Department of Pharmacology, Faculty of Medicine, Khon Kaen University, 40002, Thailand; Research Institute for Human High Performance and Health Promotion, Khon Kaen University, 40002, Thailand.
Merz Healthcare (Thailand) Company Limited, Bangkok 10110 Thailand.
Biomed Pharmacother. 2021 Jul;139:111628. doi: 10.1016/j.biopha.2021.111628. Epub 2021 Apr 30.
Pinus kesiya Royle ex Gordon (PK), widely found in Southeast Asia, has been traditionally used for the treatment of several illnesses. Our previous studies showed that PK was highly cytotoxicity against liver cancer cells. The detailed mechanism of anticancer action of 50% hydro-ethanolic extract of PK's twig was, therefore, investigated in hepatocellular carcinoma HepG2 cells. Cytotoxicity of PK was determined by using NR assay, followed by determination of the mode of cell death by flow cytometry. The apoptosis-inducing effect was determined based on caspases activity, mitochondria membrane potential change, and expression of proteins related to apoptosis by western blot. The biomolecular alteration in the PK-treated HepG2 cells was investigated by FTIR microspectroscopy. Inhibition of topoisomerase I enzyme was determined by using DNA relaxation assay. Results showed that PK displayed high selective cytotoxicity and induced apoptosis against HepG2. FTIR microspectroscopy indicated that PK altered major biomolecules in HepG2 different from melphalan (a positive control), indicating a different mechanism of anticancer action. PK induced apoptotic cell death through the intrinsic pathway by increasing caspases 9 and 3/7 activity, increasing Bax, and decreasing Bcl-2 expression leading to mitochondrial membrane potential changes. PK also inhibited Top I and PARP activity that triggered an intrinsic apoptotic pathway. The phytochemical test presented terpenoids (i.e., α-pinene confirmed by GC-MS), alkaloids, steroids, xanthone, reducing sugar, and saponin. α-Pinene exhibited low cytotoxicity against HepG2, therefore, several terpene derivatives may work synergistically for inducing apoptosis. Our data demonstrated that PK has the potential for further study with chemotherapeutic purposes.
松科植物云南松（PK）广泛分布于东南亚，传统上用于治疗多种疾病。我们之前的研究表明，PK 对肝癌细胞具有高度的细胞毒性。因此，我们研究了 PK 嫩枝 50%水-乙醇提取物在肝癌 HepG2 细胞中的抗癌作用机制。通过 NR 测定法测定 PK 的细胞毒性，然后通过流式细胞术测定细胞死亡方式。通过测定 caspase 活性、线粒体膜电位变化和与凋亡相关蛋白的表达来确定诱导凋亡的作用。通过傅里叶变换红外（FTIR）微光谱研究 PK 处理的 HepG2 细胞中的生物分子变化。通过 DNA 松弛测定法测定拓扑异构酶 I 酶的抑制作用。结果表明，PK 对 HepG2 具有高选择性细胞毒性和诱导凋亡作用。FTIR 微光谱表明，PK 改变了 HepG2 中的主要生物分子，与美法仑（阳性对照）不同，表明其抗癌作用机制不同。PK 通过增加 caspase 9 和 3/7 活性、增加 Bax 和减少 Bcl-2 表达导致线粒体膜电位变化，通过内在途径诱导凋亡细胞死亡。PK 还抑制拓扑异构酶 I 和 PARP 活性，触发内在凋亡途径。植物化学测试显示萜类化合物（即通过 GC-MS 确认的 α-蒎烯）、生物碱、类固醇、蒽酮、还原糖和皂苷。α-蒎烯对 HepG2 的细胞毒性较低，因此，几种萜烯衍生物可能协同诱导凋亡。我们的数据表明，PK 具有进一步研究用于化学治疗的潜力。
