Author(s): Sheetal Ramachandran and Darsan Balakrishnan Menon

Abstract: The genus Pinaceae is found to be rich in bioactive metabolites is used in Ayurveda for treating respiratory ailments. The aim of the study was to determine the effect of different protein fractions isolated from the leaves of the tree Abies webbiana Lindl, on adenocarcinomic human alveolar basal epithelial A549 cells. MTT assay was performed to check the ability of the fraction to induce cell death in cancerous A549 cells and normal NIH3T3 fibroblast cells. Acridine Orange/Ethidium Bromide staining, Hoechst staining and DNA fragmentation assays were performed. RT-PCR studies of pro-apoptotic BAX and anti-apoptotic Bcl-2 along with western blot of p-ATM, CHK-2, CDC25A, BAK and BAD were performed. Caspase-9 and 3 activities were determined using calorimetric assays. The fraction at lower concentrations was found to induce cell death in the lung cancer cells. Acridine Orange/Ethidium Bromide staining, Hoechst staining and DNA fragmentation assays were used to confirm the apoptosis inducing ability of the fraction on A549 cells. The transcriptional expression studies of the pro-apoptotic BAX and anti-apoptotic Bcl-2; and the western blot analysis of proteins that regulate apoptosis showed that the fraction upregulated the expression of pro-apoptotic factors and down regulated the expression of anti-apoptotic factors in the cancer cells. Caspase-9 and 3 activities were found to be upregulated in the calorimetric studies. With the above results, it could be concluded that the protein fraction (PF-2) induced apoptosis in the lung cancer cells via the intrinsic or the mitochondrial pathway.