Identification and validation of cancerous targets of kusunokinin

Abstract

Trans-(-)-kusunokinin hampers breast cancer growth by suppressing many target proteins, such as CFS1R and AKT. Previous results showed that it could bind other targets involved in cancer proliferation and migration. To fill in more information on the trans-(-)-kusunokinin target protein, this study used computational simulations and validated its target on breast and ovarian cancer cells. The results from molecular docking showed that AKR1B1 and MEK2 were potential targets. AKR1B1 represented the strongest binding affinity. Trans-(-)-kusunokinin indicated comparable binding affinity, interaction and orientation in the binding site to AKR1B1 inhibitors. Then, these results were indirect proof on breast (Hs578T and BT549) and ovarian (SKOV3 and A2780) cancer cells. Trans-(±)-kusunokinin had the cytotoxic effect on breast and ovarian cancer cells that were markedly stronger than well-known AKR1B1 inhibitors (zopolrestat and epalrestat). Moreover, trans-(±)-kusunokinin inhibited AKR1B1 enzyme activity with an IC50 value of 9.72 ± 0.18 uM which was stronger than trans-(-)-arctiin (13.65 ± 0.49 uM) but weaker than epalrestat (0.77 ± 0.01 uM) and zopolrestat (31.03 ± 1.40 nM). Moreover, binding between trans-(±)-kusunokinin and intracellular AKR1B1 protected the degradation of AKR1B1 at 75 C and 60 C on Hs578T and SKOV3 cells, respectively. Notably, the inhibitory effect of trans-(±)-kusunokinin on AKR1B1 led to the protection of glucose-induced cellular lipid peroxidation in a dose-dependent manner, which was stronger than epalrestat on Hs578T cells. In addition, trans-(±)-kusunokinin suppressed AKR1B1, resulting in the suppression of its signaling molecules, including PKCδ, NF-κB, AKT, Nrf2, COX2, Twist2. Trans-(±)-kusunokinin also altered epithelial mesenchymal transition (EMT) markers by increasing E-cadherin levels and decreasing N-cadherin levels on Hs578T cells. In conclusion, trans-(-)-kusunokinin exhibited a strong binding affinity with AKR1B1, thereby mitigating oxidative stress and changing EMT protein levels on aggressive breast cancer.