Department of Food and Nutrition, Providence University, Taichung 43301, Taiwan, R.O.C.
Faculty of Food Technology, Soegijapranata Catholic University, Semarang 50234, Indonesia.
Oncol Rep. 2024 Apr;51(4). doi: 10.3892/or.2024.8720. Epub 2024 Mar 8.
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.
α-蒎烯（α-PA）是草药中的一种天然成分，能够抑制癌细胞的活力和增殖。5-氟尿嘧啶（5-FU）是一种常用于治疗结肠癌的化疗药物，通过触发癌细胞凋亡发挥作用。本研究旨在探讨α-PA 与 5-FU 联合使用如何通过促进细胞凋亡来抑制人结肠癌细胞。通过 MTT 测定法、免疫细胞化学、Western blot 和实时定量 PCR 评估了这种治疗方法对 HT-29 细胞活力、凋亡以及 Bcl-2 家族成员、半胱天冬酶家族成员和线粒体相关分子表达水平的影响。通过评估联合指数值，确定 5-FU 和 α-PA 的组合对细胞活力具有协同抑制作用。与单独使用 5-FU 的组相比，50、100 或 250 μM α-PA 联合 5-FU 组的 Bax 蛋白表达水平更高（P<0.05）。相比之下，与单独使用 5-FU 的组相比，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（ΔΨm）更低（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使用 5-FU 的组（P<0.05）。与单独使用 5-FU 相比，用 50、100 或 250 μM α-PA 联合 5-FU 处理 HT-29 细胞后，外源性诱导的凋亡分子，包括 caspase-8 和 Bid 的 mRNA 表达水平更高（P<0.05）。用 50、100 或 250 μM α-PA 联合 5-FU 处理还增加了细胞色素 c、caspase-9 和 caspase-3 的 mRNA 表达水平，调节了细胞内凋亡（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凋亡途径对降低人结肠癌细胞 HT-29 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 Bax、Bcl-2 和 Bid 等 Bcl-2 家族成员的表达水平，调节 MMP 和 HK-2 表达水平，并增加半胱天冬酶级联分子，包括 caspase-9 和 caspase-3 的表达。此外，它可能涉及激活 caspase-8 和 caspase-3 导致凋亡的外源性凋亡途径。
