Differential gene regulation by 9-cis and all-trans retinoic acid in neuroblastoma cells

BACKGROUND: 9-cis retinoic acid (RA) is more effective than all-trans RA at inducing neuroblastoma differentiation in vitro, and has distinct biological properties with respect to its ability to promote apoptosis in N-type neuroblastoma cells. The cellular effects of 9-cis RA may, in part, result from activation of retinoid X receptor (RXR) homodimers. If this hypothesis is correct, 9-cis RA may control the expression of a different subset of retinoid-regulated genes compared to all-trans RA. PROCEDURE: We have therefore used differential mRNA display to identify genes differentially expressed in neuroblastoma cells in response to all-trans and 9-cis RA. RESULTS: The majority of cDNAs differentially expressed in response to all-trans or 9-cis RA matched to nonredundant Genbank sequences or EST database sequences. Differential-display profiles were similar in SH SY 5Y and SH S EP cells, clonal derivatives of the mixed neuroblastoma cell line SK N SH, although there were apparent differences between these cell lines with respect to the retinoid-regulation of specific RT-PCR cDNA fragments. CONCLUSIONS: These data support the view that 9-cis and all-trans RA act via different receptor mechanisms.     

Differentiation of rhabdomyosarcoma cell lines using retinoic acid

BACKGROUND: Rhabdomyosarcoma (RMS) is the most frequent sporadic soft tissue sarcoma of childhood and adolescence. The overall 5-year survival rate for patients with RMS is 70% with the use of surgery, radiation, and chemotherapy. Novel therapeutic approaches are necessary to improve on these outcomes particularly among the more aggressive alveolar RMS (ARMS) and late stages of disease, where 5-year survival is less than 20%. Retinoids have been successfully used in the treatment of acute promyelocytic leukemia (APML) and neuroblastoma. PURPOSE: However, analysis of retinoids as a differentiating agent for RMS has been incomplete. This work examined the ability of retinoic acid (RA) to promote differentiation of RMS cell lines by examining the expression of myogenic proteins in five RMS cell lines in response to All-trans Retinoic Acid (ATRA) or 9-cis retinoic acid (CRA). RESULTS: Analysis of growth curves indicates that both retinoids suppress cell growth of Rh4 and Rh28. RD cells only responded to-CRA whereas Rh30 and Rh18 did not respond. Following treatment with ATRA FACS analysis showed an altered cell cycle with the same pattern as the growth curves. ATRA altered cellular morphology of two cell lines, Rh4 and Rh28, and induced Troponin T expression in these cells suggesting a differentiating effect. CONCLUSIONS: These studies suggest that retinoids are effective inducers of growth arrest and differentiation in some RMS cell lines, and offer a basis for further in vivo testing in mice of ATRA as a potential approach to ARMS treatment.     

Premature physeal closure following 13‐cis‐retinoic acid and prolonged fenretinide administration in neuroblastoma

Retinoid therapy has contributed to improved outcomes in neuroblastoma. Clinical trials of fenretinide report favorable toxicity and disease stabilization in patients with high risk (HR) neuroblastoma. Skeletal effects have been described with other retinoids, but not with fenretinide to date. Two patients with HR, metastatic, refractory neuroblastoma received protracted courses of oral fenretinide for more than 5 years’ duration. Both developed premature long bone physeal closure, causing limb length discrepancies; their neuroblastoma remains in remission. The radiographic and clinical findings reported suggest these skeletal abnormalities may be a consequence of treatment with 13‐cis‐retinoic acid (13cisRA) followed by prolonged oral fenretinide exposure.     

