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...

Purpose: Survivin is one of the apoptosis inhibitor genes and is rarely expressed in adult ­tissues. However, survivin expression has been detected in various human cancers and ­correlations have been recognized between the level of expression of this gene in tumors and prognosis. In this study, we investigated the correlations between survivin mRNA expres­sion in osteosarcoma tissues and clinicopathological parameters.Methods: There were 22 osteosarcoma patients in our hospital with paraffin-embedded ­tissues which could be extracted from biopsy specimens. We used the RT-PCR method after extracting total RNA and conducted a densitometric analysis to determine the ratio of survivin relative to h-GAPDH as an internal marker.Results: Expression of survivin mRNA was detected in all osteosarcoma samples. Patients with metastasis had high survivin mRNA levels in initial biopsy specimens (p<0.01). Moreover, there was a statistically significant difference in survivin mRNA expression between ­patients with and without metastasis (p<0.01).Conclusion: We concluded that high levels of survivin mRNA expression suggest poor prognosis for osteosarcoma patients.

Neuroblastoma (NB) is a very common malignant solid tumor in childhood. Prognosis in NB patients tends to vary greatly, and many studies have demonstrated that both clinical and molecular biological factors are correlated with outcome. 1) For example, patients under the age of 1 year at diagnosis usually have good prognoses, but those diagnosed over the age of 1 year have poor prognoses.2) Increased expression of the molecular biological factors MYCN, H-ras and trkA is well known in NB. [3][4][5][6][7][8][9][10][11] Recently, there has been great interest in apoptosis, or programmed cell death, the mechanism by which cells essentially suicide.12) Many inhibitors of apoptosis are known to contribute to tumorigenicity and increased spread of tumor cells.13) Survivin is a recently described member of the inhibitor of apoptosis protein (IAP) family.14) This gene exists on chromosome 17q and inhibits apoptosis by blocking the effects of caspase-9, which is activated in extrinsic and intrinsic pathways.14-17) Survivin is expressed in many malignant tumors, including breast, lung, stomach, colon and pancreatic cancers, bladder tumors, malignant lymphoma, and NB.18) It is not usually present in normal tissues and is rarely found in mature tissues.17) Thus, survivin expression is likely to be an important prognostic factor in tumor malignancy, and we considered that survivin mRNA expression would be useful in determining tumor malignancy and prognosis in NB.We therefore used reverse transcription-polymerase chain reaction (RT-PCR) to investigate the expression of survivin mRNA in NB cell lines, normal blood cell samples, and clinical NB tumor samples.Here, we describe how the degree of expression of survivin mRNA is a very useful prognostic indicator. MATERIALS AND METHODSCell Lines, Clinical NB Tumor Samples, and Normal Blood Cell Samples Three NB cell lines 19,20) SK-N-SH, 20,21) and NB- 39 20) ) were examined. They were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 g/l sodium bicarbonate under 5% CO 2 at 37°C. Two normal adult blood cell samples and 13 clinical NB tumor samples were also examined. Two of the tumor samples were from recurrent tumors. These tumor tissues had been stored at Ϫ80°C since collection. The clinical diagnoses for these patients had been made by histopathology. Informed consent was obtained from all patients before the study began.RNA Extraction Total RNA from the three cell lines and 13 NB tumor samples was extracted with TRI ZOL reagent (Gibco BAL) by the acid-guanidium-phenol chloroform extraction method.22) Total RNA from the two normal blood cell samples was extracted with TRI ZOL LS Reagent (Gibco BAL) by acid-guanidium-phenol chloroform extraction method. 16)Reverse Transcription-Polymerase Chain Reaction For determination of survivin mRNA expression, total RNA (1 mg) was reverse-transcribed in a 10 ml reaction mixture with a first strand cDNA synthesis kit (Rever Tra-a-TM , Toyobo). RT was performed with Oligo...

Survivin expression has been detected in various cancers and correlations have been recognized between the level of expression of this gene in tumors and prognosis. However, the aforementioned authors did not evaluate correlations between prognosis and survivin expression levels using surgically resected samples. In this study, we retrospectively investigated outcomes by examining the correlations between expression of this gene and clinicopathological parameters. Biopsy and resected specimens from which paraffin-embedded tissues could be extracted, were available from 16 patients in our hospital. We used the RT-PCR method and conducted a densitometric analysis to determine the ratio of survivin relative to h-GAPDH as an internal marker. Expression of survivin mRNA was detected in all samples. There was a significant negative correlation between survivin expression levels and duration of follow up, in months, using the Spearman's rank for the initial biopsy samples (rho¼À0.775, p<0.01) and those obtained after chemotherapy (rho¼À0.687, p<0.01). Moreover, Cox multivariate regression identified the survivin expression levels in both biopsy and post-chemotherapy samples as independent predictors of survival. We conclude that survivin levels in both initial biopsy and post-chemotherapy samples are useful prognostic indicators. ß

Neuroblastoma (NB), which is a malignant tumor of young children derived from neural crest cells that occurs in children, exhibits a wide range of clinical behaviors, from spontaneous regression to rapid progression. Advanced NB patients have a poor prognosis, and recently, autologous bone marrow transplantation (BMT) and autologous peripheral blood stem cell transplantation (PBSCT) have been attempted to improve the prognosis of these patients. In this study, we attempted to detect the expression of tyrosine hydroxylase (TH), neuroendocrine protein gene product (PGP) 9.5, ELAVL-4 and GD2 synthetase (GALGT), all of which are highly expressed in NBs, by the reverse transcription-polymerase chain reaction (RT-PCR) technique in order to detect minimal residual disease (MRD) in the bone marrow (BM) and peripheral blood (PB). Analysis of various tumor cell lines (Ewing's sarcoma, hepatoma, leukemias, and breast cancer cell lines in addition to NBs), and human normal samples (BM and PB cells) revealed that TH was the most specific marker for the detection of NB. On the other hand, PGP9.5 was the most sensitive marker, and was detected even when there was only one positive cell per 10 7 negative cells. We concluded that TH is a better marker before the diagnosis of NB while PGP9.5 is a better marker to detect MRD after the diagnosis. Here, we describe our results on useful markers to detect MRD in patients with NB.