LW6 (HIF-1α inhibitor) is a novel HIF-1 inhibitor.

HIF:4.4 μM, MDH2:6.3 μM

LW6 decreases the expression of HIF-1α protein without affecting HIF-1β expression. LW6 affects the stability of the HIF-1α protein. LW6 promotes the degradation of wild type HIF-1α while 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. The knockdown of VHL does not abolish HIF-1α protein accumulation in the presence of LW6 which indicates 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 μM[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].

LW6 exhibits potent anti-tumor activity in vivo, leading to reduced HIF-1α expression as observed through immunohistochemical staining of frozen tissues in mice with xenografts derived from human colon cancer HCT116 cells[2].

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].