Expression of stathmin in esophageal squamous cell carcinoma and its biological significance / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the expression of stathmin gene in esophageal squamous cell carcinoma (ESCC) and its correlation to oncogenesis of ESCC.</p><p><b>METHODS</b>Three ESCC cell lines, 75 ESCC samples, 25 tumor-adjacent samples and 30 normal esophageal mucosa samples were examined for the expression of stathmin mRNA and protein by in situ hybridization and immunohistochemistry, respectively. The correlations of stathmin expression to the clinicopathological features of the patients were analyzed.</p><p><b>RESULTS</b>Overexpression of stathmin mRNA and protein was found in 3 ESCC cell lines EC9706, Eca109 and EC-1, with the positive expression rates exceeding 80%. The positive rates of stathmin mRNA and protein in ESCC samples were 82.7% and 81.3%, respectively. There were significant differences in the relative contents of stathmin mRNA and protein among normal mucosa tissue, tumor-adjacent tissue and cancer tissue (chi2=19.204 and 25.03, respectively, P<0.01). In addition, a positive correlation was noted between stathmin mRNA and protein expressions in ESCC (r=0.413, P=0.000). The relative contents of stathmin mRNA and protein were significantly correlated to the differentiation degree, lymph node metastasis, invasive depth and TNM stage of ESCC (P<0.05).</p><p><b>CONCLUSIONS</b>The expression of stathmin mRNA and protein is upregulated in ESCC with correlation to the differentiation degree, lymph node metastasis, invasive depth and TNM stage of ESCC, suggesting the possible involvement of stathmin in the oncogenesis of ESCC. Combined detection of stathmin mRNA and protein may prove valuable for early diagnosis and prognosis of ESCC, and stathmin may serve as a potential molecular target for biotherapy of the tumor.</p>

Construction and expression of human stathmin gene eukaryotic expression vector and its effect on esophageal cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector of human stathmin gene and to assess its effect on esophageal cancer EC9706 cells.</p><p><b>METHODS</b>Stathmin cDNA coding sequence was amplified by RT-PCR from Eca109 cells and was cloned into pMD18-T vector. After identifying and sequencing, the correct inserting stathmin gene was sub-cloned into eukaryotic expression vector pEGFP-C2. EC9706 cells were transfected with this recombinant plasmid and control plasmid using Lipofectamine 2000, and the stable intergrant was selected with G418 medium. The expression of enhanced green fluorescent protein (EGFP) protein was detected by fluorescence microscopy and EGFP/stathmin fusion protein by Western blot assay in transfected EC9706 cells. The growth curve of the two stably transfected cells was protracted with cell counting. FACS was used to detect the cell cycle. The clone formation rate in plate and in nude mice was tested to investigate the tumorigenic characteristics of the two stably transfected cells in vitro and vivo.</p><p><b>RESULTS</b>A 450 bp coding sequence of stathmin cDNA was amplified by RT-PCR, which was cloned into pMD18-T vector. After identified with restriction enzyme the recombinant plasmid pMD18-T-stathmin containing reverse inserting sequence was constructed successfully. Then, the sub-clone pEGFP-stathmin was sequenced, confirming that the recombinant vector was right. The recombinant plasmid pEGFP-stathmin and pEGFP-C2 vector were transfected separately into EC9706 cells. After selecting with G418, the cells were transfected steadily. EGFP in EC9706 cells was observed after transfection by fluorescence microscopy. The expressed product was proved to be 46,000 EGFP/stathmin fusion protein by Western blot. Compared with those transfected with pEGFP-C2, the growth of cells transfected with pEGFP-stathmin became slow, the cells were swelled, the cell cycle was blocked at G2/M phase, the average clone formation rate decreased in vitro, and the tumorigenicity of inoculated cells in nude mice was decreased.</p><p><b>CONCLUSION</b>The recombinant eukaryotic expression vector pEGFP-stathmin has been constructed successfully. It expresses steadily in esophageal cancer cells and inhibits the proliferation and tumorigenicity of transfected cells.</p>

Apoptosis in esophageal cancer cells induced by all-trans retinoic acid / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the anti-tumor effects of all-trans retinoic acid (ATRA) and mechanisms of its action.</p><p><b>METHODS</b>Human esophageal carcinoma cell line EC9706 cells were treated with ATRA at different concentration. The proliferation inhibition was examined by MTT assay. Morphological examination, TUNEL method and flow cytometry were used to detect the apoptosis and changes of cell cycle. Immunohistochemical method was used to detect the expression of apoptosis-related genes caspase-3 and bcl-2. The semi-quantification of protein expression was analyzed by pathological image analysis.</p><p><b>RESULTS</b>ATRA inhibited the proliferation of EC9706 cells moderately. Apoptosis in EC9706 cells was induced by ATRA treatment. The morphology of EC9706 cells showed changes such as nuclear chromatin condensation and fragmentation. Sub-G1 peak was found by flow cytometry. The maximal apoptosis rate was 32.6%. The expression of caspase-3 gene was enhanced. The expression of bcl-2 gene was decreased. All these effects were presented in a dose-dependent and time-depend manner.</p><p><b>CONCLUSION</b>Apoptosis is one of the key mechanisms of ATRA action on EC9706 cells.</p>

Demethylation of estrogen receptor gene and its re-expression in estrogen receptor-negative breast / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the correlation between the lack of estrogen receptor (ER) gene expression and hypermethylation of ER gene, and detect whether re-expressed ER protein is activated.</p><p><b>METHODS</b>The methylation status of ER gene promoter in the ER-negative breast cancer cells was evaluated by methylation specific PCR (MSP) and genomic sequencing. The expression of ER and progesterone receptor (PR) mRNA as well as the production of ER protein were detected by RT-PCR and Western blot method, respectively. MTI assay was used to examine the function of re-expressed ER protein.</p><p><b>RESULTS</b>The ER gene promoter was highly methylated, while ER mRNA and ER protein were not expressed in the ER-negative breast cell line MDA-MB-231. The ER-negative breast cells treated with demethylating agent 5 -aza-2'-deoxycytidine (5-AZA-2'-deoxyC) restored the expression of ER mRNA and ER protein. Expression of the endogenous ER-responsive PR gene was activated and the methylation of ER gene was simultaneously decreased. After MDA-MB-231 was treated with 5-AZA-2'-deoxyC, the protein of ER was re-expressed and the growth of cells treated with tamoxifen were inhibited significantly (P < 0.05).</p><p><b>CONCLUSION</b>inactivation of ER gene has a close relationship with the abnormal methylation of ER gene promoter. 5-AZA-2'-deoxyC may effectively cause demethylation and restore functional expression of ER silenced by aberrant hypermethylation. The result may offer a new measure and theory for breast cancer patients with ER-negative expression to receive endocrine therapies.</p>