Background Heterotheca inuloides, traditionally employed in Mexico, has demonstrated anticancer activities. Although it has been proven that the cytotoxic effect is attributed to cadinane-type sesquiterpenes such as 7-hydroxy-3,4-dihydrocadalene, the mechanism of action by which these agents act in tumor lines and their regulation remain unknown. This study was undertaken to investigate for first time the cytotoxic activity and mechanism of action of 7-hydroxy-3,4-dihydrocadalene and two semi-synthetic cadinanes derivatives towards breast cancer cells. Methods Cell viability and proliferation were assayed by thiazolyl blue tetrazolium bromide (MTT) assay and Trypan blue dye exclusion assay. Cell migration measure was tested by wound-healing assay. Moreover, the reactive oxygen species (ROS) and lipid peroxidation generation were measured by 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay and thiobarbituric acid reactive substance (TBARS) assay, respectively. Furthermore, expression of caspase-3, Bcl-2 and GAPDH were analyzed by western blot. Results The results showed that 7-hydroxy-3,4-dihydrocadalene inhibited MCF7 cell viability in a concentration and time dependent manner. The cytotoxic potency of semisynthetic derivatives 7-(phenylcarbamate)-3,4-dihydrocadalene and 7-(phenylcarbamate)-cadalene was remarkably lower. Moreover, in silico studies showed that 7-hydroxy-3,4-dihydrocadalene, and not so the semi-synthetic derivatives, has optimal physical-chemical properties to lead a promising cytotoxic agent. Further examination on the action mechanism of 7-hydroxy-3,4-dihydrocadalene suggested that this natural product exerted cytotoxicity via oxidative stress as evidenced in a significantly increase of intracellular ROS levels and in an induction of lipid peroxidation. Furthermore, the compound increased caspase-3 and caspase-9 activities and slightly inhibited Bcl-2 levels. Interestingly, it also reduced mitochondrial ATP synthesis and induced mitochondrial uncoupling. Conclusion Taken together, 7-hydroxy-3,4-dihydrocadalene is a promising cytotoxic compound against breast cancer via oxidative stress-induction.

中文翻译：

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来自菊花异藻和半合成卡达烯衍生物的 7-羟基-3,4-二氢卡达烯对乳腺癌细胞的细胞毒性作用：参与氧化应激介导的细胞凋亡

背景 Heterotheca inuloides 传统上在墨西哥使用，已显示出抗癌活性。尽管已证明细胞毒性作用归因于杜松烷型倍半萜烯，例如7-羟基-3,4-二氢杜松烯，但这些药物在肿瘤细胞系中的作用机制及其调节仍然未知。本研究首次研究了7-羟基-3,4-二氢杜松烯和两种半合成杜松烷衍生物对乳腺癌细胞的细胞毒活性和作用机制。方法采用噻唑蓝四唑溴化物（MTT）法和台盼蓝染料排除法测定细胞活力和增殖能力。通过伤口愈合测定来测试细胞迁移测量。此外，通过 2' 测量活性氧 (ROS) 和脂质过氧化的产生，分别进行7'-二氯荧光素二乙酸酯（DCFH-DA）测定和硫代巴比妥酸反应物（TBARS）测定。此外，通过蛋白质印迹分析caspase-3、Bcl-2和GAPDH的表达。结果结果显示7-羟基-3,4-二氢卡达林以浓度和时间依赖性方式抑制MCF7细胞活力。半合成衍生物7-(苯基氨基甲酸酯)-3,4-二氢卡达烯和7-(苯基氨基甲酸酯)-卡达烯的细胞毒性效力明显较低。此外，计算机研究表明，7-羟基-3,4-二氢卡达烯（而不是半合成衍生物）具有最佳的物理化学性质，可成为有前途的细胞毒剂。进一步考察7-羟基-3的作用机制，4-二氢卡达林表明，这种天然产物通过氧化应激发挥细胞毒性，细胞内 ROS 水平显着增加并诱导脂质过氧化就证明了这一点。此外，该化合物还能增加 caspase-3 和 caspase-9 活性并轻微抑制 Bcl-2 水平。有趣的是，它还减少了线粒体 ATP 合成并诱导线粒体解偶联。结论 综上所述，7-羟基-3,4-二氢卡达烯是一种有前途的通过氧化应激诱导对抗乳腺癌的细胞毒性化合物。