Ethanolic extracts of Pakistani euphorbiaceous plants induce apoptosis in breast cancer cells through induction of DNA damage and caspase-dependent pathway

Background: Numerous plants of Euphorbiaceae, the spurge family are traditionally used for the treatment of different diseases and recent studies also reported anti-oxidant, anti-inflammatory, and anti-tumor activities of these plants. However, the medicinal potential of several indigenous euphorbiaceous plants of Pakistan is not described yet. Therefore, we intended to evaluate the in vitro anti-breast cancer potential of 10 euphorbiaceous plants of Pakistan. Methods: Cytotoxic screening of ethanolic extracts of selected plants was performed by MTT assay. The qualitative phytochemical analysis was performed to find the major groups of chemicals responsible for cytotoxic activity. To determine the genotoxic effect of plant extracts, microscopic analysis was carried out. Flow cytometry and fluorescent microscopic analysis were done to detect apoptosis. To find out the expression analysis of cell cycle and cell death regulatory genes, quantitative real-time polymerase reaction (qRT-PCR) was performed. Results: Among the 10 tested plants, ethanolic extracts of Croton tiglium (CTL) and Euphorbia royleana (ERA) were found to possess the highest anti-proliferative activity against breast cancer cells (MDA-MB-231, MCF-7), with IC50 values 100 and 80 mu g/mL respectively. The phytochemical analysis confirmed the presence of phenols, flavonoids, and steroids in both plant extracts, whereas, glycosides and saponins were found only in CTL and ERA, respectively. The cellular aberrations and nuclear morphologies with a distinct DNA laddering pattern substantiated the genotoxic effects. Furthermore, our data showed that CTL and ERA induce cell cycle arrest at the G1/S phase by down-regulating the CDK4 and Cyclin D1 expression followed by caspase-dependent induction of apoptosis in both MCF-7 and MDA-MB-231 cells. However, based on the activation of initiator and executioner caspases, two distinct types of apoptotic pathways are proposed for these plants. The CTL prompted extrinsic while ERA triggered the intrinsic pathways of apoptosis. Conclusion: Our data demonstrate the strong anti-proliferative and caspase-dependent apoptotic potential of CTL and ERA against breast cancer cells. Further studies are suggested to find clinical implications of these plants in breast cancer therapeutic.