Flavokawain B and Doxorubicin Work Synergistically to Impede the Propagation of Gastric Cancer Cells via ROS-Mediated Apoptosis and Autophagy Pathways

Chalcone flavokawain B (FKB) possesses a chemopreventive and anti-cancer activity. Doxorubicin is a chemotherapeutic DNA intercalating agent widely used in malignancy treatment. The present study investigated whether synergistic effects exist between the combination of FKB (1.25-5 µg/mL) and doxorubicin (0.5 µg/mL) on the apoptosis and autophagy in human gastric cancer (AGS) cells, and the possible in vitro and in vivo mechanisms. The MTT assay measured cell viability. Various apoptotic-, autophagy-associated protein expression was determined by the Western blot technique. FKB+doxorubicin synergy was estimated by the Chou-Talalay combination index (CI) method. In vivo studies were performed on BALB/c mice. Results showed that compared to FKB/doxorubicin treatments, low doses of FKB+doxorubicin suppressed AGS cell growth. FKB potentiated doxorubicin-induced DNA fragmentation, apoptotic cell death, and enhanced doxorubicin-mediated mitochondrial, death receptor pathways. FKB+doxorubicin activated increased LC3-II accumulation, p62/SQSTM1 expression, and AVO formation as compared to the FKB/doxorubicin alone treatments indicating autophagy in these cells. The death mechanism in FKB+doxorubicin-treated AGS cells is due to the activation of autophagy. FKB+doxorubicin-mediated dysregulated Bax/Bcl-2, Beclin-1/Bcl-2 ratios suggested apoptosis, autophagy induction in AGS cells. FKB+doxorubicin-induced LC3-II/AVOs downregulation was suppressed due to an apoptotic inhibitor Z-VAD-FMK. Whereas, 3-methyladenine/chloroquine weakened FKB+doxorubicin-induced apoptosis (decreased DNA fragmentation/caspase-3). Activation of ERK/JNK may be involved in FKB+doxorubicin-induced apoptosis and autophagy. FKB+doxorubicin-triggered ROS generation, but NAC attenuated FKB+doxorubicin-induced autophagic (LC3 accumulation) and apoptotic (caspase-3 activation and PARP cleavage) cell death. FKB+doxorubicin blocked gastric cancer cell xenografts in nude mice in vivo as compared to FKB/doxorubicin alone treatments. FKB and doxorubicin wielded synergistic anti-tumor effects in gastric cancer cells and is a promising therapeutic approach.

Keywords: ROS; apoptosis; autophagy; doxorubicin; flavokawain B.