A study comparing endogenous protoporphyrin IX induced by 5-ALA and ALA-methyl ester with exogenous PpIX and PpIX dimethyl ester in photodynamic diagnosis of human nasopharyngeal carcinoma xenografts

5-Aminolevulinic acid (5-ALA) and its esters have been under intense investigation to enhance the endogenous production of protoporphyrin IX (PpIX) in tumour cells for the purpose of photodynamic diagnosis. In this study we have investigated the use of exogenous PpIX and its dimethyl ester (PME) and compared the results with endogenous PpIX produced via 5-ALA and ALA methyl ester (AME) in poorly differentiated NPC/CNE-2 nasopharyngeal carcinoma cells in both in vivo and in vitro systems. All prodrugs and photosensitizers were administered to tumour bearing balb/c nude mice either intravenously or topically. in vitro results show that 5-ALA induced more PpIX fluorescence when compared with AME in NPC/CNE-2 cells and PME showed better uptake than PpIX. in vivo results show that exogenous PpIX and PME show promise as good candidates as photosensitizers for photodynamic diagnosis as they exhibit significant selectivity between tumour tissue and normal tissue at 3 h. Modification of delivery vehicle used for application of exogenous PpIX and PME could allow for rapid uptake; better selectivity and localization of the photosensitizer.     

Macro-microscopic fluorescence imaging of human NPC xenografts in a murine model using topical vs intravenous administration of 5-aminolevulinic acid

The use of 5-aminolevulinic acid to induce endogenous porphyrins for the purpose of detection of epithelial cancers is being studied extensively in many centres around the world. The challenge is to prepare an efficacious formulation of 5-ALA for the purpose of cancer detection. In this study, we compared two formulations of topical 5-ALA applications with intravenous administration in NPC/CNE-2 xenografts on balb/c nude mice. One of the formulations was a gantrez muco-adhesive patch and the other was a polyvinyl-pyrolidone muco-adhesive patch. The Karl Storz fluorescence endoscopy system was used to obtain macroscopic fluorescence images. Microscopic fluorescence imaging was done by laser confocal microscopy. The macroscopic images were further analysed for fluorescence intensity distribution. It was found that between the two formulations of topical application of 5-ALA; there was very little difference in the fluorescence biodistribution. When the topical applications were compared with the intravenous administration, the tumour to normal differential in biodistribution was significantly higher with the topical application compared to the intravenous application.     

Monoclonal antibody A7-superparamagnetic iron oxide as contrast agent of MR imaging of rectal carcinoma

Superparamagnetic iron oxide (SPIO)-based colloid has been used clinically as a tissue-specific magnetic resonance contrast agent. We coupled monoclonal antibody A7 (Mab A7), which reacts specifically with human colorectal carcinoma, to Ferumoxides (SPIO) and examined the accumulation of this conjugate in xenografted tumours in nude mice. We examined in vitro immunoreactivity of Mab A7 coupled to Ferumoxides and its in vivo distribution in nude mice with human colorectal carcinoma. Magnetic resonance imaging of tumour-bearing nude mice was performed 72 h after injection of A7-Ferumoxides. A7-Ferumoxides retained binding activities that were nearly identical to intact Mab A7. More of the radiolabelled A7-Ferumoxides accumulated in the tumour than normal mouse IgG-Ferumoxides from 12 h onwards after injection (P<0.05). Both A7-Ferumoxides and normal mouse IgG-Ferumoxides disappeared from blood linearly over time. The accumulation levels in normal tissue decreased linearly over time but were lower than levels in tumours from 6 h. In magnetic resonance T2-weighted imaging of the tumour-bearing nude mice, signal intensity was reduced at the margin of the tumour by injection of A7-Ferumoxides. Mab A7 coupled to Ferumoxides is potentially suitable as a magnetic resonance contrast agent for detecting local recurrence of rectal carcinoma.     

Telomerase activation in nasopharyngeal carcinomas.

