Transforming growth factor-ß1-induced epithelial-mesenchymal transition in human esophageal squamous cell carcinoma via the PTEN/PI3K signaling pathway.

Epithelial-mesenchymal transition (EMT) is a crucial step for the invasive and metastatic properties of malignant tumor cells during tumor progression. Numerous signaling pathways are involved in the process of EMT in cancer, such as the EMT-inducing signal transforming growth factor (TGF)-ß and the recently demonstrated PTEN/PI3K signaling pathway. To date, no data have been reported concerning the influence of PTEN/PI3K signaling pathway on EMT in human esophageal squamous cell carcinoma (ESCC) and how TGF-ß1 and PTEN/PI3K act through multiple interconnected signaling pathways to trigger events associated with EMT and tumor progression. Our data showed that the PTEN/PI3K pathway was active in human ESCC tissues in vivo, particularly in ESCC with decreased E-cadherin and increased vimentin protein expression, poor differentiation, deep invasion and lymph node metastasis, which are responsible for EMT and tumor progression. In addition, in the human ESCC cell line (EC-1) in vitro, TGF-ß1 treatment markedly induced EMT, including morphological alterations, a decrease of E-cadherin and an increase of vimentin levels and enhanced mobility and invasiveness. Furthermore, the PTEN/PI3K pathway was also activated in the process of TGF-ß1-induced EMT in EC-1 cells in vitro, whereas inhibition of the PTEN/PI3K pathway by using pcDNA3.1 PTEN partially blocked TGF-ß1-induced EMT and reduced mobility and invasiveness. These studies suggest that TGF-ß1 and the PTEN/PI3K signaling pathway contribute to EMT and the PTEN/PI3K signaling pathway is a key regulator of TGF-ß1­induced EMT in ESCC. Disruption of the PTEN/PI3K pathway involved in TGF-ß1-induced EMT may provide possible routes for therapeutic intervention to ESCC.

Stathmin is a marker of progression and poor prognosis in esophageal carcinoma.

Stathmin, also called oncoprotein 18, is a founding member of the family of microtubule-destabilizing proteins that play a critical role in the regulation of mitosis. At the same time stathmin has been recognized as one of responsible factors in cancer cells. The aim of this study was to assess stathmin status, its correlations with clinicopathological parameters and its role as a progosnostic marker in EC patients. The protein and mRNA levels of stathmin were examined by immunohistochemistry (IHC) and in situ hybridization in 100EC tissues and adjacent noncancerous tissues. mRNA and protein expression of stathmin in three EC cell lines(EC9706, ECa109, EC1 commonly used in research) were also analyzed using immunocytochemistry, western blot and in situ hybridization. The prognostic value of Stathmin expression within the tumor tissues were assessed by Cox regression and Kaplan-Meier analysis. We showed that stathmin expression was significantly higher in EC tissues than in adjacent noncancerous tissues. High stathmin immunostaining score in the EC was positively correlated with tumor differentiation, Tumor invasion, Lymph node metastases, and TNM stage. In addition, we demonstrated that three EC cell lines examined, were constitutively expressing a high level of stathmin. Of those, EC-1 showed the strongest mRNA and protein expression for the stathmin analyzed. Kaplan-Meier analysis showed that significantly longer 5-year survival rate was seen in EC patients with high Stathmin expression, compared to those with low expression of Stathmin expression. Furthermore, multivariate Cox proportional hazard analyses revealed that Stathmin was an independent factors affecting the overall survival probability. In conclusion, our data provide a basis for the concept that stathmin might be associated with EC development and progression.. High levels of Stathmin expression in the tumor tissues may be a good prognostic marker for patients with EC.

Influence of human myasthenia gravis thymus on the differentiation of human cord blood stem cells in SCID mice.

The normal thymus contributes to T lymphocytes differentiation and induction of tolerance to self-antigens. Myasthenia gravis (MG) is characterized by abnormal thymic hyperplasia. To assess the potential influence of MG-thymus on the differentiation of T lymphocytes differentiation, we used the MG-thymus transplanted severe combined immunodeficiency (SCID) mice model to evaluate the human cord blood stem cells differentiation. Thymus fragments from MG patient and human cord blood stem cells were transplanted into SCID mice successively. SCID mice were observed to develop sustained human T lymphocytes and a functional anti-tumor immune. The levels of various T cell subsets in SCID mice with MG-thymus were different from that of control group. Among that, the frequency of CD4(+)CD25(+) T cells was significant lower in SCID mice with MG-thymus. The deficiency of CD4(+)CD25(+) T cells seens to contribute to the pathogenesis of MG.

Roles of GST-π and polß genes in chemoresistance of esophageal carcinoma cells.

