Antitumor effects of flavokawain-B flavonoid in gemcitabine-resistant lung cancer cells are mediated via mitochondrial-mediated apoptosis, ROS production, cell migration and cell invasion inhibition and blocking of PI3K/AKT Signaling pathway.

PURPOSE

Drug resistance in lung cancer is a growing and challenging problem affecting the overall treatment and quality of the patient's life. The main purpose of the current study was to investigate the anticancer effects of flavokawain-B in gemcitabine-resistant non-small lung cancer cells (NSCLC) along with evaluating its mode of action by studying its effects on programmed cell death, ROS production, cell migration and invasion and PI3K/AKT signalling pathway.

METHODS

Cell proliferation rate was studied using MTS cell viability assay while apoptosis induction by flavokawain-B was studied by fluorescence microscopy using DAPI staining as well as flow cytometry using Annexin V-FITC/propidium iodide (PI). Effects on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were studied by flow cytometry using Rh-123 and DCH-DA dyes respectively. Effects on cell migration and cell invasion were examined by in vitro wound healing assay and transwell assay respectively. Changes in PI3K/AKT protein expressions were evaluated by western blot.

RESULTS

Flavokawain-B selectively inhibited the viability of the human NSCLC cell line A549, indicating lower toxicity compared with normal lung cancer (NLC) CCL-151 cells and both showed dose-dependent inhibition. DAPI and annexin V-FITC/PI staining showed that flavokawain-B led to a dose-dependent onset of apoptosis in lung cancer cells characterized by shrunken cells, fragmented nuclei and chromatin condensation. Western blot showed that flavokawain-B resulted in downregulation of Bcl-2 and upregulation of Bax in a dose dependent manner. Flavokawain-B treatment led to increase of intracellular ROS concentration and decrease of mitochondrial membrane potential (MMP) both showing dose-dependence. It also led to suppression of cell migration and invasion along with blocking PI3K/AKT signalling pathway.

CONCLUSIONS

Flavokawain-B targets gemcitabine-resistant NSCLC cells selectively without inducing any significant toxicity in normal cells and these effects are mediated via apoptosis induction, ROS production, loss of MMP, suppression of cell migration and invasion and blocking PI3K/AKT signalling pathway.