[目的]探讨局部晚期鼻咽癌患者治疗前血浆EBV-DNA水平与肿瘤负荷及预后的关系.[方法] 165例初治局部晚期鼻咽癌患者,均采用适形调强放射治疗(IMRT).收集患者治疗前EBV-DNA拷贝数水平及肿瘤靶区体积,并对患者生存结果进行随访总结.[结果]治疗前血浆EBV-DNA中位拷贝数3790copies/ml,鼻咽原发灶(GTV nx)、淋巴结病灶(GTVnd)及总GTV体积中位值分别为72.46、23.26、106.25cm3; EBV-DNA水平与GTVnd及总GTV体积显著相关(P＜0.01).治疗前血浆EBV-DNA表达阴性及阳性患者2年总生存率分别为100.0％和98.4％(P＞0.05),无病生存率94.4％和80.8％ (P＜0.05).[结论]局部晚期鼻咽癌患者治疗前血浆EBV-DNA水平与鼻咽癌患者颈部淋巴结转移负荷有关,治疗前血浆EBV-DNA表达阴性的患者无病生存率优于阳性患者.

BACKGROUND:N-stage is related to distant metastasis in nasopharyngeal carcinoma (NPC) patients. The purpose of this study was to evaluate the efficacy and toxicity of different nedaplatin-based chemotherapy regimens in advanced N2-3 stage NPC patients treated with intensity modulated radiation therapy (IMRT).PATIENTS AND METHODS:Between April 2005 and December 2009, a total of 128 patients with N2-3 advanced NPC were retrospectively analyzed. Patients were treated with IMRT concurrent with 2 cycles of chemotherapy consisting of either nedaplatin plus paclitaxel (NP group, n = 67) or nedaplatin plus fluorouracil and paclitaxel (NFP group, n = 61). Two to four cycles of adjuvant chemotherapy were then administered every 21 days following concurrent chemoradiotherapy.RESULTS:With a median follow-up of 60 months, the 5-year overall survival (OS), progression-free survival (PFS), local-regional recurrence-free survival (LRRFS), and distant metastasis-free survival (DMFS) for all patients were 81.4%, 71.5%, 87.8% and 82.0%, respectively. No significant difference in PFS (66.6% vs. 76.7%, P = 0.212) and LRRFS rates (89.0% vs. 86.3%, P = 0.664) was observed between the NP and NFP groups. The 5-year OS (75.4% vs. 88.5%, P = 0.046) and DMFS (75.1% vs. 89.0%, P = 0.042) rate were superior in the NFP group compared with the NP group. The NFP group had a higher incidence of grade 3-4 acute toxicities including bone marrow suppression (leukopenia: χ2 = 3.935, P = 0.047; anemia: χ2 = 9.760, P = 0.002; thrombocytopenia: χ2 = 8.821, P = 0.003), and both liver and renal dysfunction (χ2 = 5.206, P = 0.023) compared with the NP group. Late toxicities were moderate and no difference was observed between the two groups.CONCLUSION:IMRT concurrent with nedaplatin-based chemotherapy is an advocated regimen for patients with advanced N2-3 stage NPC. Patients with advanced N2-3 stage may be better candidates for the NFP regimen although this regimen was associated with a high acute toxicity rate.

Objectives: The purpose of this study was to investigate the relationship between huntingtin-associated protein1 (HAP1) gene and radiation therapy of breast cancer cells.Methods: HAP1 gene was transfected into breast cancer MCF-7 cells, which was confirmed by quantitative reverse transcription-polymerase chain reaction analysis (qRT-PCR) and Western blot in vitro. The changes of cell radiosensitivity were assessed by colony formation assay. Apoptosis were examined by flow cytometry. The expressions of two radiation-induced genes were evaluated by Western blot. Tumor growth was investigated in nude mice xenograft models in vivo.Results: Our data showed that HAP1 gene expression was significantly increased in HAP1-transfected MCF-7 cells in comparison with the parental cells or negative control cells. The survival rate in MCF-7/HAP1 cells was significantly decreased after irradiation (0, 2, 4, 6, 8 Gy), compared to cells in MCF-7 and MCF-7/Pb groups in vitro. HAP1 gene increased apoptosis in MCF-7 cells after irradiation. Additionally, the tumor volume and weight in MCF-7/HAP1 + RT group were observably lower than in MCF-7/HAP1 group and MCF-7/Pb + RT group.Conclusion: The present study indicated that HAP1 gene expression was related to the radiosensitivity of breast cancer cells and may play an important role in the regulation of cellular radiosensitivity. (C) 2014 Elsevier Inc. All rights reserved.

