Screening of antiproliferative activity mediated through apoptosis pathway in human non-small lung cancer A-549 cells by active compounds present in medicinal plants

Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptoticrelated genes. LC-MS and 1 H-NMR techniques were used to identify the active compounds present. EnzCheck caspase-3 assay kit was used to measure caspase-3 activity. Results: Methanolic extract of Vitex negundo (V. negundo) was selected as a potent fraction. Among all fractions screened, ethylacetate fraction of V. negundo was selected as the most potent antiproliferative fraction and phytochemical analysis of the extract revealed the presence of secondary metabolites. Ethaylacetate fraction of V. negundo was found to cause characteristic apoptotic morphological changes and generation of ROS in A-549 cells. Ethaylacetate fraction of V. negundo also induced apoptosis in A-549 which was supported by DNA fragmentation and DAPI staining. To investigate the molecular mechanism behind the cytotoxic effect of ethaylacetate fraction of V. negundo, quantitative real-time PCR was used to measure expression levels of p53, bax, bcl2, casp-3 and casp-9. Using LC-MS and 1 H-NMR techniques, cytotoxic compounds (luteolin and p-hydroxy benzoic acid) were identified which increased casp-3 activity in a dose and time-dependent manner in A-549 treated cells. Conclusions: It is concluded from the present study that V. negundo is capable of triggering growth-inhibitive and apoptosis effects in A-549 cells, signifying that V. negundo may possesses anti-lung cancer activity.