Effects of retinoic acid on proliferation,apoptosis, cytotoxicity,migration, and invasion of neuroblastoma cells

BACKGROUND: Because of the known property of less aggressiveness of differentiated cells compared to immatured cells all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. We are interested in examining the influence of retinoic acid (RA) on proliferation, apoptosis, cytotoxicity, migration, and invasion in dependence of the differentiation of neuroblastoma cells classified into N-type (SK-N-FI, SH-SY5Y), I-type (SK-PN-DW), and S-type (SK-N-LO, SK-N-MC) cells. PROCEDURE: Neuroblastoma cells were exposed to 10(-5) M RA and 200 ng/ml camptothecin (CAM) (control substance for apoptosis). Proliferation, apoptosis, and cytotoxicity were quantified by photometric assays. The influence on migration and invasion of neuroblastoma cells was examined by a scratch-test and by the measurement of the invasion through matrigel coated chamber inserts. RESULTS: In general, RA treatment induced proliferation inhibition predominantly in the cell lines SK-PN-DW (16%, P < 0.05) and SK-N-MC (8%, (P < 0.001), respectively. In the N-type cell lines SK-N-FI (P > 0.05) and SH-SY5Y (P < 0.001) no proliferation inhibition was determined conforming with no detection of apoptosis. CAM confirmed its capability to induce apoptosis in the cell lines SH-SY5Y (43.6%, P < 0.05), SK-PN-DW (54.8%, P > 0.05), and SK-N-MC (28.9%, P < 0.0 01) except for SK-N-FI with only 9.3% (P > 0.05), but after 24 hr of treatment. Minor signs of restricted migration were observed, while RA treatment reduced significantly the invasion rate through Matrigel of SK-N-FI to 13.3% (P < 0.01), SH-SY5Y to 19.2% (P < 0.05), SK-N-MC to 27.8% (P < 0.05), and SK-N-LO to 17.7% (P < 0.01). CONCLUSIONS: It is demonstrated that RA treatment can interfere with cell growth and in invasion by inducing neuronal differentiation in N-type and apoptosis in S-type neuroblastoma cell lines.     

强烈化疗和自体外周血造血干细胞移植及维甲酸治疗晚期神经母细胞瘤

目的 探讨强烈化疗、自体造血干细胞移植及维甲酸对晚期神经母细胞瘤的治疗效果方法 研究Ⅳ期神经母细胞瘤患儿6例,年龄4～8岁,发病时间1个月～1年;原发部位腹部5例、胸部1例,均有骨髓转移,1例有多发性骨转移及球后病变。成立多学科参与的治疗小组,采用术前化疗、手术切除、强烈化疗、局部放疗、自体外周血造血干细胞移植及维甲酸生物治疗等。结果 经综合治疗造血干细胞移植前6例均达到完全缓解,骨髓中肿瘤细胞消失,骨转移及球后病变被控制。化疗中骨髓抑制明显,血象恢复较慢,达3～4周。感染明显,三分之一疗程后发热,用头孢他定(商品名复达欣)、亚胺培南(商品名泰能)等感染被控制。造血干细胞移植过程顺利。术后随诊4～18个月疾病处于完全缓解状态,心、肝、肾器官功能正常,骨髓恢复正常或在恢复中。结论 强烈化疗、自体外周血造血干细胞移植及维甲酸是治疗Ⅳ期神经母细胞瘤的有效方法。     

A phase 2 trial of all-trans-retinoic acid in combination with interferon-alpha2a in children with recurrent neuroblastoma or Wilms tumor: A Pediatric Oncology Branch, NCI and Children's Oncology Group Study

BACKGROUND: The combination of the antiproliferative and differentiation-inducing effects of retinoids together with the antiproliferative, immunostimulatory, and differentiation-potentiating effects of interferon-alpha (IFN-alpha) were the basis for the development of this combination in pediatric patients with refractory neuroblastoma or Wilms tumor. PROCEDURE: A phase 2 trial of all-trans-retinoic acid (ATRA), administered orally at a dose of 90 mg/m(2)/day in three divided doses for 3 consecutive days per week, and IFN-alpha2a, administered subcutaneously daily at a dose of 3 x 10(6) U/m(2)/day for 5 consecutive days per week, in 4 week cycles was performed. A two-stage design was used for each disease stratum. RESULTS: Seventeen patients (16 evaluable) with neuroblastoma, median age 9 years, and 15 patients (14 evaluable) with Wilms tumor, median age 6 years, were enrolled. Overall, the combination was well tolerated, with headache being the most common toxicity observed. There were no complete or partial responses. The median number of cycles administered was 1 (range 1-9). Four patients with neuroblastoma had stable disease for 12 or more weeks. CONCLUSIONS: The combination of ATRA and IFN-alpha2a was inactive in children with relapsed or refractory neuroblastoma and Wilms tumor. The lack of activity with this combination in children with refractory neuroblastoma is similar to the disappointing phase 2 results of single agent 13-cis-retinoic-acid (13cRA) and does not support further development of ATRA for children with relapsed neuroblastoma.     

