P S Pavithra1, Alka Mehta1, Rama S Verma2. 1. School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore-632 014, India. 2. Department of Biotechnology, Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600 036, India. Electronic address: vermars@iitm.ac.in.
Abstract
BACKGROUND: Pamburus missionis (Wight) Swingle (Rutaceae) is traditionally used in the treatment of swellings, chronic rheumatism, paralysis and puerperal diseases. In a previous study the authors demonstrated apoptotic activity of Pamburus missionis essential oil (EO) on A431 and HaCaT cells. The major components of EO were β-caryophyllene (25.40%), 4(14),11- eudesmadiene (7.17%), aromadendrene oxide 2 (14.01%) (AO-(2) and phytol (6.88%). PURPOSE OF STUDY: To investigate the role as well as the interactions among EO components inducing apoptosis in A431 and HaCaT cells. METHODS: Isobolographic analysis and combination index methods were used to detect the type of interactions among the essential oil (EO) components. Cell viability was used to detect cytotoxic activity. Mechanism of cell death was studied using Annexin V-FITC/PI binding assay, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis associated proteins was investigated by western blot. RESULTS: Combination of P. missionis EO components: β-caryophyllene/ aromadendrene oxide 2 (β-C/AO-(2)), β-caryophyllene/phytol (β-C/P) and aromadendrene oxide 2 /phytol (AO-(2)/P) inhibited growth and colony formation ability of skin epidermoid A431 and precancerous HaCaT cells. Synergistic interaction was observed between β-C/AO-(2) and β-C/P combination while AO-(2)/P exhibited an additive effect. Combination of components induced chromatin condensation, phosphatidylserine externalisation, increase in sub-G1 DNA content, cell cycle arrest at G0/G1 phase and intracellular ROS accumulation. Inhibition of intracellular ROS by N-acetyl cysteine treatment blocked apoptosis induced by the combinations. The combinations induced apoptosis in A431 and HaCaT cells mediated by loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratio, release of cytosolic cytochrome c and activation of caspases (cleaved form of caspase-3, caspase-8, caspase-9) and by PARP cleavage. CONCLUSION: The present study demonstrates interactions among β-C, AO-(2) and P in the induction of apoptosis on A431 and HaCaT cells. These data suggest the combination of β-caryophyllene with aromadendrene oxide 2 and phytol could be potential therapeutics for the treatment of skin epidermoid cancer and precancerous cells.
BACKGROUND:Pamburus missionis (Wight) Swingle (Rutaceae) is traditionally used in the treatment of swellings, chronic rheumatism, paralysis and puerperal diseases. In a previous study the authors demonstrated apoptotic activity of Pamburus missionisessential oil (EO) on A431 and HaCaT cells. The major components of EO were β-caryophyllene (25.40%), 4(14),11- eudesmadiene (7.17%), aromadendrene oxide 2 (14.01%) (AO-(2) and phytol (6.88%). PURPOSE OF STUDY: To investigate the role as well as the interactions among EO components inducing apoptosis in A431 and HaCaT cells. METHODS: Isobolographic analysis and combination index methods were used to detect the type of interactions among the essential oil (EO) components. Cell viability was used to detect cytotoxic activity. Mechanism of cell death was studied using Annexin V-FITC/PI binding assay, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis associated proteins was investigated by western blot. RESULTS: Combination of P. missionisEO components: β-caryophyllene/ aromadendrene oxide 2 (β-C/AO-(2)), β-caryophyllene/phytol (β-C/P) and aromadendrene oxide 2 /phytol (AO-(2)/P) inhibited growth and colony formation ability of skin epidermoid A431 and precancerous HaCaT cells. Synergistic interaction was observed between β-C/AO-(2) and β-C/P combination while AO-(2)/P exhibited an additive effect. Combination of components induced chromatin condensation, phosphatidylserine externalisation, increase in sub-G1 DNA content, cell cycle arrest at G0/G1 phase and intracellular ROS accumulation. Inhibition of intracellular ROS by N-acetyl cysteine treatment blocked apoptosis induced by the combinations. The combinations induced apoptosis in A431 and HaCaT cells mediated by loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratio, release of cytosolic cytochrome c and activation of caspases (cleaved form of caspase-3, caspase-8, caspase-9) and by PARP cleavage. CONCLUSION: The present study demonstrates interactions among β-C, AO-(2) and P in the induction of apoptosis on A431 and HaCaT cells. These data suggest the combination of β-caryophyllene with aromadendrene oxide 2 and phytol could be potential therapeutics for the treatment of skin epidermoid cancer and precancerous cells.