Inhibition of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Decreases DNA Methylation in Colon Cancer Cells

Shanghai Jiao Tong University, Shanghai, Shanghai Shi, China
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2007; 282(16):12249-59. DOI: 10.1074/jbc.M608525200
Source: PubMed


The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

  • Source
    • "Expression of mTOR was inhibited using the commercial SignalSilence TM mTOR siRNA kit (Cell Signaling Technology, Danvers, MA, USA) as previously described (Lu et al., 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Deregulation of the mammalian target of rapamycin pathway (mTOR pathway) is associated with human cancer. The relationship between mTOR pathway and histone acetylation is still unclear in gastric cancer. Immunohistochemistry was used to examine the phosphorylation of mTOR and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in gastric cancer (GC) tissues. MKN45 and SGC7901 cells were treated with the mTOR inhibitor rapamycin (RAPA) alone or in combination with the phosphatidylinositol 3-kinase inhibitor LY294002 and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Small interfering RNA (siRNA) technology was also used to knockdown mTOR. Phosphorylated mTOR and phosphorylated 4E-BP1 were expressed in 71.1% and 68.4% of the human gastric cancer tissues tested, respectively; significantly higher than the levels in para-cancerous tissues (50% and 57.9%) and normal tissues (44.6% and 29%). RAPA markedly inhibited cell proliferation, induced G1 cell cycle arrest, and reduced phosphorylation of p70 S6 protein kinase (p70S6K) and 4E-BP1 in gastric cancer cells, particularly when used in combination with LY294002 or TSA. The mRNA expression of the tumor suppressor gene p21(WAF1) increased significantly in gastric cancer cells treated with both RAPA and TSA. Histone acetylation also increased after RAPA and TSA treatment or siRNA knockdown of mTOR. Our findings suggest that the mTOR pathway is activated in gastric cancer, and also that inhibition of mTOR enhances the ability of TSA to suppress cell proliferation and lead to cell cycle arrest via increasing histone acetylation and p21(WAF1) transcription in human MKN45 and SGC7901 gastric cancer cells.
    Preview · Article · Jan 2014 · Cell Biology International
  • Source
    • "In particular, MMP-2 and MMP-9 play an important role in the angiogenic response as demonstrated in ECs (endothelial cells) as well as in vivo animal models [44]. Overexpressions of MMP-2 and MMP-9 have been demonstrated in human colorectal cancers [45]. Several experiments have confirmed the key role of these enzymes in angiogenesis [46], [47]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.
    Full-text · Article · Dec 2012 · PLoS ONE
  • Source
    • "In comparison, DNMT2 does not appear to have significant methylation activity and DNMT3L is likely to be limited to DNA methylation during germline development [5]. Finally, DNMT3A and DNMT3B are known to be de novo methylators of CpG sites [6], which have higher methyltransferase activity for unmethylated DNA than DNMT1 and can contribute to de novo methylation during embryogenesis [7], [8]. Although DNMT is reported to be associated with some aggressive cancers like hepatocellular carcinomas, stomach cancers, non-small cell lung cancers, lymphoma and prostate cancers [9], [10], [11], [12], [13], its role remains controversial and the overall regulation, coordination and activity of DNMTs is unclear with different cancers. "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA methyltransferase (DNMT) is one of the major factors mediating the methylation of cancer related genes such as TGF-β receptors (TβRs). This in turn may result in a loss of sensitivity to physiologic levels of TGF-β in aggressive prostate cancer (CaP). The specific mechanisms of DNMT's role in CaP remain undetermined. In this study, we describe the mechanism of TGF-β-mediated DNMT in CaP and its association with clinical outcomes following radical prostatectomy. We used human CaP cell lines with varying degrees of invasive capability to describe how TGF-β mediates the expression of DNMT in CaP, and its effects on methylation status of TGF-β receptors and the invasive capability of CaP in vitro and in vivo. Furthermore, we determined the association between DNMT expression and clinical outcome after radical prostatectomy. We found that more aggressive CaP cells had significantly higher TGF-β levels, increased expression of DNMT, but reduced TβRs when compared to benign prostate cells and less aggressive prostate cancer cells. Blockade of TGF-β signaling or ERK activation (p-ERK) was associated with a dramatic decrease in the expression of DNMT, which results in a coincident increase in the expression of TβRs. Blockade of either TGF-β signaling or DNMT dramatically decreased the invasive capabilities of CaP. Inhibition of TGF-β in an TRAMP-C2 CaP model in C57BL/6 mice using 1D11 was associated with downregulation of DNMTs and p-ERK and impairment in tumor growth. Finally, independent of Gleason grade, increased DNMT1 expression was associated with biochemical recurrence following surgical treatment for prostate cancer. Our findings demonstrate that CaP derived TGF-β may induce the expression of DNMTs in CaP which is associated with methylation of its receptors and the aggressive potential of CaP. In addition, DNMTs is an independent predictor for disease recurrence after prostatectomy, and may have clinical implications for CaP prognostication and therapy.
    Full-text · Article · Sep 2011 · PLoS ONE
Show more