The main aim of this study was to investigate the roles of GST-π and polß genes in the chemoresistance of esophageal carcinoma cells. Eukaryotic expression vectors containing each gene were constructed and transfected into EC9706 cells, and the biological effects of the two genes assessed based on a resistance index. We additionally investigated the in vitro and in vivo anti-resistance effects of GST-π and polß genes using recombinant lentiviruses carrying siRNAs against the two genes. Our results showed that upregulation of GST-π and polß genes suppresses chemosensitivity of esophageal carcinoma cells to cisplatin, while downregulation of these two genes with RNAi technology reverses this chemoresistance. Multi-site injection of recombinant lentivirus targeting the GST-π gene into transplanted cDDP tumors effectively reversed their chemoresistant phenotype. However, the same treatment against the polß gene did not lead to significant efficacy against chemoresistance.

Relationship between TWIST expression and epithelial-mesenchymal transition of oesophageal squamous cell carcinoma.

We have investigated mRNA and protein expression of TWIST, Vimentin and E-cadherin in ESCC (oesophageal squamous cell carcinoma) and explored their relationship with tumour's infiltration and metastasis. RT-PCR (reverse transcriptase-PCR) was used to evaluate mRNA expression of TWIST, E-cadherin and Vimentin in 40 cases of ESCC. The protein expression of the genes was examined by immunohistochemical staining in each specimen. Expression of TWIST, E-cadherin and Vimentin mRNA and protein with clinicopathologic parameters were analysed. mRNAs of TWIST, Vimentin and E-cadherin were expressed in 75, 55 and 35% respectively of ESCC, i.e. significantly different from that in normal oesophageal mucosa (15, 0 and 85% respectively; P<0.01). In ESCC with LN (lymph node) metastasis, expression of TWIST and Vimentin mRNA, but not E-cadherin mRNA was significantly higher (100 and 83%) than in ESCC without LN metastasis (64 and 43%, P=0.018) respectively. Levels of mRNA expression of the 3 genes followed similar patterns to their above-mentioned frequencies. Protein expression of TWIST, E-cadherin and Vimentin were observed in 70, 35 and 50% respectively of ESCC, which were significantly different from normal mucosa (15, 80 and 0%; P<0.001). In ESCC with LN metastasis, protein expression of TWIST and Vimentin, but not E-cadherin, were significantly higher (100 and 75%) than in ESCC without LN metastasis (61 and 39%). Protein expression of TWIST was positively correlated with Vimentin (r=0.327, P=0.039), but negatively correlated with E-cadherin (r= -0.633, P=0.000). Thus, both mRNAs and proteins of TWIST and Vimentin were significantly overexpressed in ESCC, especially ESCC with LN metastasis. The mRNA and protein of E-cadherin were down-regulated in ESCC. These results suggest potential roles of TWIST as the promoter of tumour invasion and metastasis associated with down-regulation of E-cadherin.

S100A4 mediated cell invasion and metastasis of esophageal squamous cell carcinoma via the regulation of MMP-2 and E-cadherin activity.

It is well documented that S100A4 is upregulated in a large amount of invasive tumors and plays a pivotal role in tumor invasion and metastasis. However, the precise role and mechanism S100A4 exerts in the invasion and metastasis of esophageal squamous cell carcinoma (ESCC) have not been fully elucidated to date. Our data demonstrated that S100A4 was overexpressed in human ESCC tissues, especially in ESCC with poor differentiation, deep invasion and lymph node metastasis. Subsequently, the knockdown of S100A4 by RNAi in ESCC cell line (EC-1) could reduce cell invasion, metastasis and proliferation ability in vitro. Most importantly, S100A4 regulated MMP-2 positively and E-cadherin negatively in vivo and in vitro to some extent. Our results suggest that S100A4 is an important factor in the invasion, metastasis and proliferation of ESCC and may control invasion and metastasis at least in part through the regulation of MMP-2 and E-cadherin activity. S100A4 may serve as a biomarker for progression of ESCC and a potential molecular target for biotherapy of ESCC.

[Expression of S100A4 in esophageal squamous cell carcinoma and its relation to tumor invasion and metastasis].

OBJECTIVE: To study the role of S100A4 in the carcinogenesis, development, invasion and metastasis of esophageal squamous cell carcinoma. METHODS: Immunohistochemistry was used to detect the expressions of S100A4, MMP-2 and E-cadherin proteins in 100 cases of surgically resected esophageal squamous cell carcinoma specimens. RT-PCR and Western blot were used to detect the expressions of S100A4 mRNA and protein in esophageal squamous cell carcinoma line EC-1 and TE-1. Boyden-chamber model in vitro was utilized to detect the invasion ability of EC-1 and TE-1 cells. RESULTS: The positivity rate of S100A4 protein was 52.0% was in esophageal carcinoma tissues, significantly higher than that in normal tissues (26.0%) (P<0.01). The expression of S100A4 was related to tumor grading, invasive depth and lymph node metastasis (P<0.05). In esophageal carcinoma, the expression of S100A4 was positively correlated to MMP-2 expression (P<0.01), but inversely to E-cadherin expression (P<0.05). The expressions of S100A4 mRNA (0.894-/+0.021) and protein (0.897-/+0.053) in EC-1 cells were significantly higher than those in TE-1 (0.812-/+0.040 and 0.645-/+0.089, respectively, P<0.01), and the invasion ability of EC-1 cells was significantly higher than that of TE-1 cells (91.00-/+17.44 vs 61.80-/+11.10, P<0.01). CONCLUSION: The overexpression of S100A4 in esophageal squamous cell carcinoma tissue and highly invasive EC-1 cells may contribute to the carcinogenesis, development, invasion and metastasis of esophageal squamous cell carcinoma.