This retrospective study aims to examine the association of plasma Epstein-Barr virus- (EBV-) DNA levels with the tumor volume and prognosis in patients with locally advanced nasopharyngeal carcinoma (NPC). A total of 165 patients with newly diagnosed locally advanced NPC were identified from September 2011 to July 2012. EBV-DNA was detected using fluorescence quantitative polymerase chain reaction (PCR) amplification. The tumor volume was calculated by the systematic summation method of computer software. The median copy number of plasma EBV-DNA before treatment was 3790 copies/mL. The median gross tumor volume of the primary nasopharyngeal tumor (GTVnx), the lymph node lesions (GTVnd), and the total GTV before treatment were 72.46, 23.26, and 106.25 cm(3), respectively; the EBV-DNA levels were significantly correlated with the GTVnd and the total GTV (P < 0.01). The 2-year overall survival (OS) rates in patients with positive and negative pretreatment plasma EBV-DNA were 100% and 98.4% (P = 1.000), and the disease-free survival (DFS) rates were 94.4% and 80.8% (P = 0.044), respectively. These results indicate that high pretreatment plasma EBV-DNA levels in patients with locally advanced NPC are associated with the degree of lymph node metastasis, tumor burden, and poor prognosis.

Background: Epithelial-mesenchymal transition (EMT) is associated with invasion and metastasis of cancer cells. High-mobility group AT-hook 2 (HMGA2) has been found to play a critical role in EMT in a number of malignant tumors. However, whether HMGA2 regulates the EMT in human nasopharyngeal carcinoma (NPC) is unclear.Objective: The aim of this study was to investigate the effect and mechanism of HMGA2 in inducing invasion and migration in NPC.Methods: In NPC tissues samples, the association of HMGA2 mRNA expression with clinicopathological characteristics were estimated by real-time quantitative RT-PCR(qRT-PCR). In vitro, following the silencing of HMGA2 in CNE-1 and CNE-2 cell lines, the viability and metastatic ability were analyzed using Cell Counting Kit-8 (CCK8), colony formation assay, and transwell assay. EMT and transforming growth factor-beta (TGF beta)/Smad3 signaling pathway-related protein expression changes were evaluated using western blot.Results: HMGA2 was upregulated in NPC cell lines and clinical specimens (P < 0.01), and HMGA2 expression correlated significantly with metastasis (P = 0.02) and disease-free survival of NPC (hazard ratio: 3.52; 95% confidence interval: 1.34-7.79; P = 0.01). In addition, following in vitro knockdown of HMGA2, the aggressiveness of cells was markedly inhibited, Vimentin and Snail level was downregulated and E-cadherin expression was upregulated. Moreover, the expression of key proteins TGF beta RII and p-Smad3 of the TGF beta/Smad3 signaling pathway was inhibited by the downregulation of HMGA2.Conclusion: HMGA2 might maintain EMT-induced invasion and migration through the TGF beta/Smad3 signaling pathway in NPC cell lines. (C) 2015 Elsevier Inc. All rights reserved.

MiR-145 is downregulated and functions as a tumor suppressor in many malignancies. In this study, the biological function, molecular mechanism, and direct target genes of miR-145 in nasopharyngeal carcinoma (NPC) cells were investigated. Cell survival was detected by cell viability assay, and cell cycle was determined through flow cytometry. Invasion and migration of NPC cells were examined using cell invasion and wound healing assays, respectively. A disintegrin and metalloproteinase 17 (ADAM17) was verified as the target of miR-145 through luciferase reporter assay, qRT-PCR, and Western blot analysis. In NPC cell lines, miR-145 expression was significantly downregulated and ADAM17 protein expression was upregulated. ADAM17 was downregulated at the post-transcriptional level by miR-145 via the binding site of ADAM17-3′UTR. Transfection with miR-145 mimic suppressed cell growth and induced cell cycle arrest in the G0/G1 phase by upregulating key G0/G1 phase regulators, namely, p53 and p21. MiR-145 also inhibited cellular migration and invasion through targeting ADAM17 involving the regulation of EGFR and E-cadherin. Knockdown of ADAM17 elicited similar effects to that of miR-145 on NPC cells. This study reveals that miR-145 suppressed the invasion and migration of NPC cells by targeting ADAM17. Thus, miR-145 could be a therapeutic target for NPC.

