目的 探讨Ki-67在下咽癌及喉癌中表达情况与肿瘤分期的相关性.方法 回顾分析35名下咽癌及喉癌患者资料,其中下咽癌患者23例,喉癌12例,患者均为男性,年龄46～79岁,平均(61.7±7.6)岁.本研究利用SPSS 13.0软件进行Spearman相关性分析,绘制ROC曲线并计算曲线下面积.结果 根据肿瘤TNM分期:T1期1例,T2期10例,T3期13例,T4期11例;N0期11例,N1期2例,N2期10例,N3期12例;所有患者均为M0期.Ki-67表达百分率及等级与肿瘤TNM分期中T分期无明显相关性(P=0.600、P=0.958);Ki-67表达指数与肿瘤TNM分期中N分期存在相关性(P=0.017),相关系数为r=0.402;ROC曲线下面积0.75(0.58-0.93).Ki-67表达等级与肿瘤TNM分期中N分期有相关性(P=0.004),相关系数为r=0.478;ROC曲线下面积0.72(0.54-0.899).结论 下咽癌及喉癌中Ki-67表达水平与肿瘤TNM分期中N分期存在相关性,Ki-67表达水平越高,肿瘤越易发生区域淋巴结转移.

目的 研究下咽癌强化CT纹理分析相关参数与Ki-67表达情况之间的相关性.方法 搜集行手术病理证实的23例下咽癌患者.所有患者在外科手术前进行颈部增强CT检查,在肿瘤最大截面勾画感兴趣区,分别测量CT平扫、动脉期、静脉期病灶纹理分析相关参数值(平均值、标准差、熵值、不均匀度、偏度及峰度),采用Spearman非参数相关分析,P＜0.05时认为存在相关关系,对有统计学意义的纹理参数进行受试者操作特征曲线(ROC)分析,评估纹理参数对Ki-67低、高表达组的鉴别诊断效能,确定诊断阈值,并计算曲线下面积、敏感性、特异性.结果 平扫期平均值与Ki-67表达情况之间呈负相关(r=-0.446,P=0.033),动脉期平均值、偏度与Ki-67表达情况之间呈负相关(r=-0.554,P=0.006;r=-0.463,P=0.026),静脉期平均值与Ki-67表达情况之间呈负相关(r=-0.514,P=0.012).平扫期平均值的阈值为1056.738时,其ROC曲线下面积、敏感性和特异性分别为0.639、100％和38.5％.动脉期平均值的阈值为1069.593时,其ROC曲线下面积、敏感性和特异性分别为0.908、100％和53.85％.动脉期偏度的阈值为-1.034时,其ROC曲线下面积、敏感性和特异性分别为0.762、90.0％和46.2％.静脉期平均值的阈值为1087.047时,其ROC曲线下面积、敏感性和特异性分别为0.862、100％和61.54％.结论 下咽癌CT纹理参数可以有效反映Ki-67表达水平,对临床了解肿瘤的增殖活性有一定的参考价值.

Multidrug resistance (MDR) is a major impediment to cancer therapy. MG‑132 has been identified to be effective against MDR in several types of cancer. However, the mechanism of MG‑132 in head and neck squamous cell carcinomas remains unknown. Based on our previous study, the present detected P‑gp and P‑gp expression in hypopharyngeal carcinoma FaDu cells, revealing that their expression was lower than that observed in the MDR cell line FaDu/T. To reverse the MDR of FaDu/T cells, the present study introduced MG‑132 and demonstrated that the high expression of P‑gp/P‑gp in FaDu/T cells was attenuated in a time‑dependent manner. MG‑132 also strengthened the sensitivity of FaDu/T cells to multidrugs. c‑Jun N‑terminal kinase (JNK) activation was further observed in FaDu/T cells. However, P‑gp/P‑gp did not decrease when FaDu/T cells were pretreated with SP600125. These results indicated that MG‑132 reversed the MDR of hypopharyngeal carcinoma by downregulating P‑gp/P‑gp, and the underlying mechanism may be associated with the activation the of the JNK signaling pathway.

