Nordin Noraziah, Majid Nazia Abdul, Hashim Najihah Mohd, Rahman Mashitoh Abd, Hassan Zalila, Ali Hapipah Mohd
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia.
Drug Des Devel Ther. 2015 Mar 10;9:1437-48. doi: 10.2147/DDDT.S77727. eCollection 2015.
Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent.
美丽黄肉楠是一种来自马来西亚的热带植物，属于番荔枝科。这种植物富含异喹啉生物碱。在本研究中，异喹啉生物碱莲叶桐碱作为一种潜在的抗癌剂进行了检测，尤其是在卵巢癌方面。通过制备型高效液相色谱法分离出莲叶桐碱。通过细胞活力检测来确定细胞毒性，同时通过吖啶橙/碘化丙啶检测来观察形态学变化。分别通过膜联蛋白V-异硫氰酸荧光素和DNA梯状条带检测来检测早期和晚期凋亡。通过半胱天冬酶-3、半胱天冬酶-8和半胱天冬酶-9分析来检测信号通路的参与情况。使用细胞毒性3检测法在 mitochondria 中进一步确认信号通路。在蛋白质水平上确认了凋亡，包括 Bax、Bcl-2和生存素，同时细胞周期的中断用于凋亡的最终验证。结果表明，在暴露24小时后，37.3 μM的莲叶桐碱可抑制CAOV-3细胞的增殖。通过细胞膜起泡、染色质浓缩和凋亡小体的形成检测到细胞形态的变化。早在24小时就通过与细胞膜结合的膜联蛋白V-异硫氰酸荧光素观察到早期凋亡。莲叶桐碱通过诱导半胱天冬酶-3和半胱天冬酶-9激活内源性途径。可以看到内源性途径在线粒体中的参与，线粒体通透性和细胞色素c释放显著增加，而线粒体膜电位降低。暴露于莲叶桐碱72小时后发生DNA片段化。DNA片段化的存在表明CAOV-3细胞经历晚期凋亡或凋亡的最后阶段。在蛋白质水平上对凋亡的确认显示Bax过表达以及Bcl-2和生存素受到抑制。莲叶桐碱抑制CAOV-3细胞周期在S期的进展。这些发现表明莲叶桐碱可被视为一种有前景的抗癌剂。
