Expression of multidrug resistance proteins in retinoblastoma

AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.     

microRNA-125b对人视网膜母细胞瘤多药耐药性影响的实验研究

目的 探讨microRNA-125b（miR-125b）在人视网膜母细胞瘤细胞中多药耐药的作用及其机制。设计 实验研究。 研究对象 人视网膜母细胞瘤SO-RB50细胞。方法 用RT-PCR方法检测miR-125b视网膜母细胞瘤细胞株SO-RB50和耐药细胞株SO-Rb50/VCR中的表达变化;化学合成的miR-125b过表达（miR-125mimic 组）和抑制载体（miR-125inhibitor组）转染SO-Rb50细胞株,用MTT法和Annexin V-FITC法检测在药物长春新碱、依托泊苷和卡铂,依次作用上述转染细胞后,细胞增生力和细胞凋亡的变化;用蛋白印迹法检测miR-125b过表达和抑制表达后细胞株SO-RB50中 MAGE-A/P53蛋白的表达变化。主要指标 细胞存活率和细胞凋亡率。结果 SO-Rb50/VCR组与SO-RB50组相比,miR-125b的表达显著增高（P=0.002）;长春新碱、依托泊苷和卡铂依次作用于转染后的SO-RB50细胞株后,miR-125mimic组与miR-125inhibitor组相比,细胞存活率显著增高（P=0.000）,细胞凋亡率显著下降（P=0.000）,P53蛋白表达水平显著下降（P=0.001）,MAGE-A蛋白表达水平显著增高（P=0.004）。结论 在SO-RB50细胞中,下调miR-125b后提高肿瘤细胞对化疗药物敏感性,且miR-125b可能是通过MAGE-A/P53通路调控视网膜母细胞瘤多药耐药性。     

Retinoblastoma CSF Metastasis Cured By Multimodality Chemotherapy Without Radiation

Objective: Cerebrospinal fluid (CSF) metastasis is the most difficult type of retinoblastoma metastasis to cure, even with bone marrow transplant. Most metastatic retinoblastoma cells express P-glycoprotein causing multidrug resistance (MDR). P-glycoprotein-rich blood vessels form blood-brain and blood-eye barriers, inhibit drug entry into central nervous system (CNS) and eyes. High-dose craniospinal radiation is too morbid for treatment of young children. To cure CSF metastasis without radiation, we designed an intensive multimodality chemotherapy regimen. Method: After left eye enucleation, a 4-month-old boy with bilateral International Intraocular Retinoblastoma Classification Group E eyes and CSF metastasis was treated with 7-cycle high-dose carboplatin and etoposide, standard-dose vincristine, and high-dose/short-infusion cyclosporine to inhibit P-glycoprotein. Intraventricular drugs, non-substrate of P-glycoprotein (cytarabine), or less susceptible to MDR (topotecan), contributed to treatment of the metastasis. On achieving complete response, he was consolidated with supralethal-dosage carboplatin, etoposide, and cyclophosphamide, and his bone marrow rescued with autologous cord blood stem cells. Results: Following 1-cycle systemic chemotherapy and 2-dose intraventricular chemotherapy, the CSF metastasis cleared. The right eye tumor regressed completely. The patient remains in remission 8.3 years after diagnosis and 7.8 years post-transplant. Conclusion: Intensive multimodality chemotherapy can cure CSF metastasis in retinoblastoma without incurring extreme morbidity from craniospinal radiation.     

人腺样囊性癌多药耐药细胞系的建立

目的:建立人腺样囊性癌耐药细胞系,为阐明腺样囊性癌的多药耐药机制及逆转尉药提供模型及研究依据.方法:以长春新碱(VCR)为诱导剂,通过浓度递增间断刺激法对人腺样囊性癌细胞系(ACC)进行体外诱导耐药,建立耐VCR的腺样囊性癌细胞系ACC/VCR.细胞计数法绘制细胞生长曲线,噻唑蓝(MTT)比色法检测细胞对化疗药物敏感性.结果:ACC经体外诱导后,ACC/VCR细胞对长春新碱(VCR)、阿霉素(ADM)和平阳霉素(PYM)的耐药性明显增强,具有交叉耐药,对环磷酰胺(CTX)、氟尿嘧啶(5-FU)和顺铂(DDP)耐药性无明显变化.耐药前后ACC细胞的生长曲线、群体倍增时间和光镜下的形态无明显改变.结论:VCR可诱导腺样囊性癌细胞产生多药耐药.     

化疗药物诱导视网膜母细胞瘤细胞凋亡模型的建立

目的 检测不同类型肿瘤化疗药物对视网膜母细胞瘤（ｒｅｔｉｎｏｂｌａｓｔｏｍａ ＲＢ）细胞系ＨＸＯ－ＲＢ４４的诱导凋亡作用,建立该细胞系的细胞凋亡模型,作为今后研究化疗药物诱导ＲＢ细胞凋亡机理的工作基础。方法 将长春新碱、阿糖胞苷、氨甲喋呤、环磷酰胺、噻替派、米托蒽醌、阿克拉霉素、吡喃阿霉素、顺铂、卡铂、丝裂霉素、足叶乙甙第十二种化疗药物分别以１０＾－９、１０＾－８、１０＾－７、１０＾－６、１０＾－     