Macrophages play a critical role in tumor invasion and metastasis, which remain major causes of mortality in patients with hypopharyngeal cancer. Here we investigate the effect of an oncogene, AEG-1 expressed in macrophages on the invasion of hypopharyngeal cancer cells. AEG-1 is more highly expressed in macrophages of human hypopharyngeal cancer samples compared with adjacent non-tumor controls. Using matrigel invasion assay system, THP-1-derived macrophages with forced AEG-1 overexpression enhance FaDu cell invasion whereas macrophages with AEG-1 silence inhibit. Matrix metalloproteinase 9 (MMP-9), which is important in tumor invasion and metastasis through degrading extracellular matrix, is up-reulated by AEG-1 partly through NF-κB p65 in macrophages. Intriguingly, macrophage AEG-1 also induces MMP-9 up-regulated expression in FaDu cells. Furthermore, macrophage AEG-1 activates signal transducer and activator of transcription 3 (STAT3) in FaDu cells, which is responsible for macrophage AEG-1-induced an increase in MMP-9 expression and invasion of FaDu cells. This is the first to demonstrate that macrophage AEG-1 promotes tumor invasion through up-regulation of MMP-9 in both macrophages and cancer cells. Thus, the results provide evidences that macrophage AEG-1 contributes to promotion of tumor invasion, and represents as a potential target in hypopharyngeal cancer therapy.

Afatinib is the second generation of irreversible inhibitor of EGFR, HER2 and HER4, which has shown encouraging phase II and III clinical outcomes in the treatment of head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanism of afatinib-induced apoptosis in HNSCC is poorly understood. In the present investigation, we discovered that down-regulation of MCL-1, an anti-apoptotic member of BCL-2 family, was responsible for afatinib-triggered apoptosis. And the inactivation of AKT-mTOR signaling caused by afatinib lead to translational inhibition of MCL-1 expression. As a crucial branch of ER stress, PERK-eIF2α-ATF4 axis was also stimulated in HNSCC cells after afatinib incubation. Silencing either eIF2α or ATF4 by siRNA transfection relieved afatinib-caused suppression of AKT-mTOR activity, attenuating MCL-1 down-regulation as well as subsequent apoptosis. Collectively, the results show that afatinib hampers AKT-mTOR activation by stimulating PERK-eIF2α-ATF4 signaling pathway, giving rise to MCL-1 down-regulation mediated apoptosis in HNSCC cells. Therefore, our findings reveal the elaborate molecular network of afatinib-induced apoptosis in HNSCC, which would provide substantial theoretical underpinnings for afatinib clinical application and highlight its promising prospect in HNSCC treatment.

Multidrug resistance (MDR) is one of the most important obstacles affecting the efficacy of chemotherapy treatments for numerous types of cancer. In the present study, we have demonstrated the possible function of Twist1 in the chemosensitivity of head and neck squamous cell carcinoma (HNSCC) and have identified that its mechanism maybe associated with MDR1/P-gp regulation. To investigate this, the hypopharyngeal cancer cell line, FaDu, and its MDR cell line induced by taxol, FaDu/T, were employed. Stable transfectants targeted to Twist1 overexpression and Twist1 silencing based on FaDu were also conducted. Morphological observation, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), western blotting and laser scanning confocal microscope detection were utilized to detect the associations between Twist1 and the chemosensitivity of FaDu cells. Our results demonstrated that Twist1 and MDR1/P-gp were upregulated in FaDu/T cells in a MDR dose-dependent manner. The anti-apoptotic capabilities of FaDu/T cells were enhanced during MDR progression, with apoptosis-related proteins (Bcl-2, Bax, activated caspase-3 and caspase-9) changing to resist apoptosis. Twist1 overexpression decreased the sensitivity of cells to taxol as revealed by a significant increase in MDR1/P-gp and IC50 (P<0.05). This overexpression also enhanced the resistance to apoptosis, with apoptotic proteins changing to resist cell death, and inhibited Ca2+ release induced by taxol (P<0.05). Detections in Twist1 silencing cells also confirmed this result. This study provided evidence that alterations of Twist1 expression modulates the chemosensitivity of FaDu cells to taxol. Therefore, Twist1 knockdown may be a promising treatment regimen for advanced hypopharyngeal carcinoma patients with MDR.

