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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.

Neuroblastoma is the most common solid pediatric tumor and remarkable for its clinical heterogeneity. Despite recent advances in chemotherapy, the prognosis of advanced neuroblastoma is still very poor. However, some favorable types of neuroblastoma, especially in infants under 1 year of age, are known to regress spontaneously or mature even if widespread metastases to bone marrow, skin and/or liver (special stage: stage IVS) are present. Apoptosis is known to occur in normal development of nervous systems, and neuroblastoma is generated from neural crest cells when the apoptotic systems do not carry out. Delayed implementation of the normal apoptotic pathway has been proposed as an explanation for the spontaneous regression of favorable neuroblastoma.1) It is reported that resistance to apoptosis plays a contributory role in the mechanism of the aggressive behavior shown by advanced neuroblastoma.2) Acute lymphocytic leukemia, like advanced neuroblastoma, is also a pediatric disease that is difficult to treat, especially in older children or those with a high amount of leukemic cells in the peripheral blood.Angelica keiskei has been used traditionally in Japan as a diuretic, laxative, analeptic and galactagogue, and an A. keiskei extract was previously reported to affect metabolic activity 3,4) and vasoconstriction 5) in rats. Moreover, A. keiskei and a major chalcone constituent of this plant, xanthoangelol, reportedly have inhibitory effects against tumor promoter activity 6,7) and metastasis. 8) Xanthoangelol possesses a chalcone structure, and some compounds related to calchones are known to have antitumor activity and to induce apoptosis. Quercetin chalcone was reported to reduce the size of implanted colon-25 tumors in vivo. 9) However, there has been no report on the effects of chalcones, including xanthoangelol, on neuroblastoma.In this study, we examined the antitumor effect and apoptosis-inducing activity of xanthoangelol against a human neuroblastoma cell line (IMR-32), and also a leukemia cell line (Jurkat) which have been widely used in previous studies of apoptosis. MATERIALS AND METHODS Materials Xanthoangelol(3Ј-C-geranyl-2Ј,4,4Ј-trihydroxychalcone) was isolated from the stem exudate of A. keiskei 6) and dissolved in dimethyl sulfoxide (DMSO) (final concentration 0.2%). IMR-32 and Jurkat were maintained in RPMI-1640 medium (Invitrogen) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum (FBS) (Invitrogen). The cells were maintained at 37°C/5% CO 2 in a humid environment.Trypan Blue Exclusion Assay The cells ( 1ϫ10 6 ) were plated into a 60-mm dish and maintained for 24 h. Xanthoangelol (final concentrations 10 Ϫ6 , 10 Ϫ5, 10 Ϫ4 M) and vehicle were applied for 48 h. For the IMR-32 cell protocol, the cells were stripped using 0.05% trypsin-EDTA solution after washing them in phosphate-buffered saline (Ϫ). They were then washed in RPMI-1640 medium (with 10% FBS) and counted with a phase-contrast microscope immediately after addition of an equal volume of 1% tr...

AimsTo determine the long-term outcomes of ocular adnexal lesions in immunoglobulin G4-related ophthalmic disease (IgG4-ROD).MethodsThis retrospective, non-randomised exploratory study included 82 patients with ocular adnexal lesions. We evaluated the long-term outcomes in 71 patients during the median follow-up period of 30 months, who underwent either watchful waiting (n=20; range 12–90 months) or systemic corticosteroid treatment, delivered according to consensus guidelines (n=51; range 9– 115 months). We also analysed factors that might contribute to recalcitrance to treatment.ResultsOf 82 patients, 40 (49%) were male, and the median patient age was 60 years old. Twenty-one (26%) patients with extraocular muscle (EOM) and/or trigeminal nerve branch (CN V) enlargements had a significantly high frequency of multiple ocular adnexal lesions (p<0.0001, Fisher’s exact test). In addition, two patients developed EOM and/or CN V enlargements de novo over time. Twenty patients with solitary lacrimal gland enlargements preferred watchful waiting, due to mild symptoms. Of these, 18 (90%) lesions remained dormant throughout a median follow-up of 27 months. Among 51 patients treated with corticosteroids, 31 (61%) experienced relapses after treatment and required systemic low-dose maintenance treatment. A multivariate analysis indicated that EOM and/or CN V enlargements comprised a risk factor for relapse (HR 2.7; 95% CI 1.1 to 6.7).ConclusionsThis exploratory study showed that different types of ocular adnexal lesions in IgG4-ROD displayed distinct proliferative activities. Our results suggested that EOM and/or CN V enlargements might be secondary lesions that confer refractoriness to systemic corticosteroid treatment recommended by consensus guidelines.