lipofectamine and Carcinoma--Squamous-Cell

To explore the role of S100A4 in the epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma and its possible molecular mechanism.. Three chemically synthesized S100A4 siRNA sequences were transiently transfected into esophageal carcinoma EC9706 cells. EC9706 cells transfected with negative siRNA, lipofectamine 2000, and vacant EC9706 cells were used as control. Fluorescence quantitative RT-PCR and Western blot were used to detect the inhibition rate of S100A4 siRNA. S100A4 siRNA2 with the best inhibition rate was chosen to transiently transfect into EC9706 cells under the same conditions. The EC9706 cells transfected with negative siRNA, lipofectamine 2000 and vacant EC9706 cells were also used as control. Fluorescence quantitative RT-PCR and Western blot were used to detect the mRNA and protein expressions of E-cadherin, vimentin and snail. The morphology of EC9706 cells was observed under an inverted microscope. Boyden chamber and scratch test were used to detect the invasion and migration ability of EC9706 cells, and CCK8 assay was used to detect the proliferation ability of EC9706 cells. EC9706 cells transfected with S100A4 siRNA2 were further transfected with snail eukaryotic expression vector. The EC9706 cells transfected with S100A4 siRNA, EC9706 cells transfected with snail eukaryotic expression vector and vacant EC9706 cells were used as control. The above indexes of all the groups were observed, too.. The S100A4 mRNA and protein expression levels of the S100A4 siRNA2 group were 0.417 ± 0.041 and 0.337 ± 0.039, the transmembrane cell number was 61.608 ± 8.937, the scratch healing distance was (0.216 ± 0.064) mm, the A value was 0.623 ± 0.084, the E-cadherin mRNA and protein levels were 0.619 ± 0.032 and 0.495 ± 0.034, the vimentin mRNA and protein levels were 0.514 ± 0.032 and 0.427 ± 0.028, the snail mRNA and protein levels were 0.573 ± 0.029 and 0.429 ± 0.041. These data were significantly different with the liposome group, the negative control group and the blank group (P < 0.05 for all). After the S100A4 siRNA2 treatment for 24 h, the appearance of EC9706 cells changed to epithelial cell morphology. The transmembrane cell number and the scratch healing distance of the S100A4 siRNA2+snail eukaryotic expression vector group were (69.382 ± 9.666) cells and (0.274 ± 0.029) mm, the A value was 0.823 ± 0.101, the snail mRNA and protein levels were 0.704 ± 0.037 and 0.625 ± 0.031, the vimentin mRNA and protein levels were 0.712 ± 0.046 and 0.609 ± 0.038, and these data were significantly higher than those of the Sl00A4 siRNA2 group (P < 0.05 for all). The E-cadherin mRNA and protein levels of the S100A4 siRNA2+eukaryotic expression vector group were 0.437 ± 0.038 and 0.381 ± 0.031, significantly lower than those of the S100A4 siRNA2 group (P < 0.05 for all). However, snail had no effect on the morphology of EC9706 cells.. S100A4 may be involved in the EMT process of esophageal squamous-cell carcinoma by regulating the expression of snail and then plays a role in the invasion and metastasis of esophageal carcinoma.

Topics: Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Humans; Indicators and Reagents; Lipids; RNA, Messenger; RNA, Small Interfering; S100 Calcium-Binding Protein A4; S100 Proteins; Snail Family Transcription Factors; Transcription Factors; Transfection; Vimentin

This study used RNA interference (RNAi) to explore the effect of NO and inducible nitric oxide synthase (iNOS) on apoptosis and proliferation in the tongue squamous carcinoma cell line Tca8113. Tca8113 cells were transfected with the plasmid pGenesil-1, which expresses iNOS short hairpin RNA (shRNA), or the negative control plasmid pSilencer-HK, and the transfected cells were compared with untransfected cells. The expression of iNOS was detected by histochemistry, and apoptosis was detected by flow cytometry. The expression of iNOS was significantly lower in the pSilencer-iNOS group than in the pSilencer-HK and empty control groups. The apoptosis rate was significantly higher in the pSilencer-iNOS group than in the pSilencer-HK and empty control groups. Growth monitoring showed that proliferation was also inhibited in cells transfected with pSilencer-iNOS. RNAi gene silencing decreased iNOS gene expression, induced apoptosis, and suppressed proliferation in Tca8113 cells.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Coloring Agents; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Histocytochemistry; Humans; Indicators and Reagents; Lipids; Nitric Oxide; Nitric Oxide Synthase Type II; Plasmids; RNA Interference; RNA, Small Interfering; Tetrazolium Salts; Thiazoles; Tongue Neoplasms; Transfection