In order to reverse the malignant characteristics of hypopharyngeal cancer, the proteasome inhibitor MG132 was introduced into FaDu/T cells and the mechanisms underlying its effects were investigated. The multi-drug resistance (MDR) sensitivities of FaDu/T and FaDu/T-MG132 cancer cells to several chemotherapeutics were investigated by a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured by staining cells with Annexin V and propidium iodide (PI) double staining. Reverse transcription-polymerase chain reaction and western blot analysis were conducted to detect mRNA and corresponding protein levels of the MDR- and apoptosis-related genes P-glycoprotein (P-gp), caspase-3, Bcl-2 and Bax. The nuclear protein of nuclear factor κ-light-chain-enhancer of activated B cells. (NF-κB) and p53 were also investigated via western blot analysis. Compared with FaDu/T cells, the drug resistance of FaDu/T + MG132 cells to cisplatin (DDP), 5-fluorouracil (5-FU), doxorubicin (Dox) and vincristine (VCR) decreased. With increased expression of caspase-3 and Bax and decreased expression of Bcl-2, the anti-apoptotic ability markedly decreased in FaDu/T + MG132 cells. P-gp and NF-κB significantly decreased; however, p53 increased in FaDu/T + MG132 cells. These results suggested that the proteasome inhibitor MG132 reversed the malignant characteristics of FaDu/T by enhancing apoptosis and inhibiting P-gp. MG132 was also able to inhibit the nuclear translocation of NF-κB and increase the expression of p53.

BACKGROUND:The objective of this study was to evaluate the efficacy of Nimesulide, a selective cyclooxygenase-2 (COX-2) inhibitor, on the growth of hypopharyngeal carcinoma cells (FaDu) in vitro, and investigate its potential mechanism.METHODS:After FaDu cells were treated with graded concentrations of Nimesulide for divergent time, sensitivity of cells to drug treatment was analyzed by MTT assay. Morphological changes of FaDu cells in the presence of Nimesulide were observed by acridine orange cytochemistry staining. Proliferating cells were detected using the 5-Bromo-2'-deoxy-uridine (BrdU) incorporation assay. Following cells were subjected to Nimesulide (500 μmol/l) for 6 h, 12 h and 24 h, the percentage of apoptosis was examined by flow cytometry. We detected COX-2 and Survivin expression change by RT-PCR and Western blot, and analyzed the correlation of them with the growth of FaDu cells. Additionally, we also analyzed Caspase-3, Bcl-2 and Bax expressions as markers to investigate the related pathway of Nimesulide-indued apoptosis.RESULTS:Compared with the control group, the viabilities rates were decreased by Nimesulide in time- and dose-dependent manners, typical morphological changes of apoptotic cells were observed in the Nimesulide-treatment groups, Nimesulide could suppress the proliferation of FaDu cells significantly. The percentage of apoptosis in FaDu cells were markedly increased after Nimesulide-treatment for 6 h, 12 h and 24 h. Nimesulide down-regulated the Survivin and COX-2 expressions at mRNA and protein levels in FaDu cells. Additional analyses indicated that Bcl-2 expression was significantly decreased and the expressions of Caspase-3 as well as Bax were increased at both mRNA and protein levels.CONCLUSIONS:Based on the induction of apoptosis and suppression of proliferation, Nimesulide could inhibit the growth of FaDu cells. Furthermore, the suppression of Survivin expression may play an important role in Nimesulide-induced growth inhibition. Nimesulide could act as an effective therapeutic agent for hypopharyngeal carcinoma therapy.

OBJECTIVE:To investigate the expression of multidrug resistance gene ABCB1 and ABCG2 in FaDu cells (human hypopharyngeal carcinoma cell line) and the multidrug resistance (MDR) cell lines FaDu/T transformed from FaDu cells by taxol and underlying mechanisms of MDR. METHODS:The multidrug resistance sensitivities of FaDu and FaDu/T to cisplatin (DDP), 5-fluorouracil (5-FU), doxorubicin (Dox), and vincristine (VCR) were examined by methyl-thiazolyl-tetrazolium (MTT) assay. The mRNA and protein expressions of multidrug resistance genes ABCB1 and ABCG2 were analysed with RT-PCR, Western blot and laser confocal microscopy. JNK signal proteins were detected through Western blot. RESULTS:The multidrug resistance of FaDu/T cells to Taxol, DDP, 5-FU, ADM and VCR was more than that of FaDu cells. The expression of ABCB1 in FaDu/T cells was significantly higher than that in FaDu cells (t = 22.42, P < 0.05), but the expression of ABCG2 in FaDu/T cells was significantly lower than that in FaDu cells (t = 10.06, P < 0.05). JNK signal was inhibited in FaDu or FaDu/T cells and the inhibited JNK was reactivated by taxol or anisomycin (an activator for MAPK signal transduction pathways). Anisomycin down-regulated the expression of ABCB1 (F = 33.72, P < 0.05) and up-regulated the expression of ABCG2 (F = 220.16, P < 0.05) in FaDu/T cells, but not in FaDu/T cells pretreated by JNK inhibitor SP600125 (P > 0.05). CONCLUSION:The overexpression of ABCB1 and the down-regulation of ABCG2 in FaDu/T cells were the main features of MDR in hypopharyngeal carcinomas, in which JNK signal transduction pathways could play an important role.