Nasopharyngeal carcinomas (NPC) are common in Hong Kong and southern China but rare in Western countries. Telomerase activation is common in human cancers but has not been reported previously in NPC. Telomerase activation in NPC was determined using the sensitive TRAP (telomerase rapid amplification protocol) assay in 45 nasopharyngeal biopsies (36 NPC, nine normal nasopharyngeal mucosae) in four xenografted NPC tumours established in nude mice and in five in vitro NPC cell lines. Telomerase activation is common in NPC and can be detected at high frequencies (85% in primary tumours and 100% in recurrent tumours). The frequency of telomerase activation was lowest in NPC biopsies without lymph node involvement (60%) compared with those with positive lymph node involvement (100%), and the difference is statistically significant (P < 0.05; Fisher exact test). All the xenografted NPC tumours and in vitro NPC cell lines were strongly positive for telomerase activity. Our results suggest that telomerase activation is common in NPC and it may be useful as a diagnostic marker in the detection of tumour cells in nasopharyngeal biopsies. The high frequency of telomerase activation in stage I NPC (80% positive) suggests that it is an early event in tumour progression.     

赫赛汀对鼻咽癌细胞株体内外生长的影响

 目的 探讨赫赛汀（Herceptin）对鼻咽癌细胞株体内外生长的影响. 方法 采用免疫组化技术检测鼻咽癌组织HER-2/neu基因表达;采用3H-TdR细胞掺入法测定Herceptin对鼻咽癌细胞株体外生长的影响;用荷瘤裸鼠模型观察Herceptin对鼻咽癌细胞株体内生长的影响. 结果 HER-2/neu基因在鼻咽癌组织中有过表达; Herceptin可抑制HER-2/neu基因过表达的鼻咽癌细胞株CNE-2Z 3H-TdR掺入和影响荷瘤裸鼠肿瘤的生长. 结论 Herceptin可抑制CNE-2Z细胞株体外3H-TdR掺入,并影响CNE-2Z细胞株体内的生长.     

CK、Tubulin-β和PCNA在鼻咽癌放疗后复发组织中的表达及意义

 目的 研究鼻咽癌中CK、Tubulin-β及PCNA的表达,探讨其与鼻咽癌放疗后复发和转移的可能机制。方法裸鼠体内移植瘤实验观察鼻咽癌细胞F1、S1放射治疗前后的细胞形态、体内增殖和转移能力,采用免疫组织化学SP法检测裸鼠移植瘤组织、鼻咽癌及放疗后复发组织中CK、Tubulin-β及PCNA的表达。结果放射后F1、S1裸鼠移植瘤瘤体质量分别较未放射组的F1、S1裸鼠移植瘤瘤体质量低（P<005）。各组均无肺转移,放射治疗后S1裸鼠移植瘤淋巴结转移率高于F1组,但差异无统计学意义（P>005）。未放射组F1裸鼠移植瘤组织中PCNA表达高于未放射S1裸鼠移植瘤（P<0.05）;放射治疗后S1裸鼠移植瘤中CK和PCNA表达均高于放射后的F1裸鼠移植瘤（P<0.05/0.01）。鼻咽癌放疗后复发组织中PCNA表达高于鼻咽癌组（P<0.05）。Spearman相关分析显示,鼻咽癌组织中CK与Tubulin-β、Tubulin-β与PCNA表达呈正相关关系,放疗后复发组织中CK与Tubulin-β和PCNA表达呈正相关（P<0.05）。鼻咽癌及放疗后复发组织中CK、Tubulin-β及PCNA的表达与患者年龄、性别、颈部淋巴结转移和临床分期无关。结论CK和Tubulin-β参与鼻咽癌放疗后复发的过程,并与细胞增殖关系密切,但不能独立作为鼻咽癌放疗后复发的预测指标。     

Anti-tumor effect of oncolytic herpes simplex virus G47delta on human nasopharyngeal carcinoma