Steroid hormones such as 17beta-estradiol (E2) are critical to diverse cellular processes including tumorigenesis. A number of cofactors such as nuclear receptor corepressor (NCoR), CREB-binding protein (CBP), and steroid receptor coactivator 1 (SRC-1) interact with estrogen receptors (ERs) to regulate transcriptional repression or activation of target genes. Estrogen signaling in non-reproductive tract tissues such as skin is less well characterized and the effectiveness of anti-estrogen therapy for cancer arising from these tissues is unknown. We show that tamoxifen (TAM) treatment inhibited cell cycle progression and proliferation of human cancer lines derived from stratified squamous epithelium squamous cell carcinoma (SCC). E2 had no effect on proliferation of these lines despite low levels of ERalpha expression. The E2 treatment promoted displacement of the NCoR from ERalpha and recruitment of CBP to the receptor. SRC-1 expression was not detected in these SCC lines; however, transient transfection of SRC-1, CBP, or both coactivators enhanced transactivation of an estrogen responsive promoter in cancer cells treated with E2 or TAM. In stable clones expressing SRC-1, the coactivator was recruited to ERalpha along with CBP in E2 but not in TAM-treated cells. SRC-1 expression restored the E2-mediated proliferative response to human SCC lines. This increased proliferation correlated with increased extracellular signal regulated kinase 1 (ERK1) expression. SRC-1 and CBP were recruited to the proximal ERK1 promoter region in E2 but not in TAM-treated cells. We concluded that SRC-1 was a key molecular determinant of estrogen-mediated proliferation in human SCC lines.

Topics: Blotting, Western; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; CREB-Binding Protein; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Extracellular Signal-Regulated MAP Kinases; Histone Acetyltransferases; Humans; Lipids; Nuclear Receptor Coactivator 1; Reverse Transcriptase Polymerase Chain Reaction; Tamoxifen; Transcription Factors; Transfection

To investigate the effects of caveolin-1 on the biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line in vitro.. Eukaryotic expression vector of human caveolin-1 gene was constructed and transfected into HEp2 cells by Lipofectamine. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western blotting. Cell proliferation viability was tested by MTT assay. Anchorage-independent growth was determined by assaying colony formation in soft agar. Flow cytometry was used to assess the cell cycle and apoptosis. The relative phosphorylation level of EGFR and ERK1/2 were detected by Western blotting. Localization of caveolin-1 and EGFR were studied by laser confocal laser scanning microscopy.. The expression vector of caveolin-1 was constructed and three clones stably overexpressing caveolin-1 were obtained. Comparing with the parental HEp2 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of FACS analysis revealed that overexpression of caveolin-1 resulted in the cell cycle arrest at G0/G1 phase and increased the apoptotic cell fraction. EGFR was found to colocalize with caveolin-1 in transfected cells by confocal laser scanning microscopy and Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR and Erkl/2.. Overexpression of caveolin-1 suppresses the growth of HEp2 cells and induces apoptosis and inhibition of EGFR-MAPK signaling pathway may be involved in its mechanism.

Topics: Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caveolin 1; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; ErbB Receptors; Flow Cytometry; Genetic Vectors; Humans; Laryngeal Neoplasms; Lipids; Microscopy, Confocal; Mitogen-Activated Protein Kinase 3; Phosphorylation; Polymerase Chain Reaction; Signal Transduction; Transfection

The purpose of this study was to establish transfergeneic Tca8113 cell and evaluate the expression of human endostatin (hES) gene in the cell colone in vitro.. To transfect hES gene into Tca8113 cells, lipofectamin was complexed with plasmid encoding hES gene, and blasticidin S antibiotic was adopted to select Tca8113--hES cell clone. Immunohistochemistry S-P method was adopted to detect the expression of hES in the transfergenic Tca8113 cell in vitro.. Transfected by hES, the transfergenic Tca8113 cells could grow and proliferate in RPMI--1640 culture medium containing blasticidin S antibiotic. The expression rate of hES reached 100%.. hES gene can express in hES-transfected Tca8113 cell in vitro.

Topics: Carcinoma, Squamous Cell; Cell Division; Cloning, Molecular; Endostatins; Humans; Lipids; Tongue Neoplasms; Transfection; Tumor Cells, Cultured

To investigate the immunological mechanism of mouse IL-2 gene therapy and the optimal lipid to DNA ratios of lipid-DNA complexed (lipoplexes) by using polycationic liposome-mediated Tumors were established in the transduction for head and neck squamous cell carcinoma (HNSCC).. floor of mouth in C3H/HeJ immunocompetent mice with SCCVII cell line. Lipoplexes with various lipid to DNA ratios (L:D) and naked DNA were transducted in vivo by direct intratumoral gene transfer and in vitro. The supernatants of SCCVII cell and tumour tissues were collected for IL-2 expression by enzyme-linked immunosorbent assay. Natural killer (NK) cell activity and cytotoxic T-lymphocyte (CTL) activity were also The optimal L:D ratio for IL-2 expression in vitro was not assayed by lactate dehydrogenase method.. consistent with that in vivo. By use of lipoplexes with L:D = 3:1, higher IL-2 expression of tumor tissues and greater activities of NK cell and CTL of murine spleen were noted in the treated group as compared with those A comparison of naked plasmid and lipid-complexed found in naked DNA and empty plasmid (EP).. DNA revealed that lipoplexes were more effective for intratumoral gene transfer to HNSCC. The results indicate that the formulation and dosage of polycationic L:D complexes play a key role in determining the level of intratumoral transgene expression.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Drug Carriers; Genetic Therapy; Head and Neck Neoplasms; Interleukin-2; Killer Cells, Natural; Lipids; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Polyamines; Polyelectrolytes; T-Lymphocytes, Cytotoxic; Transfection