The transcription factor TWIST is an important factor in regulation of the epithelial–mesenchymal transition (EMT), which represents the primary stages during the metastasis of tumors. To identify the role of TWIST in the regulation of metastasis in laryngeal carcinoma Hep-2 cells, we investigated whether the alteration of TWIST has an effect on the Hep-2 cells morphology and whether the alteration of TWIST has an effect on the expression of E-cadherin, N-cadherin as well as the ability of cell motion, migration, and invasion.

BACKGROUND:Twist is a highly conserved epithelial-mesenchymal transcription factor that has been reported to be a key factor in tumor malignancy, including lymph node metastasis. It represents the major step of dissemination and serves as a chief prognostic indicator of disease progression. However, the mechanism by which Twist regulates lymph node metastasis remains incompletely understood. Studies on the mechanism of metastasis are thus required for determining appropriate therapeutic strategies.METHODS:Immunohistochemistry for lymphatic vessel endothelial receptor 1 (LYVE-1), Ki-67, Twist, vascular endothelial growth factor C (VEGF-C), and vascular endothelial growth factor receptor 3 (VEGFR-3) was performed to detect lymphatic vessel density (LVD), cell proliferation levels and the expressions of Twist, VEGF-C, and VEGFR-3 were determined from 66 primary supraglottic carcinoma tissue samples from 36 patients with lymph node metastasis (pathological N+, pN+) and 30 patients without metastasis (pathological N0, pN0). Western blotting analysis of the proteins in pN+ and pN0 primary tumors was used to characterize the expressions of Twist, VEGF-C, and VEGFR-3 further.RESULTS:The LVD was 22.4 ± 10.3 in pN+ patients and 6.8 ± 4.1 in pN0 ones. For Ki-67, the number of proliferous cells in pN+ patients was greater than that in pN0 ones. Both, however, were associated with their clinical nodal stages. In pN+ patients, Twist, VEGF-C, and VEGFR-3 expressions were 86.11% (31/36), 80.56% (29/36), and 58.33% (21/36), respectively. These values were higher than those found for pN0 patients (i.e., 13/30, 11/30, and 7/30, respectively) (P < 0.05). Among the samples with Twist expression, 88.64% were VEGF-C-positive and 59.09% were VEGFR-3-positive. The pN0 counterparts were 4.55% and 9.09%, respectively (P < 0.05). The expressions of Twist, VEGF-C, and VEGFR-3 in pN+ patients obtained through Western blotting analysis were significantly higher than those in pN0 patients, and the levels of VEGF-C and VEGFR-3 were positively correlated with that of Twist.CONCLUSIONS:Twist expression correlates with lymph node metastasis. The mechanism involved in such a correlation may be related to lymphangiogenesis.

The transcription factor TWIST is an important factor in regulating epithelial-mesenchymal transition (EMT) and tumor metastasis. To explore the functions of TWIST in hypopharyngeal cancer, we investigated if overexpression of TWIST has an effect on FaDu cell morphology, and if alteration of TWIST has an effect on E-cadherin, N-cadherin, c-fos, MMP-9, as well as in cell migration, and the invasion ability of FaDu cells. Moreover, we also studied the relationship between TWIST overexpression and clinicopathological characteristics in hypopharyngeal cancer tissue samples by immunohistochemical assays. The results showed that overexpression of TWIST-induced morphological changes, such as occurrence of EMT. TWIST overexpression also increased cell migration and invasion ability, accompanied by an alteration of E-cadherin, N-cadherin, c-fos and MMP-9 expression. Furthermore, immunohistochemical assays showed that TWIST overexpression was related with tumor differentiation (P=0.038), tumor size (P=0.048) and lymph node metastasis (P=0.044). The data presented reveal that overexpression of TWIST plays a significant role in the metastasis of hypopharyngeal tumors, and alteration of TWIST has an effect on the EMT, c-fos and MMP-9 expression in FaDu cells. We conclude that TWIST promotes hypopharyngeal carcinoma metastasis, and the TWIST/c-fos/MMP-9 signaling pathway may play an important role in the metastasis of FaDu cells.

