α‑Phellandrene (α‑PA), a natural constituent of herbs, inhibits cancer cell viability and proliferation. 5‑Fluorouracil (5‑FU) is a frequently utilized chemotherapeutic medicine for the treatment of colon cancer, which works by triggering cancer cell apoptosis. The present study examined how the combination of α‑PA and 5‑FU affects the suppression of human colon cancer cells by promoting apoptosis. The impact of this treatment on cell viability, apoptosis, and the expression levels of Bcl‑2 family members, caspase family members and mitochondria‑related molecules in HT‑29 cells was assessed by the MTT assay, immunocytochemistry, western blotting and quantitative PCR. The combination of 5‑FU and α‑PA had a synergistic inhibitory effect on cell viability, as determined by assessing the combination index value. Bax protein expression levels were higher in the 50, 100 or 250 µM α‑PA combined with 5‑FU groups compared with those in the 5‑FU alone group (P<0.05). By contrast, Bcl‑2 protein expression levels and mitochondrial membrane potential (MMP, ΔΨm) were lower in the 100 or 250 µM α‑PA combined with 5‑FU groups than those in the 5‑FU alone group (P<0.05). In addition, hexokinase‑2 (HK‑2) protein expression levels were lower in the 50, 100 or 250 µM α‑PA combined with 5‑FU groups than those in the 5‑FU alone group (P<0.05). Compared with 5‑FU alone, after HT‑29 cells were treated with 50, 100 or 250 µM α‑PA combined with 5‑FU, the mRNA expression levels of extrinsic‑induced apoptotic molecules, including caspase‑8 and Bid, were higher (P<0.05). Treatment with 50, 100 or 250 µM α‑PA combined with 5‑FU also increased the mRNA expression levels of cytochrome c, caspase‑9 and caspase‑3, regulating intrinsic apoptosis (P<0.05). These results showed that α‑PA and 5‑FU had a synergistic effect on reducing the viability of human colon cancer HT‑29 cells by inducing extrinsic and intrinsic apoptosis pathways. The mechanism by which apoptosis is induced may involve the intrinsic apoptosis pathway that activates the mitochondria‑dependent pathway, including regulating the expression levels of Bcl‑2 family members, including Bax, Bcl‑2 and Bid, regulating MMP and HK‑2 expression levels, and increasing the expression of caspase cascade molecules, including caspase‑9 and caspase‑3. In addition, it may involve the extrinsic apoptosis pathway that activates caspase‑8 and caspase‑3 leading to apoptosis.

中文翻译：

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α-水芹烯通过调节线粒体依赖性途径增强 5-氟尿嘧啶诱导的 HT-29 细胞凋亡。

α-水芹烯 (α-PA) 是草药的天然成分，可抑制癌细胞的活力和增殖。5-氟尿嘧啶 (5-FU) 是一种常用的治疗结肠癌的化疗药物，通过触发癌细胞凋亡发挥作用。本研究探讨了 α-PA 和 5-FU 的组合如何通过促进细胞凋亡来抑制人类结肠癌细胞。通过 MTT 测定、免疫细胞化学、蛋白质印迹和定量 PCR 评估这种处理对 HT-29 细胞中细胞活力、细胞凋亡以及 Bcl-2 家族成员、caspase 家族成员和线粒体相关分子表达水平的影响。通过评估组合指数值确定，5-FU 和 α-PA 组合对细胞活力具有协同抑制作用。50、100或250 µM α-PA联合5-FU组Bax蛋白表达水平高于单独5-FU组（P<0.05）。相比之下，100或250 µM α-PA联合5-FU组的Bcl-2蛋白表达水平和线粒体膜电位（MMP，ΔΨm）低于单独5-FU组（P<0.05）。此外，50、100或250 µM α-PA联合5-FU组的己糖激酶-2（HK-2）蛋白表达水平低于单独5-FU组（P<0.05）。与单独使用5-FU相比，50、100或250 µM α-PA联合5-FU处理HT-29细胞后，外源性诱导凋亡分子（包括caspase-8和Bid）的mRNA表达水平更高（P<0.05）。50、100或250 µM α-PA联合5-FU治疗也增加了细胞色素c、caspase-9和caspase-3的mRNA表达水平，调节内在细胞凋亡（P<0.05）。这些结果表明，α-PA 和 5-FU 通过诱导外源性和内源性细胞凋亡途径，对降低人结肠癌 HT-29 细胞的活力具有协同作用。诱导细胞凋亡的机制可能涉及激活线粒体依赖性途径的内在凋亡途径，包括调节Bcl-2家族成员（包括Bax、Bcl-2和Bid）的表达水平，调节MMP和HK-2的表达水平，并增加 caspase 级联分子的表达，包括 caspase-9 和 caspase-3。此外，它可能涉及激活 caspase-8 和 caspase-3 导致细胞凋亡的外源性凋亡途径。