Recognition of an in vivo immune response to human neuroblastoma modulation of antigen expression by retinoic acid

Neuroblastoma is one of the most common solid tumors of childhood and is notable for its ability to spontaneously regress and, in some instances, to differentiate to less malignant ganglioneuromas. Since immune mechanisms may account for these phenomena, identification of in vivo immune responses to tumor cell surface antigens may be important to the progression of the disease. As determined by analysis on the fluorescence-activated cell sorter, sera from 10 of 18 neuroblastomas patients were found to contain antibodies to a cell surface antigen present on subpopulations of cells from human neuroblastoma cell lines maintained in vitro. Eight human neuroblastoma cell lines were examined and found to vary in reactivity with sera. Induction of differentiation of cell lines with retinoic acid (RA) in vitro resulted in most cell lines bearing higher percentages of positive cells but with a decreased mean cell fluorescence. Preliminary Western blot analysis of lysates of the human cell lines NMB/N7, SMS-KAN, and SK-N-MC showed two principal antigen bands on reducing gels. Comparison of sera from different individuals on lysates of cell lines showed reactivity principally with bands of 105-110 kD and 65-70 kD and an additional minor band of slightly lower molecular weight with the higher titer sera. The ability of different sera to recognize a common antigen pattern suggests that this represents an immunodominant cell surface antigen. Examination of reactivity of other cell lines in this system showed that positive sera reacted with all neuroblastoma lines examined, one neuroepithelioma (SK-N-MC), two melanoma lines (MeWo, G361), and one adrenal-derived adenocarcinoma (SW-13).     

13-顺维甲酸体外诱导神经母细胞瘤干细胞分化的体外实验

目的 体外观察并鉴定13-顺维甲酸诱导神经母细胞瘤（NB）干细胞的分化作用,为临床用维甲酸治疗NB微小残留病灶提供实验依据。方法 取14例伴骨髓转移的Ⅳ期NB患儿的骨髓液标本,分离出NB细胞,将原代肿瘤细胞接种于无血清干细胞培养基中悬浮培养;在含5 μmol·L-1 13-顺维甲酸的分化培养基中培养神经球细胞,观察细胞的形态变化;RT-PCR法检测神经球细胞诱导前、诱导3和9 d Oct-4表达水平的改变;利用细胞免疫荧光技术比较诱导前及诱导9 d神经球细胞nestin表达的差异。结果 14例骨髓标本中,4例分离到原代NB细胞。无血清培养基中培养4~6 d后,可见原代悬浮神经球形成,传代后成球细胞能再次分裂增殖为神经球。神经球细胞在血清培养基中呈贴壁生长,呈三角形或星形,添加5 μmol·L-1 13-顺维甲酸后,细胞生长速度降低,形态发生明显改变。RT-PCR法检测结果显示,13-顺维甲酸诱导9 d后,神经球细胞Oct-4表达水平逐渐降低;细胞免疫荧光显示诱导9 d后神经球细胞nestin表达减弱。结论 13-顺维甲酸能有效诱导NB干细胞分化。     

视黄酸对小儿淋巴结B细胞发育的作用及其途径

目的探讨视黄酸(RA)对儿童淋巴结B细胞成熟分化的影响,及其与视黄酸受体基因表达水平变化的关系。方法本组24例,按年龄分为<1岁组、1～3岁组和～5岁组,每组8例。取患儿正常淋巴结分离细胞进行体外培养,分为RA组,RA+视黄酸受体α(RARα)拮抗剂(Ro415253)组(RA+Ro),脂多糖组(LPS),(LPS+RA)组和对照组。分别加入atRA、Ro415253、LPS。培养24、48h收集细胞。采用流式细胞术分析细胞表面标志,观察B细胞的成熟分化;RT荧光定量PCR测定视黄酸受体基因mRNA。结果<1岁组,RA组成熟B细胞的百分比明显高于对照组(24h:23%±5%vs17%±3%;48h:28%±6%vs22%±4%)(P均<0.05);LPS+RA组活化B细胞数明显高于LPS组(24h:82%±10%vs76%±8%;48h:83%±8%vs78%±10%)(P均<0.05)。同时,RARα基因表达水平也显著上调,而该受体拮抗剂可抑制RA的调节作用。1～3岁组淋巴结B细胞的成熟和活化呈现完全相同的调节变化,而～5岁组RA的这些调节作用不明显。结论RA能促进体外培养的淋巴结B细胞的分化发育和活化,该作用在3岁以下小儿明显。RARα基因的表达调节可能是介导RA作用的主要途径。     

