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Abstract
Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.
Highlights
Nitric oxide (NO) is an essential signaling molecule in biological systems
To examine whether the NO synthase-soluble guanylate cyclase (sGC) signaling pathway is functional in human breast cancer cells, we measured the effects of bradykinin, a NO activator, on the production of cGMP in human 184A1 breast epithelial cells, human estrogen receptor (ER)-positive MCF-7 breast cancer cells, and ER-negative MDA-MB-231 breast cancer cells
To determine whether sGC functions properly, we measured the effect of sodium nitroprusside (SNP), a NO donor and sGC activator, on sGC activity in 184A1, MCF-7 and MDA-MB-231 cells
Summary
Nitric oxide (NO) is an essential signaling molecule in biological systems. NO functions as the primary activator of soluble guanylate cyclase (sGC) [1]. NO is synthesized by the enzyme nitric oxide synthase (NOS) [1]. NO diffuses across cell membranes and binds to the heme cofactor of sGC and activates the enzyme, which leads to significant increases in cGMP levels [1,2,3,4]. NO donors can efficiently activate sGC [5,6,7,8]. The second messenger, cGMP, directly modulates ion channels, cGMP-phosphodiesterases, or cGMP-dependent protein kinases (PKG) and regulates important physiological functions, including vasodilation, platelet aggregation, and neurotransmission [1]
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