Slug regulates proliferation and invasiveness of esophageal adenocarcinoma cells in vitro and in vivo

Abstract

Slug is a transcription factor and E-cadherin repressor, which has recently been demonstrated to be important for cancer cells to down-regulate epithelial markers and up-regulate mesenchymal markers in order to become motile and invasive. In the present study, we assessed the relevance of Slug for invasion and growth potential of esophageal adenocarcinoma (EA) cells in vitro and in vivo. Slug expression was detected in nine human esophageal cancer cell lines. OE33 cell line was infected with Slug siRNA to knockdown of Slug; TE7 cell line was infected with full Slug cDNA to increase Slug expression. Then, Bcl-2 and E-cadherin expression and Caspase-3 activity were analyzed. MTT assay was applied to detect growth curve. The flow cytometric and Hoechst33258 staining was performed to detect apoptosis. The cells invasion in vitro was detected with a Boyden chamber. A pseudometastatic model of OE33 and TE7 in immunodeficient mice was used to assess the effects of knockdown of Slug and Slug overexpression on metastasis development. A subcutaneously nude mice xenograft model of OE33 and TE7 was used to assess the effects of knockdown of Slug and Slug overexpression on tumor growth. Immunohistochemical staining was used to analyze the expression of Slug, bcl-2 and E-cadherin, and TUNEL was used to detected apoptosis in vivo. Western blotting and RT-PCR showed that Slug expression was detectable in 7 of 9 human esophageal cancer cell lines. Bcl-2 was down-regulated and E-cadherin was up-regulated significantly in Slug siRNA-infected OE33 cell line (P<0.01). Bcl-2 was upregulated and E-cadherin was downregulated significantly in Slug cDNA-infected TE7 cells (P<0.05). OE33 cells with Slug knockdown were shown to possess markedly decreased invasiveness (P<0.05) and markedly increased apoptosis (P<0.05). Slug cDNA-infected TE7 cells were shown only to possess markedly increased invasiveness (P<0.05). There was significant relationship between Slug knockdown or Slug overexpression and cells proliferation (respectively, P<0.05). Animals injected with Slug-silenced OE33 cells had fewer seeded tumor (P<0.01), more apoptosis cells (P<0.05) and significantly xenograft tumor growth regression (P<0.05). But in Slug cDNA-infected TE7 cells, more seeded tumor number and significantly xenograft tumor growth were found in xenograft tumor (respectively, P<0.05). It was showed in the subcutaneously nude mice xenograft model tumor tissue, bcl-2 expression was reduced followed by the decrease of Slug expression in Slug-silenced tumor, and bcl-2 expression was increased followed by the increase of Slug expression. In pseudometastatic model, E-cadherin overexpression was found in Slug siRNA tumor tissue, but less E-cadherin expression was found in Slug cDNA tissue. Slug is an important modulator of apoptosis, growth and invasion in EA in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of EA.

Crosstalk of proteins, miRNAs involved in metastatic and epithelial–mesenchymal transition pathways

“…Expression of SLUG has been found to be elevated in tumour metastasis in various types of tumours (Zhang et al 2010). In an experiment conducted on a breast cancer mouse model, it was found that SLUG promotes metastasis by partially inhibiting E-cadherin (Casas et al 2011).…”

Short interfering RNA directed against Slug blocks tumor growth, metastasis formation, and vascular leakage in bladder cancer

“…SiRNA transfections T24 cells at 70% confluence were stably transfected either a control vector (pDC316-EGFP-U6, mock) or Slug siRNA expressing pDC316-EGFP-U6 vector (Slug siRNA) (kindly provided by Dr. Zhang K, General surgery, the Affiliated Hospital of medical college, QingDao University, China) using Lipofectamine2000 Ò (Invitrogen) [8].…”

“…The product was subcloned into the expression vector pGEM-T and the pDC316-EGFP-U6 to construct the pMMP-2 cDNA or pEGFP expression plasmid as previous methods described [8]. Slug siRNAtransfected T24 cells were transiently transfected pMMP-2 cDNA for 48 h according to the manufacturer's instruction.…”

“…SDS-PAGE and Western blotting were performed as previously described [8]. Western blot images were captured using ImageQuant software (Amersham Pharmacia Biotech).…”

“…Several studies have reported Slug to be expressed in breast carcinoma [3], cholangiocarcinoma [4,5], bladder cancer [6], lung cancer [7], and oesophageal carcinoma [8,9] accompanied with the process of invasiveness and tumorigenecity through regulation of the expression of E-cadherin [4,7,8,10]. In addition to E-cadherin, Slug can induce the expression of genes associated with a mesenchymal and invasive phenotype, such as MMP-2 and MMP-9 [7,11], a family of proteolytic enzymes that degrade the extracellular matrix or components of the basement membrane have been associated with tumor cell invasion, angiogenesis, and metastasis in bladder cancers [12].…”