OBJECTIVE To investigate the different or similar characteristics between the hypopharyngeal carcinoma cell line(FaDu cells)and the multidrug resistance(MDR)cell lines(FaDu/T)induced by Taxol.METHODS Human FaDu cells were exposed in stepwise escalating concentration of Taxol until the resistant cell line was formed.The drug resistance was investigated by methyl-thiazolyl-tetrazolium(MTT)assay.The cell apoptosis was measured by staining cells with acridine orange(AO),Ho.33342 and PI staining.Meanwhile,the cell cycle distribution was quantified through flow cytometry.Reverse Transcription Polymerase Chain Reaction(RT-PCR)was conducted to detect mRNA levels of the drug resistance genes ABCB1 and apoptosis related genes Bax,Bcl-2,Caspase-3.The proteins levels were detected through Western blot.RESULTS Compared with parents FaDu cells,the drug resistance cells of FaDu/T had the biological characteristics as follows: ①They had a lower growth velocity;②Their mutidrug resistance ability was increased;③The percentage of cells in G0/G1 phases was increased while that in G2/M and S phase decreased,but the proportion of apoptosis cells had no prominent difference;④The expression of ABCB1 gene increased significantly;⑤The anti-apoptotic protein Bcl-2 increased prominently,while the apoptotic protein,Caspase-3 and Bax,decreased.CONCLUSION The high expression of ABCB1,Bcl-2 may be the critical reason of the chemotherapy resistance in FaDu/T;but the internal connection among various biological characteristics of tumor cells is worth to be further investigeated.

肿瘤干细胞来源于正常干细胞的突变,随着对肿瘤干细胞认识的不断深入,人们发现其比普通肿瘤细胞更具有放化疗抵抗性.研究证实这种特性与高表达多药耐药相关性有密切关系,其中三磷酸腺苷结合转运蛋白(ATP-biniding cassette,ABC)G超家族成员2(ABCG2)作为ABC家族中的一员在目前肿瘤多药耐药性机制中起着重要的作用.本文将对ABCG2介导的肿瘤干细胞多药耐药性作用机制做一综述。

The major obstacle to tumor chemotherapy is drug resistance. In the present study, we investigated the characteristics of FaDu cells (a hypopharyngeal carcinoma cell line) with multidrug resistance (MDR) induced by Taxol. The resistant cell line, FaDu/T, was grown by exposing normal FaDu cells to escalating concentrations of Taxol stepwise for over 12 months. The multidrug resistant sensitivities of the FaDu/T cells to cisplatin (DDP), 5-fluorouracil (5-FU), doxorubicin (Dox) and vincristine (VCR) were investigated by methyl-thiazolyl-tetrazolium (MTT) assay. Cell apoptosis was measured by acridine orange and Hoechst 33342/propidium iodide double staining. Cell cycle distribution and the cell apoptosis index were quantified using flow cytometry. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted to determine the mRNA levels of the MDR-related genes MDR1/ABCB1 and BCRP/ABCG2. Western blotting was used to assay the expression of MDR1/ABCB1 and BCRP/ABCG2 and the apoptosis-related proteins caspase-3, Bcl-2 and Bax. Compared with the FaDu cells, the drug resistance of FaDu/T cells to DDP, 5-FU, Dox and VCR was increased 8.99-, 21.96-, 31.42- and 10.00-fold, respectively. The percentages of FaDu/T cells in the G0/G1 and G2/M phases were increased while the cell percentage in the S phase decreased as compared with the percentages of FaDu cells. The anti-apoptotic ability increased prominently, as the index of apoptosis decreased. Furthermore, caspase-3, Bcl-2 and Bax expression was altered accordingly to resist apoptosis in the FaDu/T cells. MDR1/ABCB1 expression increased significantly at both the mRNA and protein levels, while BCRP/ABCG2 expression appeared to inversely affected, i.e. decreased, in a concentration-dependent manner. These -findings may provide theoretical support for the prevention of MDR in clinical cancer chemotherapy.