Pterospermum acerifolium (L.) wild bark extract induces anticarcinogenic effect in human cancer cells through mitochondrial-mediated ROS generation

Abstract

Plants have many medicinal properties including anticancer activity due to the presence of several secondary metabolites. Current cancer treatment policies are not much effective because of side effects and resistance development. Therefore, the discovery of new phytotherapeutics with no or fewer side effects is highly needed. Pterospermum acerifolium (L.) wild, an angiosperm has a broad application in traditional Indian medicinal system including cancer treatment. Despite, there is no study available on the cytotoxic and apoptotic effect of P. acerifolium in human cancer cells. Exploring the medicinal properties of P. acerifolium plant by its traditional use will be helpful towards developing novel cancer therapeutics. Hence, we decided to demonstrate the anti-carcinogenic property of P. acerifolium ethanolic bark extract against lung (A549) and pancreatic (PANC-1) cancer cells. The cytotoxicity was demonstrated by MTT assay, morphological changes, and scratch invasion assay. Flow cytometry, fluorescence staining techniques, and cell cycle analysis were confirmed the apoptotic property of P. acerifolium plant. The cell viability assay revealed that P. acerifolium ethanolic bark extract significantly reduced the viability of both A549 and PANC-1 cells. Moreover, PANC-1 cells showed more sensitivity towards P. acerifolium ethanolic bark extract than A549 at higher concentrations. Clear visualization of changes such as cytoplasmic condensation, cellular morphology, cell shrinkage, and augmented number of dead cells in both the cancer cells was observed after treatment. Scratch and invasion assay showed that cell migration and invasion rate of both the cancer cells were significantly reduced. Fluorescence microscopic studies using acridine orange/ethidium bromide and DAPI (4', 6-diamidino-2-phenylindole) staining showed early and late apoptotic symptoms after treatment with bark extract. Rhodamine-123 and DCFH-DA staining analysis by fluorescence and flow cytometry showed that bark extract depolarized the mitochondria membrane potential and induced reactive oxygen species (ROS) generation. Cell cycle analysis through flow cytometry using propidium iodide stain showed that P. acerifolium bark extract arrested A549 and PANC-1 cells in sub-G1 phase stated early apoptosis. These findings collectively point to the fact that P. acerifolium bark extract induced cell cytotoxicity in lung and pancreatic cancer cells by modulating mitochondrial-mediated ROS generation, and cell cycle checkpoints.

Keywords: Apoptosis; Cytotoxicity; Mitochondrial membrane potential; Pterospermum acerifolium (L.) wild; Reactive oxygen species.