Lay Ma Ma, Karsani Saiful Anuar, Malek Sri Nurestri Abd
Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia.
Int J Mol Sci. 2014 Jan 2;15(1):468-83. doi: 10.3390/ijms15010468.
1-(2,6-Dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) ethanone (DMHE) was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff.) Boerl fruits and the structure confirmed by GC-MS (gas chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) analysis. This compound was tested on the HT-29 human colon adenocarcinoma cell line using MTT (method of transcriptional and translational) cell proliferation assay. The results of MTT assay showed that DMHE exhibited good cytotoxic effect on HT-29 cells in a dose- and time-dependent manner but no cytotoxic effect on the MRC-5 cell line after 72 h incubation. Morphological features of apoptotic cells upon treatment by DMHE, e.g., cell shrinkage and membrane blebbing, were examined by an inverted and phase microscope. Other features, such as chromatin condension and nuclear fragmentation were studied using acridine orange and propidium iodide staining under the fluorescence microscope. Future evidence of apoptosis/necrosis was provided by result fromannexin V-FITC/PI (fluorescein-isothiocyanate/propidium iodide) staining revealed the percentage of early apoptotic, late apoptotic, necrotic and live cells in a dose- and time-dependent manner using flow cytometry. Cell cycle analysis showed G0/G1 arrest in a time-dependent manner. A western blot analysis indicated that cell death might be associated with the up-regulation of the pro-apoptotic proteins Bax PUMA. However, the anit-apotptic proteins Bcl-2, Bcl-xL, and Mcl-1 were also found to increase in a time-dependent manner. The expression of the pro-apoptotic protein Bak was not observed.
从大果木鳖果实的乙酸乙酯萃取物中分离出1-(2,6 -二羟基-4-甲氧基苯基)-2-(4-羟基苯基)乙酮(DMHE)，其结构经气相色谱-质谱联用仪(GC-MS)和核磁共振(NMR)分析得以确认。采用MTT(四甲基偶氮唑盐比色法)细胞增殖试验，在HT-29人结肠腺癌细胞系上对该化合物进行了测试。MTT试验结果显示，DMHE对HT-29细胞具有良好的细胞毒性作用，呈剂量和时间依赖性，但在孵育72小时后对MRC-5细胞系无细胞毒性作用。通过倒置相差显微镜检查了经DMHE处理后凋亡细胞的形态特征，如细胞皱缩和细胞膜起泡。利用吖啶橙和碘化丙啶染色，在荧光显微镜下研究了其他特征，如染色质凝聚和核碎裂。通过膜联蛋白V-异硫氰酸荧光素/碘化丙啶(FITC/PI)染色结果提供了凋亡/坏死的进一步证据，该结果显示使用流式细胞仪可呈剂量和时间依赖性地检测出早期凋亡、晚期凋亡、坏死和活细胞的百分比。细胞周期分析显示G0/G1期阻滞呈时间依赖性。蛋白质免疫印迹分析表明，细胞死亡可能与促凋亡蛋白Bax、PUMA的上调有关。然而，抗凋亡蛋白Bcl-2、Bcl-xL和Mcl-1也呈时间依赖性增加。未观察到促凋亡蛋白Bak的表达。
