Cell experiment:

Inhibition of HIF-1a is assayed by a reporter assay using dualluciferase reporter assay system. HCT116 cells in 75-90% confluence are transiently co-transfected with pGL3-HRE-luciferase plasmid containing six copies of HREs from human VEGF genes and pRLSV40 encoding firefly renilla luciferase and incubated for 24 h. Cells are treated with LW6 or 17-AAG for 16 h before report assay. Luciferase activity is integrated over a 10 second period and measured using a luminometer[2].

Animal experiment:

Mice: The mice receive the following treatments using a dosing vehicle solution, containing 10% dimethylacetamide, 10% Cremophor EL and 80% of sodium carbonate buffer (pH 10), by intraperitoneal (i.p.) injection: group1(control group; six mice), vehicle solution; group2 (six mice), LW6 at a dose of 10 and 20mg/kg (QD); and group 3 (six mice), topotecan at a dose of 2mg/kg, (Q2D), which is the dose and dosing schedule that showed more than 60% inhibition of growth of HCT116 tumors. The treatments are continued for 13 days[2].

• Front Cell Dev Biol (2021): 3048.

LW6 is a novel HIF-1 inhibitor with an IC50 of 4.4 μM.

LW6 decreases HIF-1α protein expression without affecting HIF-1β expression. LW6 affects the stability of the HIF-1α protein. LW6 promotes the degradation of wild type HIF-1α, but not of a DM-HIF-1α with modifications of P402A and P564A, at hydroxylation sites in the oxygen-dependent degradation domain. LW6 induces the expression of von Hippel-Lindau (VHL), which interacts with prolyl-hydroxylated HIF-1α for proteasomal degradation. In the presence of LW6, knockdown of VHL does not abolish HIF-1α protein accumulation, indicating that LW6 degraded HIF-1α via regulation of VHL expression[2]. In MDCKII-BCRP cells overexpressing BCRP, LW6 enhances significantly the cellular accumulation of mitoxantrone, a BCRP substrate. LW6 also down-regulates BCRP expression at concentrations of 0.1-10 &#181M[3]. LW6 inhibits the expression of HIF 1α induced by hypoxia in A549 cells at 20 mM, independently of the von Hippel Lindau protein. LW6 induces hypoxia selective apoptosis together with a reduction in the mitochondrial membrane potential[4].

In mice carrying xenografts of human colon cancer HCT116 cells, LW6 demonstrates strong anti-tumor efficacy in vivo and causes a decrease in HIF-1α expression in frozen-tissue immunohistochemical staining[2].

[1]. Naik R, et al. Synthesis and structure-activity relationship study of chemical probes as hypoxia induced factor-1α/malate dehydrogenase 2 inhibitors. J Med Chem. 2014 Nov 26;57(22):9522-38. [2]. Lee K, et al. LW6, a novel HIF-1 inhibitor, promotes proteasomal degradation of HIF-1alpha via upregulation of VHL in a colon cancer cell line. Biochem Pharmacol. 2010 Oct 1;80(7):982-9. [3]. Song JG, et al. Discovery of LW6 as a new potent inhibitor of breast cancer resistance protein. [4]. Sato M, et al. LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells. Mol Med Rep. 2015 Sep;12(3):3462-8.