[Role of ABCB1 and ABCG2 in the multidrug resistance of hypopharyngeal carcinoma FaDu cell line].

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.

Down-regulation of TWIST decreases migration and invasion of laryngeal carcinoma Hep-2 cells by regulating the E-cadherin, N-cadherin expression.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Down-regulation of TWIST expression decreases migration and invasion of laryngeal carcinoma Hep-2 cells by regulation of the E-cadherin, N-cadherin expression.

Twist modulates lymphangiogenesis and correlates with lymph node metastasis in supraglottic carcinoma.

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.