OBJECTIVE:High-mobility group protein 2 (HMGA2) and epithelial-mesenchymal transition (EMT)-associated proteins play key roles in cancer progression and metastasis. However, the clinical significance of HMGA2 and its relationship with EMT markers in nasopharyngeal carcinoma (NPC) is unclear. This study aimed to assess the clinicopathological significance and prognostic value of HMGA2, E-cadherin, and vimentin in NPC.METHODS:Using immunohistochemistry, HMGA2, E-cadherin, and vimentin expression levels were evaluated in NPC (n=124) and non-tumoral inflammatory nasopharynx (n=20) tissues. The association of HMGA2 and EMT markers with clinicopathological characteristics and relationships between the protein levels and overall survival were analyzed.RESULTS:Compared with non-tumorous tissues, HMGA2 and vimentin levels were markedly increased in NPC tissues, whereas decreased E-cadherin levels were observed (P<0.001). Moreover, HMGA2 expression was positively correlated with vimentin levels (r=0.431, P<0.001) and negatively correlated with E-cadherin amounts (r=-0.413, P<0.001) in NPC tissues. The expression of all three proteins correlated significantly with tumor N stage, TNM stage, and 2-year metastasis. Furthermore, significant correlations were found for T stage, N stage, TNM stage, HMGA2, E-cadherin, and vimentin (all P<0.013) with poor prognosis (univariate analysis). However, multivariate analyses showed that only HMGA2 (hazard ratio [HR]: 2.683, 95% confidence interval [CI]: 1.185-6.077, P=0.018) and N stage (HR: 7.892, 95% CI: 2.731-22.807, P<0.001) were independent predictors of poor prognosis.CONCLUSION:These results demonstrated that HMGA2, an independent prognostic factor, may promote NPC progression and metastasis, and is significantly associated with EMT proteins. Therefore, HMGA2 may be considered a potential therapeutic target in NPC.

This study aims to evaluate the radiosensitization effect of nedaplatin on nasopharyngeal carcinoma (NPC) cell lines with different Epstein-Barr virus (EBV) status. Human NPC cell lines CNE-2 (EBV-negative) and C666 (EBV-positive) were treated with 0-100 μg/mL nedaplatin, and inhibitory effects on cell viability and IC50 were calculated by MTS assay. We assessed changes in radiosensitivity of cells by MTS and colony formation assays, and detected the apoptosis index and changes in cell cycle by flow cytometry. MTS assay showed that nedaplatin caused significant cytotoxicity in CNE-2 and C666 cells in a time- and dose-dependent manner. After 24 h, nedaplatin inhibited growth of CNE-2 and C666 cells with IC50 values of 34.32 and 63.69 μg/mL, respectively. Compared with radiation alone, nedaplatin enhanced the radiation effect on both cell lines. Nedaplatin markedly increased apoptosis and cell cycle arrest in G2/M phase. Nedaplatin radiosensitized human NPC cells CNE-2 and C666, with a significantly greater effect on the former. The mechanisms of radiosensitization include induction of apoptosis and enhancement of cell cycle arrest in G2/M phase.

Our previous studies showed that prostaglandin E2 (PGE2) promotes hepatoma cell growth and migration, as well as invasion; however, the precise mechanism remains elusive. Snail and p65 protein levels were detected in human samples with hepatocellular carcinoma (HCC) by immunohistochemistry (IHC) staining. HCC cell lines (Huh-7 and Hep3B) were used for in vitro experiments. PGE2/Akt/NF-κB pathway was investigated in Huh-7 and Hep3B cells after treatment with PGE2, EP4 receptor (EP4R) agonist, Akt inhibitor, and NF-κB inhibitor, respectively, by real-time reverse transcription (RT)-PCR, Western blotting, and immunofluorescence (IF) staining. In vitro cell invasion assay was performed to evaluate the effect of PGE2 on tumor invasiveness. Knockdown of EP4R was carried out in Huh-7 cells through plasmid-based small interfering RNA (siRNA) approach to confirm the regulation of PGE2 on Snail by EP4R. Dual luciferase reporter assay was performed to assess Snail promoter activity in Huh-7 cell after treatment with EP4R agonist. We found that the protein levels of Snail were higher in HCC tissues than those in control and that PGE2 and EP4R agonist treatment significantly increased Snail expression in Huh-7 and Hep3B cells. EP4R agonist also profoundly promoted invasiveness of Huh-7 cells. Knockdown of the EP4R by siRNA completely blocked the PGE2-induced upregulation of Snail expression and reduced invasiveness of Huh-7 cells. We failed to find that EP4R-induced upregulation of Snail was reversed by inhibition of cAMP response element-binding protein (CREB), a canonical downstream target of EP4R. Alternatively, EP4R agonist treatment significantly increased the levels of phosphorylated EGFR and Akt both in Huh-7 and Hep3B cells. AG1478, an EGFR inhibitor, blocked the phosphorylation of Akt. The levels of phosphorylated IκB increased in Huh-7 cells after treatment with EP4R agonist for 30 min. The levels of phosphorylated p65 started to increase in Huh-7 cells treated with EP4R agonist for 4 h, and p65 translocated into the nucleus. In EP4R-agonist-treated Hep3B, the levels of phosphorylated p65 were also increased compared to the control group. The phosphorylation levels of p65 were significantly decreased in Huh-7 and Hep3B cells after treatment with the Akt signaling inhibitor LY294002 and EP4R agonist for 24 h. Treatment with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) at 10 μM for 24 h blocked EP4R-agonist-induced Snail upregulation in Huh-7 and Hep3B cells. Furthermore, we obtained human Snail promoter sequence from TRED-Promoter Database and identified a putative binding site of NF-κB in the sequence through TFSEARCH analysis. Subsequently, we treated Huh-7 cells with EP4R agonist or EP4R agonist and PDTC (NF-κB antagonist) and found significantly increased Snail promoter activity after EP4R agonist treatment for 12 h. The increased Snail promoter activity could be partially abolished by additional PDTC treatment. In addition, p65 protein levels were found increased together with Snail in HCC tissues compared to normal liver tissues. In conclusion, PGE2 activates Akt/NF-κB signaling and then upregulates Snail via the EP4R/EGFR to promote migration and invasion in hepatoma cells. These findings may help future evaluation of novel chemo-preventive strategies for HCC.

