Generation of novel affibody molecules targeting the EBV LMP2A N-terminal domain with inhibiting effects on the proliferation of nasopharyngeal carcinoma cells.
产生靶向 EBV LMP2A N 端结构域的新型附属分子，对鼻咽癌细胞的增殖具有抑制作用。
- 作者列表："Zhu J","Kamara S","Cen D","Tang W","Gu M","Ci X","Chen J","Wang L","Zhu S","Jiang P","Chen S","Xue X","Zhang L
:Nasopharyngeal carcinoma (NPC) induced by latent infection with Epstein-Barr virus (EBV) remains the most common head and neck cancer in Southeast Asia, especially in the southern part of China. It is well known that persistent expression of two EBV latent membrane proteins (LMP1/LMP2A) plays a key role in nasopharyngeal carcinogenesis. Therefore, the therapeutic approach of targeting the LMP1/LMP2A protein and subsequently blocking the LMP1/LMP2A-mediated signalling pathway has been considered for treating patients with NPC. Recently, affibody molecules, a new class of small (~6.5 kDa) affinity proteins, have been confirmed to be powerful generalisable tools for developing imaging or therapeutic agents by targeting specific molecules. In this study, three EBV LMP2A N-terminal domain-binding affibody molecules (ZLMP2A-N85, ZLMP2A-N110 and ZLMP2A-N252) were identified by screening a phage-displayed peptide library, and their high affinity and specificity for the EBV LMP2A N-terminal domain were confirmed by surface plasmon resonance (SPR), indirect immunofluorescence, co-immunoprecipitation and near-infrared small animal fluorescence imaging in vitro and in vivo. Moreover, affibody molecules targeting the EBV LMP2A N-terminal domain significantly reduced the viability of the EBV-positive cell lines C666-1, CNE-2Z and B95-8. Further investigations showed that affibody ZLMP2A-N110 could inhibit the phosphorylation of AKT, GSK-3β and β-catenin signalling proteins, leading to suppression of β-catenin nuclear translocation and subsequent inhibition of c-Myc oncogene expression, which may be responsible for the reduced viability of NPC-derived cell lines. In conclusion, our findings provide a strong evidence that three novel EBV LMP2A N-terminal domain-binding affibody molecules have great potential for utilisation and development as agents for both molecular imaging and targeted therapy of EBV-related NPC.
Eb病毒 (Epstein-bar病毒，EBV) 潜伏感染诱发的鼻咽癌 (NPC) 仍然是东南亚地区最常见的头颈部肿瘤，尤其是在中国南部地区。众所周知，两种 EBV 潜伏膜蛋白 (LMP1/LMP2A) 的持续表达在鼻咽癌变中起关键作用。因此，以 LMP1/LMP2A 蛋白为靶点，阻断 LMP1/LMP2A-mediated 信号通路治疗鼻咽癌已被广泛考虑。最近，affibody 分子，一类新的小 (~ 6.5 kda) 亲和蛋白，已经被证实是通过靶向特定分子开发成像或治疗剂的强大通用工具。本研究通过筛选噬菌体展示的肽库，鉴定了 3 个 EBV LMP2A N 端结构域结合连接体分子 (ZLMP2A-N85 、 ZLMP2A-N110 和 ZLMP2A-N252),通过表面等离子体共振 (SPR) 、间接免疫荧光、免疫共沉淀和近红外小动物荧光成像的体内外研究。此外，靶向 EBV LMP2A N 端结构域的连接体分子显著降低了 EBV 阳性细胞系 C666-1 、 CNE-2Z 和 B95-8 的活力。进一步研究表明，ZLMP2A-N110 可抑制 AKT 、 gsk-3 β 和 β-catenin 信号蛋白的磷酸化，从而抑制 β-catenin 核转位，进而抑制 c-Myc 癌基因的表达。这可能是 NPC 来源的细胞系活力降低的原因。总之,我们的研究结果提供了强有力的证据，表明三种新型 EBV LMP2A N 端结构域结合的词缀分子在作为 EBV 相关 NPC 的分子成像和靶向治疗的药物方面具有巨大的应用和开发潜力。
METHODS:INTRODUCTION:Human papillomavirus (HPV) is the most common sexually transmitted infection and is associated with several types of cancer. The number of cases of HPV-associated head and neck squamous cell carcinomas (HNSCCs), especially oropharyngeal carcinomas, has increased significantly in recent years despite decreased tobacco smoking rates. Currently, no data concerning the risk factors and prevalence of HPV in HNSCC patients in all regions of Brazil are available, making it difficult to promote advances in this field of public health. Therefore, our goal is to determine the impact of infection by HPV, including HPVs with different genotypes, on head and neck cancer