Jicui Zheng

Growth-promoting effect of bisphenol A on neuroblastoma in vitro and in vivo

PURPOSE To investigate the effect and mechanism of bisphenol A (BPA), one of the main environmental endocrine disruptors, on the proliferation of human neuroblastoma cells. METHODS In vitro, cultured SK-N-SH cells were treated with 17beta-estradiol (E(2); 1 ng/mL), BPA (2 microg/mL) with or without estrogen receptor antagonist ICI182,780 (10(-6) mol/L). Viable cell number, DNA proliferation index, and expression of cyclin-dependent kinase 4 and cyclin D1 were assessed by absorbance reading, flow cytometry, and western blotting, respectively. In vivo, ovariectomized nude mice bearing SK-N-SH tumors were administered by gavage with E(2) (500 microg/kg per day, n = 11), BPA (200 mg/kg per day, n = 10), or vehicle (n = 9) for 18 days. Mice body weight, tumor volume and weight were examined every 3 days. Tumor microvessel density, proliferating cell nuclear antigen and vascular endothelial growth factor expression were evaluated by immunohistochemical staining or western blotting. RESULTS In vitro, the BPA group had 20% higher number of viable cells, 70% higher proliferation index (both P < .01), and higher expression of cyclin-dependent kinase 4 and cyclin D1 than the nontreated group. In vivo, the BPA group had over 50% higher gross tumor volume, tumor weight, microvessel density, proliferating cell nuclear antigen (P < .05 or .01), and higher vascular endothelial growth factor protein expression than the mock control group. Both in vitro and in vivo BPA effects were comparable with those by E(2). ICI182,780 effectively abolished the promoting effect for both. CONCLUSIONS Bisphenol A can promote the growth of neuroblastoma to a level similar to that of E(2). Estrogen receptor-dependent pathway and angiogenesis may contribute to the underlying mechanisms.

Endoscopic-assisted surgery for pyriform sinus fistula in children: Experience of 165 cases from a single institution

BACKGROUND Congenital pyriform sinus fistula (PSF) is relatively rare, but often presents diagnostic and therapeutic challenges. Herein, we report our experience of endoscopic-assisted surgery of PSF in children. METHODS Since 1999, 165 children (100 males, 65 females) with PSF had been enrolled. Their clinical manifestations were recurrent lateral neck infection, cervical mass and respiratory distress. Preoperative investigations included barium swallow, ultrasound, computed tomography, and thyroid scan. After resolution of the infection, the fistulas were identified by the endoscopic-assisted technique at operation. The fistula tract was completely excised just at the apex of the pyriform sinus. More recently, the anatomic point where the fistula tract penetrated into the pharynx was specified and recorded. RESULTS Of the 165 cases, the male to female ratio was 1.54:1, the median age of onset was 3.2 years (range, 1 day to 13.8 years), and median age at operation was 5.0 years (range, 17 days to 15.0 years). One hundred fifty-six (94.6%) fistulas located on the left side, 7 right, and 2 bilateral. Twelve neonates and young infants (younger than 3 months) presented with a large cervical cyst. The fistulas were completely excised in all but 2 (98.8%) with intraoperative gastroscopy successfully conducted in 160 cases (97.0%). In 77 cases the points where fistulas penetrated into the pharynx were specified intraoperatively, which were classified into 3 types according to their anatomic relationship with the inferior cornu of the thyroid cartilage (ICTC): type I (anterior to ICTC), 22 cases (28.5%); type II (inferior to ICTC), 18 cases (23.4%); and type III (posterior to ICTC), 37 cases (48.1%). Postoperatively, 160 cases recovered well without complications. PSF recurred in 5 cases, 2 of whom were cured by fistula re-excision and 3 remained asymptomatic. Esophageal perforation was found and repaired uneventfully in 1 neonate and 1 young infant. Transient postoperative hoarseness happened in 1 neonate. All the latter 3 cases had cervical cysts. CONCLUSIONS To our knowledge, this series is the largest report of PSF in children. Our results suggested that PSF is more common in males. With the help of endoscopy and a better understanding of the anatomic relationship between ICTC and the points where the fistulas penetrated into the pharynx, PSF excision can be done successfully with minimized complications. For neonates and young infants with a cervical cyst, however, the management of PSF continues to be a challenge.

