Citrus macroptera induces apoptosis via death receptor and mitochondrial mediated pathway as prooxidant in human non-small cell lung cancer cells

The role of plant-based prooxidants as the selective anticancer agents is of a great promise. Citrus macroptera commonly known as "Satkara", is an important herbal and medicinal plant native to Southeast Asia. Its fruit is used in cooking different kinds of meat and pickle preparation for extra flavour and as folk medicine. In this study, the antiproliferative effect of ethyl acetate extract of C. macroptera peel (CPEA) was demonstrated on human non-small cell lung cancer cells through prooxidant induced apoptosis. Fatty acids, eight coumarins (three furanocoumarins) and one triterpenoid were identified from the CPEA through rigorous spectroscopic (UV, H-1 and C-13 NMR) and spectrometric (GC-MS, LC-ESI-HRMS) studies. Cytotoxicity, apoptotic rates, expression of apoptosis related genes, caspases activity and oxidative stress induced by reactive oxygen species (ROS) overproduction and mitochondrial membrane potential (MMP) damage in A549 cells were evaluated. MTT and flow cytometry analysis confirmed inhibition of A549 cell proliferation in a concentration-dependent manner with early apoptosis. qRT-PCR studies showed CPEA significantly upregulated p53, Bax, FasL, FADD and Bid mRNA expression levels and lowered Cdk1 and Bcl-2 compared to control. Caspase -9, -8 and -3 were also activated in cells treated by CPEA in both real-time and colorimetric/flurometric analysis. These results indicate that C. macroptera extract acts as prooxidant and induces cell death targeting both mitochondrial-and death receptor pathways of apoptosis in A549 cells.