Effect of epithelial cell adhesion molecule on metastasis in hypopharyngeal carcinoma FaDu cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of knockdown of EpCAM by siRNA on invasion, migration, and colony abilities in hypopharyngeal carcinoma FaDu cells.</p><p><b>METHODS</b>A siRNA against EpCAM was employed to inhibit the expression of EpCAM in FaDu cells. Measurements included the Transwell assay for invasion and migration, plate colony formation assay for cell colony ability, Western blot assay for EpCAM, E-cadherin, and β-catenin expressions in total protein, cytoplasm, and cytoskeleton, respectively.</p><p><b>RESULTS</b>mRNA and protein expressions of EpCAM were suppressed significantly in FaDu cells transfected by EpCAM siRNA (t = 6.46, P < 0.05; t = 10.25, P < 0.05) . Transwell assay showed in transwell assay, the average invasive cells in EpCAM siRNA cells (26.33 ± 3.71) was less than that in FaDu cells (61.47 ± 6.70; t = 7.95, P < 0.05)and control cells (54.13 ± 6.51; t = 6.42, P < 0.05); the average number of migration cells in EpCAM siRNA cells (79.87 ± 8.44) was lower than that in FaDu (167.53 ± 11.49; t = 10.90, P < 0.05) cells and control cells (162.13 ± 13.45; t = 8.97, P < 0.05). In plate colony formation assay, the average colony number of EpCAM siRNA cells was (78.00 ± 5.57), which was less than that of FaDu cells(177.30 ± 16.50; t = 9.78, P < 0.05) and control cells (173.67 ± 13.50; t = 11.35, P < 0.05). Western blot assays showed, silencing of EpCAM increased the expressions of E-cadherin (t = 4.58, P = 0.01) and β-catenin (t = 3.76, P = 0.02) in cytoskeleton, and decreased the expressions of E-cadherin (t = 6.60, P < 0.05) and β-catenin (t = 8.20, P < 0.05) in cytoplasm.</p><p><b>CONCLUSIONS</b>The knockdown of EpCAM inhibits the invasion, migration, and colony formation abilities of FaDu cells, which is probably related to the regulation of E-cadherin and β-catenin in cytoplasm and cytoskeleton, and EpCAM may be a promising gene therapy target for hypopharyngeal carcinoma.</p>

Role of ABCB1 and ABCG2 in the multidrug resistance of hypopharyngeal carcinoma FaDu cell line / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Twist modulates lymphangiogenesis and correlates with lymph node metastasis in supraglottic carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>Twist expression correlates with lymph node metastasis. The mechanism involved in such a correlation may be related to lymphangiogenesis.</p>

Nimesulide induced apoptosis and inhibited metastasis of hypopharyngeal carcinoma cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of Nimesulide, a selective Cox-2 inhibitor, on the apoptosis, invasion and migration of hypopharyngeal carcinoma cell line (FaDu).</p><p><b>METHODS</b>Viabilities of FaDu cells treated with various concentrations of Nimesulide were detected by MTT assay. Morphological changes were observed by acridine orange cytochemistry staining. Apoptosis was examined by flow cytometry. The ability of invasion and migration of cells was detected by Transwell chambers. The mRNA and protein expressions of Cox-2, MMP-9 and caspase-3 in response to Nimesulide were examined by RT-PCR and Western blot, respectively.</p><p><b>RESULTS</b>MTT assay showed that the cell surviving rates significantly decreased in time- and concentration-dependent manners (P < 0.05). The typical morphological changes of apoptotic cells were observed. Percent of apoptosis after 6 h Nimesulide treatment was (32.4 ± 6.1)%. The metastatic cells were decreased by Nimesulide to about 20%. Nimesulide decreased the expressions of Cox-2 and MMP-9, whereas increased expression the of Caspase-3.</p><p><b>CONCLUSION</b>Nimesulide could induce the apoptosis and inhibit metastasis of FaDu cells.</p>

Role of TWIST in the apoptosis of Hep-2 cells induced by paclitaxel / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore the relationship between the expression of transcription factor TWIST and apoptosis of Hep-2 cells induced by paclitaxel.</p><p><b>METHODS</b>Morphological changes of Hep-2 cells were observed by reserved microscopy and acridine orange cytochemistry staining. Viability of Hep-2 cells treated with various concentrations of paclitaxel was detected by MTT assay. Apoptosis was examined by flow cytometry. The expressions of transcription factor TWIST at both mRNA and protein level in response to paclitaxel at 24 h, 48 h and 72 h were then examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively.</p><p><b>RESULTS</b>Typical morphological changes of apoptotic cells, i.e., cellular rounding-up, cytoplasmic contraction, chromatin condensation and, particularly, apoptotic body, the main morphological characteristic of apoptosis, were observed by reserved microscopy and acridine orange cytochemistry staining. The cell surviving rates significantly decreased in a concentration- and time-dependent manner as evidenced by MTT assay (P < 0.05). Percent of apoptosis after 24 h, 48 h or 72 h paclitaxel-treatment was (22.6 +/- 5.3)%, (38.7 +/- 7.9)% and (52.4 +/- 14.3)%, respectively, whereas the percent of control was (9.85 +/- 5.83)%. There existed a statistically significant difference between treatment and control (F = 12.621, P < 0.05). The expression of TWIST at both mRNA and protein levels for 24 h, 48 h or 72 h in the paclitaxel-induced apoptosis of Hep-2 cells were decreased by 16.7%, 46.8%, 76.9% (F = 10.407, P < 0.05) and 16.4%, 33.6%, 69.6% (F = 18.013, P < 0.05) respectively.</p><p><b>CONCLUSIONS</b>TWIST, which is significantly decreased in expression in response to paclitaxel in Hep-2 cells, may play a pivotal role in paclitaxel-induced apoptosis of Hep-2 cells.</p>