Upon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whether heterotrimeric G proteins also undergo agonist-induced endocytosis. To investigate the intracellular trafficking of G␣s, we developed a functional G␣s-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing G␣s-GFP were treated with 10 M isoproterenol, and trafficking was assessed with fluorescence microscopy.

Although evidence has shown the regulating effect of n-3 poly-unsaturated fatty acid (n-3 PUFA) on cell signaling transduction, it remains unknown whether n-3 PUFA treatment modulates estrogen signaling. The current study showed that docosahexaenoic acid (DHA, C22:6), eicosapentaenoic acid (EPA, C20:5) shifted the pro-survival and proliferative effect of estrogen to a pro-apoptotic effect in human breast cancer (BCa) MCF-7 and T47D cells. 17 β-estradiol (E2) enhanced the inhibitory effect of n-3 PUFAs on BCa cell growth. The IC50 of DHA or EPA in MCF-7 cells decreased when combined with E2 (10 nM) treatment (from 173 µM for DHA only to 113 µM for DHA+E2, and from 187 µm for EPA only to 130 µm for EPA+E2). E2 also augmented apoptosis in n-3 PUFA-treated BCa cells. In contrast, in cells treated with stearic acid (SA, C18:0) as well as cells not treated with fatty acid, E2 promoted breast cancer cell growth. Classical (nuclear) estrogen receptors may not be involved in the pro-apoptotic effects of E2 on the n-3 PUFA-treated BCa cells because ERα agonist failed to elicit, and ERα knockdown failed to block E2 pro-apoptotic effects. Subsequent studies reveal that G protein coupled estrogen receptor 1 (GPER1) may mediate the pro-apoptotic effect of estrogen. N-3 PUFA treatment initiated the pro-apoptotic signaling of estrogen by increasing GPER1-cAMP-PKA signaling response, and blunting EGFR, Erk 1/2, and AKT activity. These findings may not only provide the evidence to link n-3 PUFAs biologic effects and the pro-apoptotic signaling of estrogen in breast cancer cells, but also shed new insight into the potential application of n-3 PUFAs in BCa treatment.

The present study was designed to determine whether the cADP-ribose-mediated Ca(2+) signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca(2+) mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca(2+) release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the alpha-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 microM) produced a rapid Ca(2+) release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca(2+) concentration ([Ca(2+)](i)). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca(2+)](i), but it had no effect on the U-46619-induced increase in [Ca(2+)](i). In the presence of 8-Br-cADPR (100 microM) and ryanodine (10 microM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1-100 microM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 microM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca(2+)](i) by inhibition of cADP-ribose-induced Ca(2+) mobilization.

Background:Currently, there is a lack of ideal biomarkers for predicting nodal status in preoperative stage of oesophageal adenocarcinoma (EAC) to aid optimising therapeutic options. We studied the potential of applying subtype macrophages to predict lymph node metastasis and prognosis in EAC.Material and Methods:Fifty-three EAC resection specimens were immunostained with CD68, CD40 (M1), and CD163 (M2). Lymphatic vessel density (LVD) was estimated with the staining of D2-40. Subsequently, we tested if M2d macrophage could promote EAC cell migration and invasion.Results:In EAC without neoadjuvant treatment, an increase in M2-like macrophage was associated with poor patient survival, independent of the locations of macrophages in tumour. The M2/M1 ratio that represented the balance between M2- and M1-like macrophages was significantly higher in nodal-positive EACs than that in nodal-negative EACs, and inversely correlated with patient overall survival. The M2/M1 ratio was not related to LVD. EAC cell polarised THP1 cell into M2d-like macrophage, which promoted EAC cell migration and invasion. Neoadjuvant therapy appeared to diminish the correlation between the M2/M1 ratio and survival.Conclusions:The ratio of M2/M1 macrophage may serve as a sensitive marker to predict lymph node metastasis and poor prognosis in EAC without neoadjuvant therapy. M2d macrophage may have important roles in EAC metastasis.

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