Department of Natural Sciences, Lebanese American University, Byblos, 1102 2801, Lebanon.
School of Pharmacy, Lebanese American University, Byblos, 1102 2801, Lebanon.
J Ethnopharmacol. 2020 May 10;253:112545. doi: 10.1016/j.jep.2020.112545. Epub 2020 Jan 7.
Cedrus libani A. Rich (C. libani) is majestic evergreen Mediterranean conifer growing in the mountains of Lebanon. The ethnobotanical and traditional uses of cedar wood oil traces back to ancient times for the treatment of various ailments including cancer. Previous work in our laboratories revealed that himachalol (7-HC), a major sesquiterpene isolated from C. libani, possesses potent cytotoxic activity against various human cancer cell lines as well as promising anti-inflammatory effect in isolated rat monocytes.
The present study aims to elucidate the mechanism of action behind the cytotoxic activity of 7-HC against murine melanoma cells (B16F-10) and evaluates its chemopreventive effect against chemically-induced skin carcinogenesis in mice.
7-HC was extracted and purified from Cedrus libani wood. Cell viability was evaluated using WST-1 kit. Cell cycle analysis and apoptosis were assessed by Flow cytometry using propidium iodide (PI) and fluorescein Isothiocyanate (FITC)-conjugated Annexin V/PI staining respectively. Apoptosis related protein were quantified using western blot. The chemopreventive activity of 7-HC was evaluated for 20 weeks using a DMBA/TPA induced skin carcinogenesis model in Balb/c mice.
7-HC displayed a potent anti-proliferative activity against the melanoma cells with an IC50 of 8.8 μg/ml and 7.3 μg/ml at 24 and 48 h, respectively. Co-treatment with Cisplatin did not show any synergistic or additive effect on cell viability. Flow cytometry analysis using PI revealed that 7-HC treatment (5 and 10 μg/ml) induces the accumulation of cells in the sub-G1 phase and causes a decline in cell populations in the S and G2/M phases. Annexin/PI staining also reveals that 7-HC treatment significantly increases the percentage of cells undergoing early and late apoptosis. Western blot analysis shows that 7-HC treatment decreases the level of the anti-apoptotic protein Bcl-2 and increases the level of the pro-apoptotic protein Bax. A reduction in the level of phosphorylated Erk and Akt was also observed. 7-HC via topical (2.5%), intraperitoneal (10, 25 and 50 mg/kg) or gavage (50 mg/kg) treatment revealed a significant decrease in papilloma volume with no adverse effect on liver and kidney function.
The present study demonstrates that 7-HC treatment protects against chemically-induced skin carcinogenesis, promotes cell cycle arrest and induces apoptosis partially through an inhibition of both the MAPK/Erk and PI3K/Akt pathways.
黎巴嫩雪松（Cedrus libani A. Rich）是一种雄伟的地中海常绿针叶树，生长在黎巴嫩山区。雪松木材油的民间植物学和传统用途可以追溯到古代，用于治疗各种疾病，包括癌症。我们实验室之前的工作表明，从黎巴嫩雪松中分离出的倍半萜 7-HC（7-HC）具有针对各种人类癌细胞系的强大细胞毒性活性，以及在分离的大鼠单核细胞中具有有希望的抗炎作用。
本研究旨在阐明 7-HC 对小鼠黑色素瘤细胞（B16F-10）的细胞毒性活性背后的作用机制，并评估其对化学诱导的皮肤癌变的化学预防作用在小鼠中。
从黎巴嫩雪松木材中提取和纯化 7-HC。使用 WST-1 试剂盒评估细胞活力。通过碘化丙啶（PI）和异硫氰酸荧光素（FITC）缀合的 Annexin V/PI 染色，使用流式细胞术评估细胞周期分析和细胞凋亡。使用蛋白质印迹法定量细胞凋亡相关蛋白。使用 DMBA/TPA 诱导的皮肤癌变模型在 Balb/c 小鼠中评估 7-HC 的化学预防活性 20 周。
7-HC 对黑色素瘤细胞表现出强大的抗增殖活性，在 24 和 48 小时时，IC50 分别为 8.8μg/ml 和 7.3μg/ml。顺铂联合治疗对细胞活力没有显示出任何协同或相加作用。使用 PI 的流式细胞术分析表明，7-HC 处理（5 和 10μg/ml）诱导细胞在 sub-G1 期积累，并导致 S 和 G2/M 期细胞群体减少。Annexin/PI 染色也表明，7-HC 处理可显著增加早期和晚期凋亡细胞的百分比。蛋白质印迹分析显示，7-HC 处理降低了抗凋亡蛋白 Bcl-2 的水平，并增加了促凋亡蛋白 Bax 的水平。还观察到磷酸化 Erk 和 Akt 水平降低。通过局部（2.5%）、腹腔内（10、25 和 50mg/kg）或灌胃（50mg/kg）给予 7-HC 治疗可显著降低乳头瘤体积，且对肝功能和肾功能无不良影响。
本研究表明，7-HC 治疗可预防化学诱导的皮肤癌变，通过抑制 MAPK/Erk 和 PI3K/Akt 通路部分促进细胞周期停滞和诱导细胞凋亡。
