Induction of apoptosis in A431 cells via ROS generation and p53-mediated pathway by chloroform fraction of Argemone mexicana (Pepaveraceae)

Abstract

Argemone mexicana(Pepaveraceae) is an important medicinal plant commonly known as 'maxican prickly poppy' and is traditionally used to treat skin diseases. In the present study, the extract/fractions of aerial parts of A. mexicana after carrying out the organoleptic characteristics were sequentially extracted with the solvents of increasing polarities. Total fractions were examined for their radical scavenging activities in DPPH and DNA nicking assays. Among all, maximum antioxidant activity was shown by chloroform fraction (AmC) in DPPH assay with IC₅₀ of 26.12 μg/ml, and DNA nicking assay showed 80.91% protective potential. The AmC fraction was analyzed for its antibacterial, cytotoxic potential, cell cycle analysis, mitochondrial membrane potential (MMP) and accumulation of reactive oxygen species (ROS) using A431 cell line. The AmC fraction exhibited remarkable antibacterial activity against bacterial strains in the order Klebsiella pneumoniae> Bacillussubtilis> Salmonella typhi> Staphylococcus epidermidis. The cytotoxic potential of the AmC fraction was analyzed in skin epidermoid carcinoma (A431) cells, osteosarcoma (MG-63) and cervical (HeLa) cell lines with a GI₅₀ value of 47.04 μg/ml, 91.46 μg/ml and 102.90 μg/ml, respectively. The AmC fraction was extended further to explore its role in cell death using A431 cell line. Phase contrast and scanning electron microscopic studies on A431 cells exhibited all the characteristics indicative of apoptosis, viz., viability loss, cell shrinkage, cell rounding-off, DNA fragmentation and formation of apoptotic bodies. Flow cytometric analysis revealed enhanced ROS level, decreased MMP and arrest cell cycle at the G₀/G₁ phase further strengthened cell death by apoptosis. Increased expressions of apoptotic markers (p53, PUMA, cyt c, Fas and Apaf-1) were confirmed by RT-qPCR analysis. Furthermore, the AmC fraction was subjected to ultra-high-performance liquid chromatography, which revealed the presence of different polyphenols in the order: caffeic acid> epicatechin> kaempferol> chlorogenic acid> gallic acid> catechin> ellagic acid >umbeliferone> quercetin> coumaric acid. A critical analysis of results revealed that the AmC fraction induced cell death in epidermoid carcinoma cells via ROS and p53-mediated apoptotic pathway which may be ascribed to the presence of polyphenols in it.

Keywords: Antibacterial; Anticancer; Antioxidant; Apoptosis; Argemone mexicana; Cell cycle arrest; Cell viability; Mitochondrial membrane potential (MMP); Phytochemicals; Reactive oxygen species (ROS).