Siripong Pongpun, Yahuafai Jantana, Shimizu Kosuke, Ichikawa Kanae, Yonezawa Sei, Asai Tomohiro, Kanokmedakul Kwanjai, Ruchirawat Somsak, Oku Naoto
Natural Products Research Section, Research Division, National Cancer Institute, Bangkok, Thailand.
Biol Pharm Bull. 2006 Oct;29(10):2070-6. doi: 10.1248/bpb.29.2070.
Rhinacanthus nasutus KURZ. (Acanthaceae) has been used as Thai traditional medicine for the treatment of various cancers. Recently, we reported that rhinacanthins, active components of the plant, had antiproliferative activity against human cancer line cells. In the present study, we investigated the growth inhibitory mechanism of rhinacanthins-C, -N and -Q, three main naphthoquinone esters isolated from the roots of R. nasutus KURZ. in human cervical carcinoma (HeLaS3) cells by means of TUNEL staining, DNA fragmentation assay, flow cytometry, and cleavage assay of Asp-Glu-Val-Asp-peptide-nitroanilide, a caspase-3 substrate. After the HeLaS3 cells was exposed with different concentrations of the drugs, rhinacanthins-C, -N and -Q exhibited antiproliferative effects on HeLaS3 cells with the IC50 values of 80, 65, 73 microM; 55, 45, 55 microM; and 1.5, 1.5 and 5.0 microM for 24, 48 and 72 h time points, respectively. Morphological changes showing nuclear fragmentation of rhinacanthins-treated cells were clearly observed after 48 h exposure. Consistent with this observation, the appearance of a ladder formation was also evident with an agarose gel electrophoresis of the extracted DNA. Flow cytometric analysis revealed that rhinacanthin-N caused G2/M arrest of HeLaS3 cells after 24 h incubation, and increased the proportion of sub-G1 hypodiploid cells, apoptotic cells, in the population of HeLaS3 cells after 48 and 72 h incubation. Moreover, the drug treatment markedly elevated the activity of caspase-3. Based on these results, our findings demonstrated for the first time that the inhibitory effects of three main naphthoquinone esters isolated from the roots of R. nasutus KURZ. on the growth of HeLaS3 cells appear to arise from the induction of apoptosis, that might be associated with the activation of caspase-3 pathway.
长穗猫须草（爵床科）已被用作泰国传统药物来治疗各种癌症。最近，我们报道了该植物的活性成分猫须草素对人类癌细胞系具有抗增殖活性。在本研究中，我们通过TUNEL染色、DNA片段化分析、流式细胞术以及对半胱天冬酶-3底物天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-肽-硝基苯胺的切割分析，研究了从长穗猫须草根部分离出的三种主要萘醌酯——猫须草素-C、-N和-Q对人宫颈癌（HeLaS3）细胞生长的抑制机制。在用不同浓度的药物处理HeLaS3细胞后，猫须草素-C、-N和-Q对HeLaS3细胞表现出抗增殖作用，在24、48和72小时时间点的IC50值分别为80、65、73微摩尔；55、45、55微摩尔；以及1.5、1.5和5.0微摩尔。在暴露48小时后，清楚地观察到经猫须草素处理的细胞出现显示核碎裂的形态变化。与该观察结果一致，提取的DNA进行琼脂糖凝胶电泳时也明显出现了梯状条带。流式细胞术分析显示，猫须草素-N在孵育24小时后导致HeLaS3细胞的G2/M期阻滞，并在孵育48和72小时后增加了HeLaS3细胞群体中亚G1期亚二倍体细胞（即凋亡细胞）的比例。此外，药物处理显著提高了半胱天冬酶-3的活性。基于这些结果，我们的研究首次证明，从长穗猫须草根部分离出的三种主要萘醌酯对HeLaS3细胞生长的抑制作用似乎源于凋亡的诱导，这可能与半胱天冬酶-3途径的激活有关。
