Induction of apoptosis by essential oil from P. missionis in skin epidermoid cancer cells

Background: The genus Pamburus (Rutaceae) comprises the only species, Pamburus missionis (Wight) Swingle. Pamburus missionis is traditionally used in the treatment of swellings, chronic rheumatism, paralysis and puerperal diseases. Purpose: The present study investigates the cancer chemotherapeutic potential of essential oil (EO) from P. missionis. Methods: EO was isolated by steam distillation and chemical composition was determined by GC-MS. Cell viability was used to detect cytotoxic activity. Mechanism of cell death was studied using Annexin V-FITC/PI binding, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis related proteins was investigated by western blot. Results: GC-MS analysis of the essential oil revealed the presence of 51 components. The major components were beta-Caryophyllene, 4(14),11-Eudesmadiene, Aromadendrene oxide-(2) and Phytol. EO inhibited the growth and colony formation ability of A431 and HaCaT cells. EO treatment induced nuclear condensation and loss of membrane integrity, DNA fragmentation, increase in sub-G1 DNA content and increase in intracellular ROS level. Inhibition of intracellular ROS by ascorbic acid and N-acetyl cysteine treatment blocked EO induced apoptosis, revealing that apoptotic activity was by ROS accumulation. EO induced apoptosis was found to be due to the loss of mitochondrial membrane potential (Delta Psi m), increase in Bax/Bcl-2 ratio, release of cytochrome c and activation of caspases (cleaved form of caspase-3, caspase-8, caspase-9) and by PARP cleavage. Conclusion: The present study revealed cancer chemotherapeutic potential of EO from P. missionis. EO induces cell death through intrinsic (mitochondrial) and extrinsic apoptotic pathway in A431 and HaCaT cells. These results suggest that EO could be used as a potential therapeutic agent for the treatment of skin epidermoid cancer.