In vitro activity: LW6 inhibits HIF-1α expression induced by hypoxia. It promotes apoptosis preferentially in hypoxic cells. Treatment with LW6 presents no additive effect on cell cycle arrest. LW6 induces ROS formation through the depolarization of MMP in hypoxic cells. Although LW6 increases mitochondrial ROS production, the combination with hypoxia induces a marked increase in ROS production and this high level is maintained up until 24 h. LW6 is also a specific inhibitor of MDH2. As MDH2 is known to serve a significant role in the citric acid cycle at the mitochondrial membrane, LW6 indirectly reduces the activity of the mitochondrial respiratory chain through the inhibition of MDH2. In MDCKII-BCRP cells overexpressing BCRP, LW6 enhances significantly (p < 0.05) the cellular accumulation of mitoxantrone, a BCRP substrate, and is more potent than Ko143, a well-known BCRP inhibitor. In contrast to BCRP, LW6 has no inhibition effect on the functional activity and gene expression of P-gp. LW6 also down-regulates BCRP expression at concentrations of 0.1-10 μM. Furthermore, cells become more susceptible to the cytotoxicity of anticancer drugs in the presence of LW6.

Kinase Assay: LW6 is a hypoxia-inducible factor 1 (HIF) inhibitor which potently inhibits HIF-1α accumulation by degrading HIF-1α without affecting the HIF-1a mRNA levels during hypoxia. It inhibits HIF-1α transcription activity with IC50 of 2.64 μM in cell-based HRE-reporter gene assays.

Cell Assay: Cells are incubated in 96-well ELISA Plates with 100 μl culture medium at 2×105 cells/ml with or without LW6 for 24 h. Cell viability is assessed by the dimethyl thiazolcarboxy-methoxyphenylsulfophenyltetrazolium (MTS) assay. The absorbance is measured at 490 and 620 nm using a microplate reader. For the trypan blue dye. exclusion test, cells are stained by phosphate-buffered saline (PBS) containing 0.1% trypan blue. Cell viability is assessed by counting the number of unstained cells.