Oct4 and Sox2 are overexpressed in human neuroblastoma and inhibited by chemotherapy.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Oct4 and Sox2 are essential transcription factors for embryonic development and play key roles in determining the fate of stem cells. In this study, we aimed to investigate the expression of Oct4 and Sox2 in NB tissues, and evaluated their relationship with various clinicopathological parameters. Oct4 and Sox2 expression in 65 samples of NB and paracancerous tissues was examined by real-time PCR. The relationship between Oct4 and Sox2 expression and clinical data was assessed. To detect Oct4 and Sox2 expression at the protein level, western blot analyses and immunohistochemical staining were employed. We found that the expression levels of Oct4 and Sox2 in NB tissues were significantly higher than those in paracancerous tissues (P<0.05). Oct4 and Sox2 expression was significantly correlated to the clinical stage of NB, but not other clinicopathological parameters including patient gender and age, tumor size, location and histological classification. In stage III and IV tumors, Oct4 and Sox2 expression was significantly decreased in the chemotherapy subgroup as compared with that of the non-chemotherapy subgroup (P<0.05). These findings suggest that the expression of Oct4 and Sox2 may correlate with the genesis and progression of NB. In addition, Oct4 and Sox2 expression can be inhibited by chemotherapy.

Identification of neuroblastoma stem cells by characterization of side population cells in the human neuroblastoma SK-N-SH cell line

PURPOSE The purpose of the study was to sort side population (SP) cells from the neuroblastoma SK-N-SH cell line and to systematically investigate whether this population has stem cell characteristics. METHODS Side population and non-SP cells were separated from the SK-N-SH cell line by flow cytometry, and their morphologic characteristics were analyzed by light and electron microscopy. We also used Western blotting to analyze marker proteins, Cell Counting Kit-8 assay for proliferative ability, series differentiation studies for differentiation properties, and Matrigel invasion study and tumorigenicity assay for malignant potential. RESULTS The SK-N-SH SP cells expressed high levels of stem cell markers, had high proliferative and malignant abilities, and had the capacity for rapid differentiation. The non-SP cells expressed differentiated cell marker proteins at high levels, had low proliferative and malignant abilities, and exhibited slow differentiation. CONCLUSIONS The SK-N-SH SP cells have cancer stem cell-like properties.

Lateral buttock and postanal congenital dermal sinus tract

We present 2 rare cases of lateral buttock and postanal congenital dermal sinus tract. These 2 children were noted to have a pit in the left upper lateral buttock and a postanal orifice after birth. Magnetic resonance imaging, computed tomography, and ultrasonography showed an inflammatory mass lesion in the buttock and a funicular structure adjacent to the rectum. Fistulography showed no connection between the pit and orifice. Surgical exploration revealed firm tracts from both the lateral buttock and postanus running deep to the tip of the coccyx. Histopathologic findings showed squamous cells in the walls of the sinus tracts. Our report broadens the clinical literature regarding congenital dermal sinus tract. We postulate that this disease may not always have a connection with the central nervous system, and our cases may provide examples of anal fistulas of congenital etiology in infants.

Antiangiogenic effect of propranolol on the growth of the neuroblastoma xenografts in nude mice

BACKGROUND Propranolol has been reported to display an antiangiogenic effect on infantile hemangiomas and also some adult cancers. Little is known, however, about whether propranolol has such effect on pediatric malignancies. METHODS Nude mice bearing BE(2) C neuroblastoma xenografts were injected intraperitoneally with propranolol and divided into groups of PROP-2 (n=11), -5 (n=11), and -10 (n=10) according to the treating dosages of 2, 5, and 10 mg kg(-1) day(-1), respectively. The tumor volume and body weight were recorded every other day. All mice were sacrificed on day 9, and the levels of angiogenic factors were measured in harvested xenografts by immunohistochemistry and western blotting. RESULTS The tumor volume and weight of PROP-2 (0.72±0.28 cm(3), 0.59±0.21 g) and PROP-5 (0.81±0.35 cm(3), 0.61±0.25 g) were significantly decreased when compared with those of CTL (1.22±0.58 cm(3), 0.93±0.15 g; P<0.01). The tumor microvessel density (MVD) scores that PROP-2, -5, and -10 groups had (49.28±17.53, 52.45±17.11, and 51.09±13.18 pixels per picture, respectively) were lower than those from the control group (65.29±17.33 pixels per picture, P<0.01). Furthermore, vascular endothelial growth factor (VEGF), metalloproteinase-2 (MMP-2), and metalloproteinase-9 (MMP-9) levels were significantly lower in the groups with propranolol treated dosage of 5 and 10 mg kg(-1)day(-1) than in the control group. CONCLUSIONS Propranolol can exhibit an inhibitory effect on the tumor growth and angiogenic factors expression in neuroblastoma xenografts, which may provide some knowledge to the role of β-blockers in the management of NB.

Modified Soave procedure for the treatment of vascular malformations involving anorectum and sigmoid colon

PURPOSE The objective of this study is to introduce a modified Soave procedure for the treatment of vascular malformations involving the anorectum and sigmoid colon (VMARS) in children. METHODS Cases of 12 children with VMARS between 2000 and 2008 were reviewed and analyzed. The confirmed diagnosis was established by barium enema, colonoscopy, computed tomography, and magnetic resonance imaging. All the patients underwent a modified Soave procedure. In 11 patients, Sarasola-Klose hemorrhoidectomy was used for the distal part of endorectal dissection. RESULTS The mean length of resected bowel was 22.5 cm, ranging from 17 to 28 cm. Histologically, the surgical specimens showed that the lesions were venous malformation. Postoperative recovery was uneventful. The patients had good continence with no rectal bleeding, but intermittent fecal soiling was noted in one case. CONCLUSIONS VMARS forms a unique subset of patients with vascular malformations who have rectal bleeding. The combination of Soave procedure and Sarasola-Klose hemorrhoidectomy is a safe and effective procedure for VMARS.

