Search

// Xiuxiu Liu 1, 2, * , Zhenghua Lv 1, * , Jidong Zou 1 , Xianfang Liu 1, 2 , Juke Ma 1 , Chengtao Sun 1 , Na Sa 1 , Wei Xu 1, 2 1 Department of Otorhinolaryngology Head and Neck Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China 2 Shandong Provincial Key Laboratory of Otology, Jinan, Shandong, China * These authors contributed equally to this work Correspondence to: Wei Xu, email: xuwhns@126.com Keywords: macrophages, cancer invasion, AEG-1, MMP-9, hypopharyngeal cancer Received: July 11, 2016 Accepted: October 14, 2016 Published: October 25, 2016 ABSTRACT 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.

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

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.

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.

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.

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. Morphological changes of Hep-2 cells that were transfected a mircoRNA against TWIST vector were observed by the reserved microscope. Reverse transcription-polymerase chain reaction was performed in order to examine the mRNA expression of TWIST, E-cadherin, and N-cadherin. Western blotting was performed to examine the protein expression of TWIST, E-cadherin, and N-cadherin. Cell motion ability was examined by Scratch-wound assay. Transwell™ chamber assays were used to determine cell migration and invasion. Transfecting a mircoRNA down-regulated TWIST expression at mRNA and protein levels. Down-regulation of TWIST expression induced morphological changes, such as the inversion of the EMT. Moreover, down-regulation of TWIST expression up-regulated E-cadherin and down-regulated N-cadherin expressions at mRNA and protein levels, respectively. Furthermore, we confirmed that down-regulation of TWIST expression decreased the motion, invasion, and migration ability of the Hep-2 cells. Down-regulation of TWIST expression decreases migration and invasion of laryngeal carcinoma Hep-2 cells by regulation of the E-cadherin, N-cadherin expression.