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Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

Xiong H, Chen ZF, Liang QC, Du W, Chen HM, Su WY, Chen GQ, Han ZG, Fang JY - J. Cell. Mol. Med. (2009)

Bottom Line: Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling.Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells.Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China.

ABSTRACT
DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

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The affects of SHP1 expression on JAK2/STAT3/STAT5 protein levels in CRC cells. CRC cells transfected with pEGFP-N1 or pEGFP-N1-SHP1 were sorted for GFP expression and extracts analysed by Western blot. Substantial decreases in expression of pJAK2, JAK2, pSTAT3 and pSTAT5 were detected. The decrease in pJAK2 levels was greater than that of JAK2. In contrast, no significant changes in STAT3 and STAT5 protein levels were detected. The data shown are from a representative experiment and detection of GAPDH was used as a loading control.
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fig03: The affects of SHP1 expression on JAK2/STAT3/STAT5 protein levels in CRC cells. CRC cells transfected with pEGFP-N1 or pEGFP-N1-SHP1 were sorted for GFP expression and extracts analysed by Western blot. Substantial decreases in expression of pJAK2, JAK2, pSTAT3 and pSTAT5 were detected. The decrease in pJAK2 levels was greater than that of JAK2. In contrast, no significant changes in STAT3 and STAT5 protein levels were detected. The data shown are from a representative experiment and detection of GAPDH was used as a loading control.

Mentions: To determine whether up-regulation of SHP1 mediates down-regulation of JAK/STAT signalling in CRC cells, SW1116 and HT29 cell lines were transfected with pEGFP-N1 or pEGFP-N1-SHP1. Transfection efficiency was determined from the expression of GFP detected by flow cytometry, and was estimated to vary between 40% and 70%, with a median of 50% for both vectors. When transfected cells were sorted, 98% of the isolated GFP-expressing cells were obtained and analysed by Western blot. As shown in Fig. 3, SHP1 expression levels were higher in both CRC cell lines transfected with pEGFP-N1-SHP1 relative to CRC cell lines transfected with pEGFP-N1. Additionally, expression of SHP1 was also associated with substantial decreases in the protein levels of phosphorylated JAK2 (pJAK2), pSTAT3 and pSTAT5, as well as levels of unphosphorylated JAK2. However, decreases in JAK2 were less than that for pJAK2. For unphosphorylated STAT3 and STAT5 protein levels, no significant changes were observed following exogenous expression of SHP1. These data demonstrate that the JAK2/STAT3/STAT5 pathway is subject to negative regulation by SHP1 in CRC cells, and negative regulation of JAK2 by SHP1 may include both dephosphorylation and others, such as protein degradation.


Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

Xiong H, Chen ZF, Liang QC, Du W, Chen HM, Su WY, Chen GQ, Han ZG, Fang JY - J. Cell. Mol. Med. (2009)

The affects of SHP1 expression on JAK2/STAT3/STAT5 protein levels in CRC cells. CRC cells transfected with pEGFP-N1 or pEGFP-N1-SHP1 were sorted for GFP expression and extracts analysed by Western blot. Substantial decreases in expression of pJAK2, JAK2, pSTAT3 and pSTAT5 were detected. The decrease in pJAK2 levels was greater than that of JAK2. In contrast, no significant changes in STAT3 and STAT5 protein levels were detected. The data shown are from a representative experiment and detection of GAPDH was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516515&req=5

fig03: The affects of SHP1 expression on JAK2/STAT3/STAT5 protein levels in CRC cells. CRC cells transfected with pEGFP-N1 or pEGFP-N1-SHP1 were sorted for GFP expression and extracts analysed by Western blot. Substantial decreases in expression of pJAK2, JAK2, pSTAT3 and pSTAT5 were detected. The decrease in pJAK2 levels was greater than that of JAK2. In contrast, no significant changes in STAT3 and STAT5 protein levels were detected. The data shown are from a representative experiment and detection of GAPDH was used as a loading control.
Mentions: To determine whether up-regulation of SHP1 mediates down-regulation of JAK/STAT signalling in CRC cells, SW1116 and HT29 cell lines were transfected with pEGFP-N1 or pEGFP-N1-SHP1. Transfection efficiency was determined from the expression of GFP detected by flow cytometry, and was estimated to vary between 40% and 70%, with a median of 50% for both vectors. When transfected cells were sorted, 98% of the isolated GFP-expressing cells were obtained and analysed by Western blot. As shown in Fig. 3, SHP1 expression levels were higher in both CRC cell lines transfected with pEGFP-N1-SHP1 relative to CRC cell lines transfected with pEGFP-N1. Additionally, expression of SHP1 was also associated with substantial decreases in the protein levels of phosphorylated JAK2 (pJAK2), pSTAT3 and pSTAT5, as well as levels of unphosphorylated JAK2. However, decreases in JAK2 were less than that for pJAK2. For unphosphorylated STAT3 and STAT5 protein levels, no significant changes were observed following exogenous expression of SHP1. These data demonstrate that the JAK2/STAT3/STAT5 pathway is subject to negative regulation by SHP1 in CRC cells, and negative regulation of JAK2 by SHP1 may include both dephosphorylation and others, such as protein degradation.

Bottom Line: Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling.Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells.Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China.

ABSTRACT
DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

Show MeSH
Related in: MedlinePlus