Siripong P*, Yahuafai J*, Shimizu K**, Ichikawa K**,
                                     Yonezawa S**, Asai T**, Kanokmedakul K***, Ruchirawat S****, Oku N**.
                                     *Natural Products Research Section, Research Division, National Cancer Institute. 
                                      **Department of Medical Biochemistry and COE Program in the 21th Century, Graduate School of Pharmaceutical                                      Sciences, University of Shizuoka
                                       ***Department of Chemistry, Khon Kaen University
                                     ****Laboratory of Medicinal Chemistry, Chulabhorn Research Institute
                                     Email: pongpun8@yahoo.com
                                                                              
                                                                                  Abstract                   


              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.

 Keywords : Rhinacanthus nasutus; naphthoquinone ester; rhinacanthin; liposome; antitumor activity
 Biol Pharm Bull. 2006 Nov;29(11):2279-83.