School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore-632 014, India.
Department of Biotechnology, Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600 036, India.
Phytomedicine. 2018 Aug 1;47:121-134. doi: 10.1016/j.phymed.2018.05.001. Epub 2018 May 9.
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%).
To investigate the role as well as the interactions among EO components inducing apoptosis in A431 and HaCaT cells.
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.
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.
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.
Pamburus missionis（Wight）Swingle（芸香科）传统上用于治疗肿胀、慢性风湿、麻痹和产后疾病。在之前的一项研究中，作者证明了 Pamburus missionis 精油（EO）对 A431 和 HaCaT 细胞的凋亡活性。EO 的主要成分是β-石竹烯（25.40%）、4（14）、11-艾杜马二烯（7.17%）、芳樟醇氧化物 2（14.01%）（AO-（2）和植醇（6.88%）。
研究 EO 成分诱导 A431 和 HaCaT 细胞凋亡的作用及其相互作用。
采用等辐射分析和组合指数法检测精油（EO）成分之间相互作用的类型。细胞活力用于检测细胞毒性。通过 Annexin V-FITC/PI 结合试验、细胞周期分析、流式细胞术检测 MMP 和 ROS 生成来研究细胞死亡机制。通过 Western blot 检测凋亡相关蛋白的表达。
P. missionis EO 成分的组合：β-石竹烯/芳樟醇氧化物 2（β-C/AO-（2））、β-石竹烯/植醇（β-C/P）和芳樟醇氧化物 2/植醇（AO-（2）/P）抑制皮肤表皮 A431 和癌前 HaCaT 细胞的生长和集落形成能力。β-C/AO-（2）和 β-C/P 组合观察到协同作用，而 AO-（2）/P 表现出相加作用。成分组合诱导染色质浓缩、磷脂酰丝氨酸外化、亚 G1 DNA 含量增加、G0/G1 期细胞周期停滞和细胞内 ROS 积累。N-乙酰半胱氨酸处理抑制细胞内 ROS 可阻断组合诱导的细胞凋亡。组合诱导 A431 和 HaCaT 细胞凋亡通过线粒体膜电位（ΔΨm）丧失、Bax/Bcl-2 比值增加、细胞质细胞色素 c 释放和半胱天冬酶（裂解形式的 caspase-3、caspase-8、caspase-9）激活以及 PARP 切割介导。
本研究表明，β-C、AO-（2）和 P 在诱导 A431 和 HaCaT 细胞凋亡中相互作用。这些数据表明，β-石竹烯与芳樟醇氧化物 2 和植醇的组合可能是治疗皮肤表皮癌和癌前细胞的潜在治疗方法。