Oncolytic herpes simplex virus (HSV) can replicate in and kill cancer cells without harming normal tissue. G47delta is a third-generation HSV vector. In this study, the therapeutic effects of G47delta on human nasopharyngeal carcinoma (NPC) were determined in vitro and in vivo. The human NPC cell lines CNE-2 and SUNE-1, primary normal nasopharyngeal epithelial cells (NPECs), and immortalized nasopharyngeal cells NP-69 and NPEC2/Bmi1 were infected with G47delta at different multiplicities of infection (MOIs). The survival of infected cells was observed daily. Two subcutaneous models of NPC were established with CNE-2 and SUNE-1 in Balb/c nude mice. G47delta or virus buffer as control was injected into the subcutaneous tumors. Tumor size was measured twice a week, and animals were euthanized when the diameter of their tumors exceeded 18 mm or when the animals appeared moribund. For the NPC cell lines CNE-2 and SUNE-1, more than 85% and 95% of cells were killed on day 5 after G47delta infection at MOI = 0.01 and MOI = 0.1, respectively. Similar results were observed for an immortalized cell line NPEC2/Bmi-1. A moderate effect of G47delta was also found on another immortalized cell line NP-69, of which only 27.7% and 75.9% of cells were killed at MOI = 0.01 and MOI = 0.1, respectively. On the contrary, there was almost no effect observed on NPECs. The in vivo experiments showed that tumors in mice in the G47delta-treated group regressed completely, and the mice exhibited much longer survival time than those in the control groups. Our results suggest that the potential therapeutic effects of G47delta would be applicable for treatment of NPC patients in the future.     

Inhibiting tumorigenic potential by restoration of p16 in nasopharyngeal carcinoma

The p16 gene, encodes a key checkpoint protein p16 in the cell cycle, has been reported inactivation in a wide variety of human cancers. We have previously demonstrated high frequency of p16 alterations in primary nasopharyngeal carcinoma (NPC), xenografts and cell lines. The finding implied that inactivation of the p16 gene may play an important role in the NPC development. To investigate the tumour suppressor function of p16 in NPC, we transfected p16-deficient NPC cell line, NPC/HK-1, with a wild-type p16 expression construct, and evaluated growth and tumorigenic properties of the clones stably expressing exogenous p16. Expression of the exogenous wild-type p16 significantly inhibited cell growth by more than 70% when compared to that of the parental and empty vector-transfected cells. This growth inhibition was attributable to a significant proportion of p16-expressing cells arrested at G1 phase in the cell cycle as revealed by flow cytometric analysis. By anchorage-independent colony forming assay, we found that the ability to form colonies in soft agar was highly reduced in cells expressing p16. NPC/HK1 cells expressing functional p16 also showed suppressed tumorigenicity in athymic nude mice. Taken together, our results provide strong evidence for a tumour suppressor role of p16 in NPC.     

Bcl-xl反义寡核苷酸对裸鼠人鼻咽癌移植瘤抑制作用的研究

目的　探讨bcl xl反义寡核苷酸 (ASODN)对裸鼠人鼻咽癌移植瘤生长和基因表达的抑制作用。方法　建立裸鼠人鼻咽癌模型 ,接种肿瘤细胞后 2 4h之内将bcl xlASODN、序列对照寡核苷酸 (ODN)皮下注射进行治疗 ,观察肿瘤大小变化和组织形态学改变 ,并检测药物治疗后肿瘤细胞中bcl xlmRNA和蛋白表达情况。结果　Bcl xlASODN治疗组裸鼠鼻咽癌的生长受到明显抑制 ,抑瘤率为 41.7% ,其对应的bcl xlmRNA和蛋白水平明显降低 ,与对照组相比 ,差异有显著性 (P <0 .0 1) ,而序列对照ODN治疗组抑瘤率仅为 19.0 % ,差异无显著性 ,对基因的表达亦无明显影响。结论　Bcl xlASODN对裸鼠鼻咽癌移植瘤的生长具有一定的抑制作用 ,为鼻咽癌的治疗提供新的方法。     

MiR-663, a microRNA targeting p21(WAF1/CIP1), promotes the proliferation and tumorigenesis of nasopharyngeal carcinoma

MicroRNAs (miRNAs) may function as either oncogenes or tumor suppressors in the malignant progression of different tumor types. MiR-663 was recently reported to be decreased and identified as a tumor suppressor in gastric cancer. We also verified its role in repressing cell proliferation of a gastric cancer cell line. In this study, however, miR-663 was found to be upregulated in nasopharyngeal carcinoma (NPC) cells compared with human immortalized nasopharyngeal epithelium cells, using a miRNA microarray, and this higher expression was confirmed in NPC tissue samples. Indeed, inhibition of miR-663 impaired the proliferation of NPC cells in vitro and the NPC tumor growth of xenografts in nude mice. Mechanistically, miR-663 directly targeted p21(WAF1/CIP1) to promote the cellular G1/S transition, as the inhibitory effects of miR-663 on the G1/S transition could be rescued by p21(WAF1/CIP1) silencing. Our results imply that miR-663 may act as an oncogene in NPC. The newly identified miR-663/p21(WAF1/CIP1) axis clarifies the molecular mechanism of NPC cell proliferation and represents a novel strategy for the diagnosis and treatment of patients with NPC.     