The somatostatin analogue octreotide inhibits neuroblastoma growth in vivo.

Neuroblastoma, a neural crest-derived childhood tumor of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. However, the majority of neuroblastomas are diagnosed as metastatic tumors with a poor prognosis despite intensive multimodal therapy. The neuropeptide somatostatin (SOM) has been shown to inhibit neuroblastoma growth and induce apoptosis in vitro. Therapeutic effects of SOM analogues are dependent on tumor expression of high-affinity receptors. In the present study, human neuroblastoma SH-SY5Y cells were grown as xenografts in nude rats. In vivo SOM receptor expression in the xenografts was identified using scintigraphy with 111In-pentetreotide. Rats were randomized to treatment with the long-acting SOM analogue octreotide (10 microg s.c. every 12 h), 13-cis-retinoic acid (4 mg orally every 24 h), or vasoactive intestinal peptide (40 microg s.c. every 24 h) and compared with controls. Tumor volume was assessed every second day and tumor weight after 10-12 d. Octreotide treatment inhibited neuroblastoma growth significantly with reduced tumor volumes at 10 and 12 d compared with untreated controls (mean 3.56 and 4.24 versus 6.48 and 8.01 mL, respectively; p < 0.01). Also, tumor weights after 10-12 d were reduced in octreotide-treated animals (n = 8, median weight 2.90 g, range 1.67-5.57 g) compared with untreated rats (n = 14, 7.54 g, 1.65-10.82 g, p = 0.005). Serum IGF-I decreased significantly over time both in rats treated with octreotide and in untreated controls. It is concluded that treatment with the SOM analogue octreotide may significantly decrease neuroblastoma tumor growth in vivo. Further studies are warranted to establish the role of SOM analogues in the treatment of children with unfavorable neuroblastoma.     

Hypercalcemia and osteoblastic lesions induced by 13‐Cis‐retinoic acid mimicking relapsed neuroblastoma

A 6‐year‐old male diagnosed with extensive neuroblastoma was treated with chemotherapy, surgery, autotransplantation, and radiotherapy. He was then enrolled on a study to assess the monoclonal antibody Ch14.18 (anti‐GD2) with 13 cis‐retinoic acid. 13‐cis‐retinoic acid therapy caused severe bone pain and hypercalcemia. Bone scans showed multiple osteoblastic lesions suggesting recurrent disease however MIBG scans were negative. Serum markers of bone turnover were increased and the patient required pamidronate therapy to treat persistent hypercalcemia. Retinoic acid toxicity needs to be considered in the differential of painful osteoblastic lesions and/or hypercalcemia. MIBG scans can assist in differentiating from recurrent disease. Pediatr Blood Cancer 2009;53:666–668. © 2009 Wiley‐Liss, Inc.     

Prenatal administration of retinoic acid upregulates connective tissue growth factor in the nitrofen CDH model

Purpose  

Recent studies have suggested that retinoids may be involved in the molecular mechanisms of pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH). Connective tissue growth factor (CTGF) plays a key role in foetal lung development and remodelling during later gestation. CTGF knockout mice exhibit PH with similar characteristics to the human and nitrofen-induced PH. Prenatal administration of retinoic acid (RA) has been shown to stimulate alveologenesis in nitrofen-induced PH. In vitro studies have revealed that RA can induce CTGF gene expression. We hypothesized that pulmonary gene expression of CTGF is downregulated during the later stages of lung development, and that prenatal administration of RA upregulates CTGF in the nitrofen CDH model.     