and the risk factors associated with the development of head and neck cancer in all regions of Brazil. METHODS AND ANALYSIS:This is a case-control study that will include 622 patients and 622 controls from all regions of Brazil. A questionnaire will be applied to gather information on sociodemographic, behavioural and health factors. Oral, cervical or penile/scrotal, and anal specimens and serum samples will be collected from all participants. Formalin-fixed paraffin-embedded tissue from tumour biopsies will be analysed only in the case group. Molecular and serological analyses will be performed to evaluate the presence and role of HPV in the development of head and neck cancer. ETHICS AND DISSEMINATION:This project was approved by the research ethical committee of the proposing institution (Hospital Moinhos de Vento, number 2.852.060). Ethical approval from the collaborators is currently under evaluation and is not yet complete. The results of this study will be presented at meetings with the Brazilian Ministry of Health through technical reports and to the scientific community at national and international events, with subsequent publication of scientific articles.
METHODS:BACKGROUND:Factors related to head and neck cancer and the treatment of the disease can affect quality of life. The aim of this study was to determine factors associated with the severity of impact on oral health-related quality of life (OHRQoL) in survivors of head and neck cancer using a multivariate analysis. METHODS:This cross-sectional study evaluated 90 volunteers who had completed radiotherapy at least 3 months earlier. OHRQoL was assessed using oral health impact profile (OHIP-14) and the data were analyzed using robust variance poisson regression models. RESULTS:The mean total OHIP-14 score was 23.98 ± 12.55. Patients with hyposalivation had 56% higher (worse) mean OHIP-14 total scores (CI:1.11-2.18) and patients with advanced stage tumors had 31% higher mean OHIP-14 total scores (CI:1.03-1.66) in multivariate analyses. CONCLUSION:OHRQoL of survivors of head and neck cancer experienced a negative impact following radiotherapy. The impact was associated with hyposalivation and advanced stage tumors.
METHODS:BACKGROUND:To immunohistochemically evaluate the association between the presence of cancer-associated fibroblasts (CAFs) and the tumour expression of podoplanin (PDPN) in head and neck squamous cell carcinoma (HNSCC) and their association with clinicopathological variables. MATERIAL AND METHODS:A tissue microarray (TMA) with biopsy sections from patients diagnosed with HNSCC was stained with antibodies against the CAFs marker, α-smooth muscle actin (α-SMA), and PDPN. We subsequently evaluated their expression to determine the association between them and with clinicopathological variables including age, primary tumour site, TNM stage, and tumour differentiation grade. RESULTS:Positive reaction to α-SMA was observed in the tumour stroma, revealing spindle-shaped cells compatible with CAFs, which showed a high expression in 62% of cases and a significant association with laryngeal carcinomas, advanced clinical stages, and lower tumour differentiation (P ≤ 0.05). PDPN staining on tumour cells showed low expression in 72% of cases, and it was not associated with any clinicopathological variable or with the presence of CAFs. CONCLUSIONS:The presence of CAFs in the tumour stroma is related to an aggressive phenotype and could increase as the disease progresses, although based on our findings, it would have no relationship, at least directly, with the expression of PDPN.