mTOR activation in immature cells of primary nasopharyngeal carcinoma and anti-tumor effect of rapamycin in vitro and in vivo

The mammalian target of rapamycin (mTOR) signaling is a key pathway in the progression of different cancers and in the homeostasis of stem cells. Here, we investigated the link between mTOR signaling and cancer stem cells (CSCs) in nasopharyngeal carcinoma (NPC). We found that human primary NPC expressed embryonic stem cell (ESC) markers: CD133, SOX2 and OCT4 as well as pmTOR and pS6. Primary ESC-positive NPC cells could form secondary NPC in BALB/c nude mice. Rapamycin, an mTOR inhibitor, significantly suppressed ESC-positive NPC cell growth in vitro and tumor formation in vivo. Our findings suggest that mTOR signaling is activated in CSC-like cells and plays an important role in NPC growth.     

茶多酚对人鼻咽癌细胞及其裸鼠移植瘤生长的抑制作用

背景与目的:许多研究报告表明,茶多酚(TP)有防癌和抗癌作用,但对鼻咽癌细胞作用的报道很少。我们最近研究证明TP对人鼻咽癌CNE2细胞有抗增殖和诱导凋亡的作用。为进一步了解TP对人鼻咽癌细胞的作用,进行TP对多种鼻咽癌细胞和人鼻咽癌裸鼠移植瘤生长影响的实验研究。方法:以MTT法检测TP对NPC/HK1、CNE1、CNE2、HNE1、HNE2、SUNE1和Fadu7种人鼻咽癌细胞株的生长抑制作用。以人鼻咽癌细胞株(CNE2)裸鼠移植瘤为模型,作体内抑瘤试验。结果:在体外抗癌细胞增殖试验中,TP对NPC/HK1、CNE1、CNE2、HNE1、HNE2、SUNE1和Fadu的IC50分别为55.83、98.43、119.21、127.74、158.07、160.72μg/ml和163.59μg/ml。在TP对人鼻咽癌细胞株(CNE2)裸鼠移植瘤的抑瘤试验的3批实验中,5mg·(kg·d)-1TP作用18天的平均抑瘤率为12.7%,P>0.05;10mg·(kg·d)-1TP作用18天的平均抑瘤率为31.0%,P<0.01;20mg·(kg·d)-1TP作用18天的平均抑瘤率为38.5%,P<0.01。结论:TP对7种人鼻咽癌细胞株均有不同程度的生长抑制作用,对人鼻咽癌CNE2裸鼠移植瘤的生长有一定的抑制作用。     

Bcl-xL反义寡核苷酸对裸鼠人鼻咽癌移植瘤化疗药物敏感性的影响

目的观察Bcl-xL反义寡核苷酸(ASODN)对裸鼠人鼻咽癌移植瘤化疗药物顺铂敏感性的影响.方法建立裸鼠人鼻咽癌移植瘤模型,分为对照组、ASODN治疗组、顺铂(DDP)治疗组、ASODN DDP联合治疗组,观察裸鼠瘤体变化及组织形态学改变.结果单独应用ASODN和DDP治疗组的抑瘤率分别为14.2%和30.9%,而在相同条件下,联合用药组效果最明显,抑瘤率为52.2%,较ASODN、DDP单独应用差异有显著性.结论 ASODN与DDP联合化疗有协同作用,ASODN可以提高肿瘤细胞对顺铂的敏感性,具有增效作用.     