高氧、维甲酸对早产鼠肺组织基质金属蛋白酶-2、-9 及特异性组织抑制物-1、-2表达的影响

目的探讨基质金属原蛋白-2(MMP-2)、MMP-9、基质金属蛋白酶特异性组织抑制物-1(TIMP-1)和TIMP-2在高氧肺损伤中的作用及维甲酸(RA)的保护作用机制。方法建立高氧(FiO285%)暴露早产SD大鼠肺损伤模型,应用RT-PCR法检测MMP-2、MMP-9、TIMP-1和TIMP-2mRNA表达,采用明胶酶谱检测MMP-2和MMP-9酶原及活酶表达,采用Western blot技术检测TIMP-1和TIMP-2蛋白表达。结果与空气组比较,高氧暴露4、7、14d,MMP-2、MMP-9和TIMP-1 mRNA的表达均显著升高(P均<0.01),MMP-2活酶、MMP-9酶原及活酶和TIMP-1蛋白的表达明显上调(P<0.05);RA对空气暴露下它们的表达均无明显影响(P均>0.05),但不同程度下调高氧暴露后MMP-2、MMP-9、TIMP-1mRNA的表达和MMP-2活酶、MMP-9酶原及活酶的表达,进一步提高TIMP-1蛋白表达;高氧、RA对TIMP-2 mRNA和蛋白的表达均无明显影响(P均>0.05)。结论高氧暴露明显改变MMPs/TIMPs的表达,在肺泡形成关键时期,MMPs/TIMPs之间平衡关系的破坏是造成肺发育受阻和纤维化的重要因素;通过协调MMPs/TIMPs之间的表达,改善肺泡结构,降低肺纤维化程度,从而逆转高氧所致肺损伤,是RA发挥保护作用的重要机制之一。     

Outcome of high-risk stage 3 neuroblastoma with myeloablative therapy and 13-cis-retinoic acid: a report from the Children's Oncology Group

Background

The components of therapy required for patients with INSS Stage 3 neuroblastoma and high‐risk features remain controversial.

Procedure

A retrospective cohort design was used to determine if intensive chemoradiotherapy with purged autologous bone marrow rescue (ABMT) and/or 13‐cis‐retinoic acid (13‐cis‐RA) improved outcome for patients with high‐risk neuroblastoma that was not metastatic to distant sites. We identified 72 patients with INSS Stage 3 neuroblastoma enrolled between 1991 and 1996 on the Phase 3 CCG‐3891 randomized trial. Patients were analyzed on an intent‐to‐treat basis using a log‐rank test.

Results

The 5‐year event‐free survival (EFS) and overall survival (OS) rates for patients with Stage 3 neuroblastoma were 55 ± 6% and 59 ± 6%, respectively (n = 72). Patients randomized to ABMT (n = 20) had 5‐year EFS of 65 ± 11% and OS of 65 ± 11% compared to 41 ± 11 (P = 0.21) and 46 ± 11% (P = 0.23) for patients randomized to CC (n = 23), respectively. Patients randomized to 13‐cis‐RA (n = 23) had 5‐year EFS of 70 ± 10% and OS of 78 ± 9% compared to 63 ± 12% (P = 0.67) and 67 ± 12% (P = 0.55) for those receiving no further therapy (n = 16), respectively. Patients randomized to both ABMT and 13‐cis‐RA (n = 6) had a 5‐year EFS of 80 ± 11% and OS of 100%.

Conclusion

Patients with high‐risk Stage 3 neuroblastoma have an overall poor prognosis despite aggressive chemoradiotherapy. Further studies are warranted to determine if myeloablative consolidation followed by 13‐cis‐RA maintenance therapy statistically significantly improves outcome. Pediatr Blood Cancer 2009;52:44–50. © 2008 Wiley‐Liss, Inc.     

