Six chalcones from Angelica keiskei KOIDZUMI (Ashitaba in Japanese) and two chalcones from Humulus lupulus L. (hop) were examined for their cytotoxicity in two human neuroblastoma cell lines (IMR-32 and NB-39) and normal cells (primary culture of rat cerebellar granule cells) by [3-(4,5)-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. All chalcones exhibited cytotoxicity against neuroblastoma cells, and two of them (isobavachalcone and xanthoangelol H) had no effect on normal cells even at high concentration (10 ؊4 M) exposure. Typical morphologic features of apoptosis, including cell shrinkage, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies, were observed in isobavachalcone-treated cells by Hoechst 33342 staining. Western blot analysis showed that isobavachalcone significantly reduced pro-caspase-3 and pro-caspase-9, and subsequently increased the level of cleaved caspase-3 and cleaved caspase-9 in both neuroblastoma cell lines. Moreover, Bax was markedly induced by isobavachalcone application. These results suggest that isobavachalcone induces apoptotic cell death in neuroblastoma via the mitochondrial pathway and has no cytotoxicity against normal cells. Therefore, isobavachalcone may be applicable as an efficacious and safe drug for the treatment of neuroblastoma.

Mulberry (Morus spp.; Moraceae), whose leaves represent indispensable food for silkworms, has been widely cultivated in China and Japan since early times. On the other hand, the root bark of mulberry (Mori Radicis Cortex) has been used in the traditional Chinese medicine as antiphlogistic, diuretic, antitussive, expectorant, antiheadache, and antipyretic. 1)Many novel compounds, regarded biogenetically as "DielsAlder-type adducts" of dehydroprenyl phenols and chalcone derivatives, have been isolated from several Morus species.2,3) Recently, albanol A 4) (mulberrofuran G 5) ) (1) and several other related compounds, isolated from the stem bark of M. australis, have been reported to show potent cytotoxic activities against four human cancer cell lines, lung (A549), stomach (BGC 823), colon (HCT-8), and ovarian (A2780) cancer cell lines, by means of the thiazolyl blue tetrazolium bromide (MTT) assay.3) In the course of our search for potential bioactive compounds from natural sources, 6,7) we were especially interested to undertake the investigation on the mechanisms of cytotoxic action of the "Diels-Alder-type adducts". In this study, we have isolated albanol A (1) and mulberofuran Q (2) from the root bark of M. alba, and have evaluated their cytotoxic activity against human leukemia (HL60) and human melanoma (CRL1579) cells. In addition, we have studied the apoptosis-inducing activity of albanol A (1), which exhibited potent cytotoxicity against HL60 cells, and the mechanisms of apoptotic cell death in HL60. Results and DiscussionAlbanol A (1) and mulberofuran Q (2) (Fig. 1) were isolated from the bark extract of Morus alba L. with ordinaland reversed-phase column chromatography and subsequent preparative HPLC. Compounds 1 5) and 2 8) were identified by MS and 1 H-NMR comparison with the literature data. On evaluation of these compounds for their cytotoxicities on HL60 and CRL1579 cell lines, compound 1 exhibited potent activity against both HL60 (IC 50 1.7 mM) and CRL1579 (9.8 mM) cell lines which were almost comparable with those of a reference compound, cisplatin (Table 1).Compound 1 was then evaluated for its inducing activity of early apoptosis on HL60 cells. Exposure of the membrane phospholipid, phosphatidylserine, to the external cellular environment is one of the earliest markers of apoptotic cell death.9) Annexin V is a calcium-dependent phospholipidbinding protein with high affinity for phosphatidylserine expressed on the cell surface. Propidium iodide (PI) does not enter whole cells with intact membranes and was used to differentiate between early apoptotic (Annexin V possitive, PI negative), late apoptotic (Annexin V, PI double positive), or necrotic cell death (Annexin V negative, PI positive). The proportion of early apoptotic HL60 cells (lower right) was significantly increased after 6 h of incubation with 1 (30 mM) Albanol A (1), isolated from the root bark extract of Morus alba (mulberry), was evaluated for the cytotoxic and apoptosis-inducing activities in human leukemia (HL60) cells, and ...

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Pulmonary arterial hypertension (PAH) is prevalent in patients with obstructive sleep apnea syndrome (OSAS). Aging induces arginase activation and reduces nitric oxide (NO) production in the arteries. Intermittent hypoxia (IH), conferred by cycles of brief hypoxia and normoxia, contributes to OSAS pathogenesis. Here, we studied the role of arginase and aging in the pathogenesis of PAH in adult (9-mo-old) and young (2-mo-old) male Sprague-Dawley rats subjected to IH or normoxia for 4 weeks and analyzed them with a pressure-volume catheter inserted into the right ventricle (RV) and by pulsed Doppler echocardiography. Western blot analysis was conducted on arginase, NO synthase isoforms, and nitrotyrosine. IH induced PAH, as shown by increased RV systolic pressure and RV hypertrophy, in adult rats but not in young rats. IH increased expression levels of arginase I and II proteins in the adult rats. IH also increased arginase I expression in the pulmonary artery endothelium and arginase II in the pulmonary artery adventitia. Furthermore, IH reduced pulmonary levels of nitrate and nitrite but increased nitrotyrosine levels in adult rats. An arginase inhibitor (N(ω)-hydroxy-nor-1-arginine) prevented IH-induced PAH and normalized nitrite and nitrate levels in adult rats. IH induced arginase up-regulation and PAH in adult rats, but not in young rats, through reduced NO production. Our findings suggest that arginase inhibition prevents or reverses PAH.