[Inhibitory effect and molecular mechanism of silencing S100A4 gene on the growth of transplanted tumor of human esophageal carcinoma EC-1 cells in nude mice].

OBJECTIVE: To study the effect of S100A4siRNA on tumor growth of xenografted human esophageal squamous cell carcinoma (ESCC) cell line EC1 in nude mice and explore its related molecular mechanism. METHODS: The xenografted tumor model was established in nude mice, and S100A4siRNA chemically synthesized was used to transfect the xenografted nude mice. The tumor growth was observed. The mRNA and protein expressions of S100A4, MMP-2 and E-cadherin in tumor after transfection with S100A4siRNA were detected by RT-PCR and immunohistochemistry. RESULTS: The tumor volume of S100A4siRNA transfection group was lower than that of nonsense siRNA transfection group and blank control group (P < 0.05), and tumor inhibitive ratio of S100A4siRNA group was higher than that of nonsense siRNA transfection group (P < 0.05). Furthermore, the mRNA and protein expressions of S100A4, MMP-2 in S100A4siRNA group were lower than those in nonsense siRNA group and control group (P < 0.05), the mRNA and protein expressions of E-cadherin in S100A4siRNA group were higher than those in nonsense siRNA group and control group (P < 0.05). CONCLUSION: S100A4siRNA could effectively lead to growth inhibition of xenografted human esophageal tumor in nude mice, with down regulation of MMP-2 and up regulation of E-cadherin, which provides theoretical basis for molecular therapy of ESCC.

[Effect of KISS-1 on invasive potential and proliferation of esophageal squamous carcinoma cell line EC-1].

OBJECTIVE: To investigate the effect of KISS-1 expression on the potential of invasion and proliferation of esophageal squamous carcinoma cell EC-1. METHODS: Protein and mRNA expressions of KISS-1 were evaluated by Western blot and RT-PCR in four esophageal carcinoma cell lines (EC-1, Eca109, EC9706 and TE-1). Using liposome-mediated transfection, an eukaryotic expression vector (pcDNA3.1-KISS-1) of KISS-1 gene was transfected into EC-1 cells. Boyden chamber model, MTT and clone formation assay were used to detect the potential of invasion and proliferation. RESULTS: Western blot and RT-PCR showed a baseline low level of expression of KISS-1 protein (0.715 +/- 0.109) and mRNA (0.670 +/- 0.176) in EC-1 cells. pcDNA3.1-KISS-1 expression vector was successfully transfected into EC-1 cells. Western blot and RT-PCR showed that the expression of KISS-1 protein (1.143 +/- 0.218) and mRNA (0.877 +/- 0.162) in EC-1 cells transfected with pcDNA3.1-KISS-1 were significantly higher than those transfected with the control vector pcDNA3.1 (0.745 +/- 0.130, 0.685 +/- 0.128; t = 3.850, 2.481, P < 0.05) and the control cells (0.855 +/- 0.184, 0.677 +/- 0.138; t = 2.275, 2.306, P < 0.05). Boyden chamber analysis showed that the invasiveness of the cells transfected with KISS-1 at 24 h (91.8 +/- 11.7), 48 h (117.8 +/- 11.1) and 72 h (139.2 +/- 11.8) were significantly reduced than that of the cells transfected with the control vector pcDNA3.1 (118.1 +/- 14.7, 141.7 +/- 13.2, 162.2 +/- 22.7; t = 3.153, 4.215, 3.569, P < 0.01) and the control cells (112.2 +/- 15.6, 138.1 +/- 13.0, 162.3 +/- 14.0; t = 4.154, 3.797, 2.702, P < 0.05). MTT showed that the proliferation potential of cells after transfection with KISS-1 at 48 h (0.517 +/- 0.127) and 72 h (0.394 +/- 0.137) were significantly reduced than that of cells transfected with the control vector pcDNA3.1 (0.636 +/- 0.186, 0.513 +/- 0.150; t = 2.054, 2.709, P < 0.05) and the control cells (0.646 +/- 0.135, 0.511 +/- 0.153; t = 2.276, 2.205, P < 0.05). Clone formation assay suggested that cells transfected with KISS-1 (157.2 +/- 36.4) showed significantly decreased clone formation than cells transfected with the control vector pcDNA3.1 (236.3 +/- 78.1; t = 3.441, P < 0.01) and the control cells (242.5 +/- 48.6; t = 2.250, P < 0.05). CONCLUSION: KISS-1 gene inhibits the potential of invasion and proliferation of EC-1 cells.