ALL-TRANS-RETINOIC ACID-INDUCED GROWTH SUPPRESSION OF BLASTEMAL WILMS' TUMOR

All-trans-retinoic acid (RA) has been used to suppress growth of malignant cells and induce epithelial differentiation. We investigated whether RA had a similar effect on Wilms' tumor, a childhood tumor of the kidney that arises from the undifferentiated metanephric blastema. W13 cells, a cell line derived from a blastemal Wilms' tumor, were exposed to RA(10-9-10-5 M) and its effects on cell proliferation, gene expression, and differentiation were examined. Treatment of W13 cells with RA resulted in a dose-dependent suppression of growth. Changes in expression of selected genes were determined by Northern analysis. After 24 h, there was a marked dose-dependent down-regulation of N-myc mRNA as well as up-regulation of insulin-like growth factor-II(IGF-II) mRNA. [125I]IGF-II ligand blotting of conditioned medium from RA-treated cultures revealed a dramatic alteration in the pattern of expression of insulin-like growth factor binding proteins (IGFBPs). Examination of RA-treated W13 cultures by light and electron microscopy did not reveal appreciable morphological changes. We conclude that RA inhibits growth and alters gene expression of W13 cells without inducing epithelial differentiation. The modulation of expression of IGF-II, IGFBP, and N-myc may play a role in RA-induced growth suppression of Wilms' tumor cells.     

13-cis-retinoic acid (NSC 122758) in the treatment of children with metastatic neuroblastoma unresponsive to conventional chemotherapy: report from the Childrens Cancer Study Group.

The Childrens Cancer Study Group evaluated daily oral 13-cis-retinoic acid to determine its therapeutic efficacy in 28 children with advanced neuroblastoma refractory to conventional therapy. Cheilitis and fissured lips were the most common side effects; however, fewer than 50% of the patients experienced any toxicity. Two of twenty-two evaluable children demonstrated positive response to therapy. In one case, a child received the drug for 11 months. Seventeen patients demonstrated progressive disease within 28 days of the start of treatment. Three other patients with stable disease, or removed from study at day 28, were considered nonresponsive. Our data demonstrate that, when given as a single daily oral dose of 100 mg/m2, 13-cis-retinoic acid does not have significant activity in children with advanced neuroblastoma.     

维甲酸对先天性马蹄内翻足相关致病基因表达影响的研究

目的 研究维甲酸对于先天性马蹄内翻足相关基因表达的影响.方法 选取的SD大鼠交配后,分为对照组和实验组.对照组随机选取孕SD大鼠2只,于怀孕10 d时以橄榄油灌胃一次.实验组选取孕SD大鼠6只,于怀孕10 d时以140 mg/kg的维甲酸剂量(40 mg/ml溶于橄榄油中)灌胃一次.于孕16～18 d待孕鼠分娩.对照组孕鼠所产幼鼠为对照组,实验组孕鼠所产幼鼠根据是否出现后肢畸形分为畸形组及非畸形组.取两组新生幼鼠后肢软骨及肌肉组织,应用RT-PCR方法测定组织中HoxA9、TBX-3、CASP10基因表达情况,并与对照组正常新生幼鼠比较.结果 2只对照组SD孕大鼠共产仔22只,未发现畸形或死胎.6只实验组SD孕大鼠中有一只在灌胃后2 d死亡,余下可利用5只孕鼠共产仔49只,其中死胎6只,无肛无尾畸形1只,后肢出现类马蹄内翻足畸形的共30只,未出现下肢畸形共19只.畸形出现率为61.2%(30/49),并以此将实验组分为畸形组与非畸形组.荧光RT-PCR结果:实验组中畸形组在HoxA9及TBX-3基因的平均拷贝数分别为(7.00±0.52、4.55±0.52)明显低于对照组(7.92±0.47、4.99±1.02)和非畸形组(8.07±0.28、4.94±0.24),P<0.001,P<0.05;而在CASP10基因的平均拷贝数4.34±0.35则明显大于对照组3.51±0.42和非畸形组3.29±0.50(P<0.001).而对照组与非畸形组在各个基因的拷贝数比较上则无明显差异(P>0.05).结论 维甲酸能成功诱导出SD大鼠新生幼鼠类马蹄内翻足畸形的模型;维甲酸对于SD大鼠的TBX-3、HoxA9、CASP10基因的表达均有影响;维甲酸可能与通过影响TBX-3、HoxA9、CASP10基因的表达导致SD新生大鼠产生类马蹄内翻足样畸形.