葡萄膜黑色素瘤对化疗药物的敏感性及影响机制的研究

目的研究探讨葡萄膜黑色素瘤体外化疗对6种药物的敏感性及其影响机制。方法葡萄膜黑色素瘤体外原代与传代培养,抗HMB45免疫组织化学估计肿瘤细胞纯度,MTT法检测对5-氟脲嘧啶、顺铂、噻替哌、阿霉素、长春新碱和足叶乙甙6种化疗药物的敏感性,蛋白印迹法检测耐药基因bcl-2和LRP的表达,分析其表达程度与耐药性的关系。结果体外药敏试验显示6种化疗药物临床常规剂量,对肿瘤细胞的抑制率均不能达到50%,蛋白印迹结果显示bcl-2在体外培养的肿瘤细胞中均有较高表达,而LRP表达在不同类型的肿瘤细胞中不同,表达的高低与耐药程度呈正相关。结论葡萄膜黑色素瘤体外对临床常用6种化疗药物具有较高耐受性,LRP和bcl-2均与其天然耐药性有关,LRP可能起主要作用。     

Chemotherapy for retinoblastoma

Retinoblastoma is the most common eye cancer in children. Pilot studies of chemotherapy for intraocular retinoblastoma have been reported by several groups, using different combinations, dosages, schedules, and durations of carboplatin, etoposide, or teniposide, with or without vincristine, and with or without cyclosporine to counteract multidrug resistance. All studies of chemotherapy for intraocular retinoblastoma have included consolidation by focal therapy, with or without radiation. Chemotherapy alone reduces tumor size but does not cure retinoblastoma. Focal therapy, consisting of photocoagulation, thermotherapy, cryotherapy, or brachytherapy, is necessary to consolidate chemotherapy response.     

Clinicopathological parameters and expression of P-glycoprotein and MRP-1 in retinoblastoma

PURPOSE: The immunohistochemical expressions of two multidrug resistance proteins, P-glycoprotein (P-gp) and multidrug resistance-related protein-1 (MRP-1), were studied in retinoblastoma and the correlations with the clinicopathological parameters were assessed. METHOD: Sixty-five enucleated eyes containing retinoblastoma were included in the study. Following hematoxylin-eosin staining, tumor differentiation, presence of choroidal invasion, optic nerve invasion, retinal invasion, necrosis and presence of calcification were evaluated with the light microscope. P-gp and MRP-1 expressions were evaluated immunohistochemically. RESULTS: Fifty-three eyes were enucleated primarily and 12 eyes were operated after failure of chemotherapy. P-gp and MRP-1 expressions were positive in 69.3 and 73.4% of specimens, respectively. There was no statistically significant relationship between the expressions of P-gp and MRP-1, and tumor differentiation, presence of tumor invasion or treatment with chemotherapy. CONCLUSION: Retinoblastoma intrinsically expresses both P-gp and MRP-1 and their expressions are not related to tumor differentiation. The expressions of P-gp and MRP-1 do not seem to be induced by chemotherapy and are not related to the degree of tumor invasion.     

视网膜母细胞瘤多药耐药相关蛋白的研究

目的 检测P-糖蛋白(P-Gp)、多药耐药基因相关蛋白(MRP)及肺耐药相关蛋白(LRP)在视网膜母细胞瘤(Rb)中的表达及临床意义;初步分析Rb患者临床病理指标与MRP间的关系;探讨Rb多药耐药现象的可能机制.方法 实验研究.应用免疫组织化学染色方法检测P-gp、MRP、LRP在75例Rb肿瘤标本中的表达情况.分析3种蛋白表达的相关性及其与患者年龄、性别、眼别、临床表现、组织病理分化程度等临床病理指标的相关性.各蛋白表达情况与一般临床特点、组织病理学特征的比较采用卡方检验,各蛋白间的相关性采用多元相关分析.结果 P-Gp、LRP、MRP蛋自在Rb中阳性表达率分别为64.0%、25.3%、36.0%.P-gp与LRP、P-gp与MRP、LRP与MRP的共表达阳性率分别为18.7%、32.0%、20.0%.P-gp、LRP、MRP在分化型Rb组织中的阳性表达率均高于杀分化型,且组间差异具有统计学意义(χ2=8.002,χ2=17.327,χ2=28.421;P<0.05).3种蛋白的表达均与年龄、性别、眼别无关(χ2=0.003～3.385,P>0.05).P-gp、LRP的表达分别与MRP的表达其有相关性(r=0.389,r=0.521;P<0.05).结论 Rb原发性多药耐药的形成是一个多因素、多步弱的复杂过程,与包括P-gp、LRP、MRP等在内的多项因素的参与有关.P-gp、LRP、MRP蛋白可以作为反映Rb耐药的分子基础,耐药相关标志的联合检测更有利于准确判断Rb的多药耐药状态,为Rb化学治疗提供科学的理论依据.     

Relationship between histopathological features of chemotherapy treated retinoblastoma and P-glycoprotein expression

BACKGROUND: P-glycoprotein (P-gp) has been identified as a possible mediator of chemoresistance in retinoblastoma. The aim of this study was to determine the expression of P-gp in retinoblastoma treated with chemotherapy prior to enucleation. METHODS: Seventeen enucleated specimens of retinoblastoma from 16 patients were studied. Nine had been treated with chemotherapy alone, and eight had been treated with chemotherapy and other forms of local treatment. Tumour differentiation as well as choroidal and optic nerve invasion were assessed. P-gp immunohistochemical staining was performed and evaluated as negative, low or high. RESULTS: Histopathological assessment of the cases showed that 14 of 17 eyes (82.3%) had viable retinoblastoma cells. Nine retinoblastomas were considered regressed with a well-differentiated component, five regressed retinoblastomas had viable cells with poor differentiation and three retinoblastomas had regressed leaving no viable cells. Sixteen of 17 retinoblastomas were P-gp positive.In the one case with optic nerve invasion and the three cases with massive choroidal invasion, P-gp expression was found in invading retinoblastoma cells. CONCLUSION: Almost all retinoblastomas expressed P-gp. High levels of P-gp expression might play a role in chemotherapy resistance of retinoblastoma or, conversely, chemotherapy might induce P-gp expression. These results might have an impact on management of bilateral retinoblastoma.