical dislocation and hindlimbs are fixed and kept for ex vivo X-ray analysis. In a second experiment, 4 groups of mice (n = 10) receive ENMD-1198 (200 mg/kg/d, per oral gavage) or ENMD-1198 + CTX (30 mg/kg/d, through the drinking water) or ENMD-1198 + CTX + risedronic acid (1.6 μmol/kg/d, by subcutaneous injection).

ENMD-119 is a 2-methoxyestradiol analogue with antiproliferative and antiangiogenic activity, and is suitable for inhibiting HIF-1α and STAT3 in human HCC cells.

ENMD-1198 inhibits the proliferation of endothelial cell HMEC-1 and BMH29L with IC50 of 0.4 μM and 3.8 μM, respectively. ENMD-1198 (0.5 μM) inhibits the formation of capillary tubes in HMEC-1 cells. ENMD-1198 also significantly inhibits capillary tube formation from 0.1 μM and suppresses endothelial cell morphogenesis at 1 μM. Moreover, ENMD-1198 at the IC50 for cell proliferation causes a significant decrease in VEGFR-2 expression both in the presence and in the absence of serum. ENMD-1198 inhibits endothelial cell motility, ENMD-1198 inhibits endothelial cell migration. ENMD-1198 rapidly induces extensive microtubule depolymerization and accumulation of actin stress fibers and large focal adhesions[1]. ENMD-1198 inhibits MDA-BO2 cancer cell viability with an IC50 of approximately 0.8 μM. ENMD-1198 has both an antiangiogenic effect and a vascular disruptive effect in vitro. ENMD-1198 reduces the viability of RAW264.7 osteoclast precursor cells and inhibits PTHrP-stimulated bone resorption. ENMD-1198 shows inhibitory activity against RAW264 with an IC50 value of approximately 0.4 μM and is shown to be 4 times more potent than 2ME2 (IC50 appro 1.6 μM)[2]. ENMD-1198 inhibits the phosphorylation of MAPK/Erk, PI-3K/Akt and FAK. Moreover, activation of HIF-1α and STAT3 is dramatically reduced by ENMD-1198, which results in lower VEGF mRNA expression. In addition, tumor cell migratory and invasive properties are significantly inhibited[3].

ENMD-1198 (200 mg/kg/d, per oral gavage)-based combination treatments reduce tumor burden but does not eradicate the tumor in mice. ENMD-1198 treatment protects the bone against tumor-induced osteolysis in vivo[2]. ENMD-1198 (200 mg/kg/day, p.o.) leads to a significant reduction in tumor growth, tumor vascularization, and numbers of proliferating tumor cells[3].

[1]. Pasquier E, et al. ENMD-1198, a new analogue of 2-methoxyestradiol, displays both antiangiogenic and vascular-disrupting properties. Mol Cancer Ther. 2010 May;9(5):1408-18. [2]. Snoeks TJ, et al. 2-methoxyestradiol analogue ENMD-1198 reduces breast cancer-induced osteolysis and tumor burden both in vitro and in vivo. Mol Cancer Ther. 2011 May;10(5):874-82. [3]. Moser C, et al. ENMD-1198, a novel tubulin-binding agent reduces HIF-1alpha and STAT3 activity in human hepatocellular carcinoma(HCC) cells, and inhibits growth and vascularization in vivo. BMC Cancer. 2008 Jul 23;8:206.