赫赛汀对鼻咽癌荷瘤裸鼠肿瘤生长影响的实验研究

目的：探讨赫赛汀（Herceptin）对HER-2/neu基因蛋白高表达的鼻咽癌荷瘤裸鼠肿瘤生长的影响.方法：用鼻咽癌CNE-2Z细胞株接种于裸小鼠右腋下建立HER-2/neu基因蛋白高表达的裸鼠移植瘤模型.将已构建模型的裸鼠24只随机分为4组,每组6只,分别设阴性和阳性对照两组,Herceptin 10mg/kg和20mg/kg两实验组,以观察Herceptin对移植瘤体内生长的影响.结果：用Herceptin处理后的裸鼠移植瘤肿瘤体积均低于阴性对照组,其中10mg/kg剂量组与阴性对照组相比有统计学差异（P〈0.05）,但无量效关系;瘤重抑制率Herceptin 10mg/kg和20mg/kg分别为33.7%和23.3%,均未达到阳性标准;对裸小鼠生长无明显影响.结论：Herceptin对HER-2/neu基因过表达的鼻咽癌细胞株移植的荷瘤裸鼠肿瘤体内的生长具有一定的抑制作用,但效果并不满意.     

DNA flow cytometric analysis of human nasopharyngeal carcinoma in nude mice

The tissue from a patient with nasopharyngeal carcinoma has been transferred to nude mice (BALB/c), and has successfully been growing through twenty passages. The tumors in the nude mice and primary human tumor have been examined for the cellular DNA content by FCM and also conventional pathological examination, chromosome analysis, and EBV test. The tumor take rate varied markedly in different passages with a mean value of approximately 70%, and showing a tendency to increase. The tumor doubling time within 6-12 weeks after transplantation of six tumors in 18 and 20 passages were 14.8 and 9.3 days respectively. However, the tumor volume at 12 weeks varied significantly, ranging from 438 to 1998 mm3. By FCM, it has been found that the values of DNA index were about the same in both primary tumor and the tumors in nude mice. The distribution of various phase cells in cell cycle was also about the same in both. In conclusion, the application of FCM to examine the cellular DNA content of the tumor in nude mice is a rapid and sensitive method, useful in the investigation on the stability of biological characteristics of human NPC in nude mice and in further studies on the effects by various therapeutic methods.     

5-杂氮-2′-脱氧胞苷对人鼻咽癌裸鼠移植瘤的抑制作用

背景与目的:5-杂氮-2′-脱氧胞苷是DNA甲基转移酶抑制剂,能使因甲基化而失活的抑癌基因重新激活,进而抑制肿瘤的生长。本文观察了5-杂氮-2′-脱氧胞苷对人鼻咽癌细胞及其裸鼠移植瘤的抑制作用,探讨其抗肿瘤效应及可能的机理,寻找鼻咽癌治疗的新靶点。方法:用5-杂氮-2′-脱氧胞苷处理人鼻咽癌细胞,观察其对细胞生长的抑制及死亡相关蛋白激酶基因(death-associated proteinkinase,DAPK)甲基化情况。建立人鼻咽癌移植瘤裸鼠模型,用5-杂氮-2′-脱氧胞苷处理裸鼠,观察移植瘤的生长情况,并用RT-PCR和免疫组织化学技术检测DAPK基因mRNA及蛋白表达的变化。结果:未经5-杂氮-2′-脱氧胞苷处理的CNE细胞中DAPKmRNA不表达。经5-杂氮-2′-脱氧胞苷处理后,不同药物浓度组的细胞中均有DAPKmRNA表达,且表达随浓度增高而增强。5-杂氮-2′-脱氧胞苷对人鼻咽癌细胞及其裸鼠移植瘤的生长均有明显的抑制作用,且能使失活的DAPK基因重新激活。药物作用4周后,用药组裸鼠体重(22.35±2.02)g与对照组(21.68±2.14)g之间无显著性差异(t=0.011,P>0.05),用药组肿瘤的体积(195.32±27.57)mm3较对照组(343.67±23.08)mm3明显减小,两者之间有显著性差异(t=10.11,P﹤0.01)。在鼻咽癌移植瘤中,对照组DAPK均不表达,而用药组出现表达。结论:5-杂氮-2′-脱氧胞苷对人鼻咽癌细胞及其裸鼠移植瘤的抑制作用可能是因为其使因甲基化而失活的抑癌基因DAPK再转录,诱导该基因的表达,从而抑制肿瘤细胞的生长和增殖。     