SOX2 promotes tumorigenicity and inhibits the differentiation of I-type neuroblastoma cells.

SOX2 is a transcription factor associated with the pluripotency, proliferative potential, and self-renewing properties observed with embryonic stem cells and germ cells. SOX2 expression has been reported in several cancers and is implicated in tumorigenesis. We previously found that SOX2 expression was correlated to the clinical stage of neuroblastoma. Recently, we found that SOX2 overexpression occurs in I-type neuroblastoma cells (BE(2)-C cells). To elucidate the tumorigenic function of SOX2, we established a SOX2 overexpressed BE(2)-C cell line. SOX2 overexpressed cells showed higher tumorigenicity than control cells and exhibited decreased expression levels of marker proteins of N- or S-type cells after agent-induced differetiation. By contrast, in cells where SOX2 mRNA expression was knocked down by gene-specific siRNA, tumorigenicty was significantly decreased and the expression levels of marker proteins of N- or S-type cells were upregulated. In conclusion, our findings indicate an important function for SOX2 in promoting tumorigenicity of I-type neuroblastoma cells and in inhibiting their differentiation, suggesting that SOX2 might be a potential therapeutic target in neuroblastoma.

Genistein inhibits estradiol- and environmental endocrine disruptor-induced growth effects on neuroblastoma cells in vitro

The aim of this study was to examine the effect of genistein on human neuroblastoma cell proliferation induced by two common environmental endocrine disruptors, bisphenol A (BPA) and Di-2-ethylhexyl phthalate (DEHP), and to investigate its underlying mechanism. SK-N-SH human neuroblastoma cells were treated with E2 (1 ng/ml), BPA (2 μg/ml) or DEHP (100 μM), with or without genistein (12.5 μM) in vitro. The number of viable cells was detected with an absorbance reader after 0, 24, 48 or 72 h treatment. The percentage of cells in different phases, and expression of Akt and its phosphorylation levels were also assessed by flow cytometry and western blot analysis at 72 h, respectively. The BPA and DEHP groups had a 30% higher number of viable cells compared to the non-treated group at 48 h (P<0.001). However, the cell numbers did not increase significantly in the groups with additional treatment with genistein (P>0.05 vs. control) and the same trend was observed at 72 h. The expression of phospho-Akt protein was increased in the groups treated with BPA or DEHP compared to the control group at 72 h (P<0.05), while no significant elevation in the expression of phospho-Akt was observed (P>0.05) in genistein-treated groups. Cells were arrested at the G2/M phase by genistein. Similar effects were observed in the E2 group with or without genistein treatment. Akt protein expression had no significant change among all the groups (P>0.05). In conclusion, estradiol- or environmental endocrine disruptor-induced proliferation of human neuroblastoma cells is effectively abolished by genistein, likely in a cell cycle- and Akt pathway-dependent manner.

Neuroblastomas in Eastern China: a retrospective series study of 275 cases in a regional center

Purpose Most studies on neuroblastoma (NB) have been conducted in Western countries or Japan. The objective of our study was to analyze clinical and pathological features, MYCN status, surgical methods, and prognosis in Chinese NB patients. Methods A retrospective, single-center case series study of 275 NBs was implemented. Clinical manifestations, pathological features, MYCN status, and surgical treatment were analyzed. Log-rank test and Cox hazards models were used to assess overall survivals (OSs). Results The cohort consisted of 105 females and 170 males, with an age range of five days to 15 years. MYCN amplification was detected in 21.5% of all cases. The median OS was 15.0 months for MYCN amplified group. The five-year OS rates were 70.8% and 18.3% for MYCN unamplified and amplified groups, respectively, and the comparison of Kaplan–Meier curves for these two groups showed statistical significance (P < .001 by log-rank test). Gross total resection (GTR, n = 111) and subtotal resection (STR, n = 58) were administered in 169 patients at stages 3 and 4 who received chemotherapy and the comparison of Kaplan–Meier curves for different groups in these patients had statistical significance (STR vs. GTR, P = .009; MYCN unamplified vs. amplified, P < .001 by log-rank test, respectively).The multivariate survival analyses showed statistical significance (STR vs. GTR, P = .047; MYCN unamplified vs. amplified, P = .001 by Cox regression model). Conclusions MYCN amplification is an independently adverse prognostic factor in Chinese NB patients at stages 3 and 4 and GTR is associated with improved OS compared with STR in these patients.