Prostaglandin E-2 (PGE(2)) has been shown to influence cell invasion and metastasis in several types of cancer, including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying it remain to be further elucidated. Snail, as one of key inducers of epithelial-mesenchymal transition (EMT), plays pivotal roles in HCC invasion and metastasis. The present study was designed to evaluate the possible signaling pathways through which PGE2 regulates Snail protein expression in HCC cell lines. PGE2 markedly enhanced Huh-7 cell invasion and migration ability by upregulating the expression level of Snail protein, and EP2 receptor played an important role in this process. Src, EGFR, Akt and mTOR were all activated and involved in the regulation of Snail protein expression. Our findings suggest that PGE2 could upregulate the expression level of Snail protein through the EP2/Src/EGFR/Akt/mTOR pathway in Huh-7 cells, which promotes HCC cell invasion and migration.

Liver cancer is a common human cancer with a high mortality rate and currently there is no effective chemoprevention or systematic treatment. Recent evidence suggests that prostaglandin E(2) (PGE(2)) plays an important role in the occurrence and development of liver cancer. However, the mechanisms through which PGE(2) promotes liver cancer cell growth are not yet fully understood. It has been reported that the increased expression of FUSE-binding protein 1 (FBP1) significantly induces the proliferation of liver cancer cells. In this study, we report that PGE(2) promotes liver cancer cell growth by the upregulation of FBP1 protein expression. Treatment with PGE2 and the E prostanoid 3 (EP3) receptor agonist, sulprostone, resulted in the time-dependent increase in FBP1 protein expression; sulprostone increased the viability of the liver cancer cells. The protein kinase A (PKA) inhibitor, H89, and the adenylate cyclase (AC) inhibitor, SQ22536, inhibited the cell viability accelerated by sulprostone. By contrast, the Gi subunit inhibitor, pertussis toxin (PTX), exhibited no significant effect. Treatment with PGE(2) and sulprostone caused a decrease in JTV1 protein expression, blocked the binding of JTV1 with FBP1, which served as a mechanism for FBP1 degradation, leading to the decreased ubiquitination of FBP1 and the increase in FBP1 protein expression. Furthermore, H89 and SQ22536 prevented the above effects of JTV1 and FBP1 induced by PGE(2) and sulprostone. These findings indicate that the EP3 receptor activated by PGE(2) may couple to Gs protein and activate cyclic AMP (cAMP)-PKA, downregulating the levels of JTV1 protein, consequently inhibiting the ubiquitination of FBP1 and increasing FBP1 protein expression, thus promoting liver cancer cell growth. These observations provide new insights into the mechanisms through which PGE(2) promotes cancer cell growth.

Here we report two unrelated Chinese families with congenital missing teeth inherited in an X-linked manner. We mapped the affected locus to chromosome Xp11-Xq21 in one family. In the defined region, both families were found to have novel missense mutations in the ectodysplasin-A (EDA) gene. The mutation of c.947A>G caused the D316G substitution of the EDA protein. The mutation of c.1013C>T found in the other family resulted in the Thr to Met mutation at position 338 of EDA. The EDA gene has been reported responsible for X-linked hypohidrotic ectodermal dysplasia (XLHED) in humans characterized by impaired development of hair, eccrine sweat glands, and teeth. In contrast, all the affected individuals in the two families that we studied here had normal hair and skin. Structural analysis suggests that these two novel mutants may account for the milder phenotype by affecting the stability of EDA trimers. Our results indicate that these novel missense mutations in EDA are associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level.