靶向纳米造影剂C225-USPIO标记裸鼠鼻咽癌移植瘤MR成像研究

  目的  制备针对EGFR的靶向纳米造影剂C225-USPIO,并检测其标记裸鼠鼻咽癌移植瘤MR成像（MRI）能力。  方法  将西妥昔单抗（C225）与USPIO结合成C225-USPIO;检测造影剂粒径;选择EGFR高表达的鼻咽癌SUNE1 5-8F细胞株,接种于12只裸鼠的大腿腹侧皮下,待瘤直径增长至5 mm时,随机分实验组和对照组各6只,将C225-USPIO或USPIO通过鼠尾静脉注射,于注射后0、8、24、72 h经磁共振成像仪行T2WI扫描;检测肿瘤组织内造影剂分布。  结果  C225-USPIO粒径约45~50 nm;MRI示实验组8 h T2值较0 h略降低（P>0.05）,24 h显著降低（P < 0.05),72 h无明显改变（P>0.05）。对照组8~72 h各时间点T2值与0 h相比无明显改变（P>0.05）;注射造影剂72 h后肿瘤组织未见明显铁颗粒。  结论  C225-USPIO颗粒可通过毛细血管,并能运用于体内磁共振分子成像,可以降低裸鼠移植瘤磁共振T2信号强度,具有一定的特异性和靶向性。      

Isolation of infectious EB virus from the epithelial tumour cells of nasopharyngeal carcinoma.

Evidence of herpesvirus replication has been found by light and electron microscopy in the malignant epithelial cells of two out of six nasopharyngeal carcinomas (NPC) examined directly after growth in nude mice to eliminate non-malignant infiltrating cells. The agent has been identified as EB virus by immunofluorescence tests for EB virus capsid antigen, and has been shown to be biologically active by its ability to infect and transform foetal cord blood lymphocytes. Lymphoblastoid cell lines which express the EB virus nuclear antigen have been established from the transformed foetal lymphocytes, and thus carry the first isolate of the virus from the actual epithelial tumour cells of NPC, in a form suitable for further investigation. The results are discussed in terms of the relationship of EB virus to NPC epithelial cells.     

鼻咽癌颅底骨质侵犯的MRI评价

目的:评价MRI检查对鼻咽癌侵犯骨质的诊断价值及其对肿瘤分期的影响.方法:回顾性分析本院350例初诊鼻咽癌患者的MRI影像资料,其中有颅底骨质侵犯196例,病理类型以鳞癌为主180例.134例采用PhilipsT5-Ⅱ型超导磁共振成像仪,场强0.5T.62例采用Philips intera型超导磁共振成像仪,场强1.5T.8例单纯平扫,188例平扫后增强,造影剂为Gd-DTPA,剂量为0.1mmol/kg体重,经前臂静脉注射.结果:196例有骨质侵犯的初诊鼻咽癌中,单部位侵犯65例(33.16%),多部位侵犯131例(66.83%).单侧侵犯178例(90.81%),双侧侵犯18例(9.19%).累计骨质破坏部位698个,骨质侵犯类型:骨质硬化型13个部位(1.86%),骨髓浸润型120个部位(17.19%),溶骨性破坏565个部位(80.95%).结论:MRI可作为评价鼻咽癌颅底骨质侵犯首选检查方法,尤其在评价鼻咽癌早期骨髓侵犯方面MRI比CT敏感,MRI检查可增加鼻咽癌分期的准确性.     

Activation in vitro by BUdR of a productive EB virus infection in the epithelial cells of nasopharyngeal carcinoma.

Material from a nasopharyngeal carcinoma (NCP) has been passaged in athymic (nude) mice to eliminate non-malignant infiltrating cells. The human origin and derivation from NPC malignant epithelial cells of the nude mouse tumours have been confirmed by chromosome examination, electron microscopy showing desmosomes and keratin fibrils, and postive EB virus nuclear antigen (EBNA) testing. Samples of the mouse-grown tumours were cultured and pure monolayers of epithelial cells were obtained which still expressed EBNA and contained desmosomes and keratin; these cultures grew well for about 3 weeks. Extensive electron microscope searches failed to reveal herpes virus particles. In contrast, cultures treated with BUdR showed typical immature and mature herpes virus particles in epithelial, keratin-containing cells, and immunofluorescence tests for virus capsid antigen with a battery of human sera identified this agent as EB virus. EB virus has thus, for the first time, been activated in NPC epithelial cells and shown to be capable of replication in a cell type other than a primate B-lymphocyte.