Ischemic stroke is a common disease with high mortality and morbidity worldwide. One of the important pathophysiological effects of ischemic stroke is apoptosis. A neuroprotective effect is defined as the inhibition of neuronal apoptosis to rescue or delay the infarction in the surviving ischemic penumbra. Resveratrol is a natural polyphenol that reportedly prevents cerebral ischemia injury by regulating the expression of PI3K/AKT/mTOR. Therefore, this study aimed to elucidate the neuroprotective effect of resveratrol on cerebral ischemia/reperfusion injury and to investigate the signaling pathways and mechanisms through which resveratrol regulates apoptosis in the ischemic penumbra. Rats were subjected to middle cerebral artery occlusion for 2 h followed by 24 h reperfusion. Cerebral infarct volume was measured using 2% TTC staining. TUNEL staining was conducted to evaluate neuronal apoptosis. Western blotting and immunohistochemistry were used to detect the proteins involved in the JAK2/STAT3/PI3K/AKT/mTOR pathway. The results suggested that resveratrol significantly improved neurological function, reduced cerebral infarct volume, decreased neuronal damage, and markedly attenuated neuronal apoptosis; these effects were attenuated by the inhibition of PI3K/AKT with LY294002 and JAK2/STAT3 with AG490. We also found that resveratrol significantly upregulated the expression of p-JAK2, p-STAT3, p-AKT, p-mTOR, and BCL-2 and downregulated expression of cleaved caspase-3 and BAX, which was partially reversed by LY294002 and AG490. These results suggested that resveratrol provides a neuroprotective effect against cerebral ischemia/reperfusion injury, which is partially mediated by the activation of JAK2/STAT3 and PI3K/AKT/mTOR. Resveratrol may indirectly upregulate the PI3K/AKT/mTOR pathway by activating JAK2/STAT3.

Resveratrol provides neuroprotection by regulating the JAK2/STAT3/PI3K/AKT/mTOR pathway after stroke in rats.

Significance: Cancer is a complex disease, which not only involves the tumor but its microenvironment comprising different immune cells as well. Nitric oxide (NO) plays specific roles with...

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Molecular Mechanisms of Nitric Oxide in Cancer Progression, Signal Transduction, and Metabolism.

NSAIDs display promising antineoplastic activity for colorectal and other cancers, but toxicity from cyclooxygenase (COX) inhibition limits their long-term use for chemoprevention. Previous studies have concluded that the basis for their tumor cell growth inhibitory activity does not required COX inhibition, although the underlying mechanism is poorly understood. Here we report that the NSAID, sulindac sulfide (SS) inhibits cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) activity to increase intracellular cGMP levels and activate cGMP dependent protein kinase (PKG) at concentrations that inhibit proliferation and induce apoptosis of colon tumor cells. SS did not activate the cGMP/PKG pathway, nor affect proliferation or apoptosis in normal colonocytes. Knockdown of the cGMP-specific PDE5 isozyme by siRNA and PDE5-specific inhibitors, tadalafil and sildenafil, also selectively inhibited the growth of colon tumor cells that expressed high levels of PDE5 compared with colonocytes. The mechanism by which SS and the cGMP/PKG pathway inhibits colon tumor cell growth appears to involve the transcriptional suppression of β-catenin to inhibit Wnt/β-catenin TCF transcriptional activity, leading to down-regulation of cyclin D1 and survivin. These observations suggest that safer and more efficacious sulindac derivatives can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP degrading isozymes.

https://mct.aacrjournals.org/content/molcanther/12/9/1848.full.pdf

Sulindac Selectively Inhibits Colon Tumor Cell Growth by Activating the cGMP/PKG Pathway to Suppress Wnt/β-Catenin Signaling

Both phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen activated protein kinase (MAPK) signaling cascades play an important role in cell proliferation, survival, angiogenesis, and metastasis of tumor cells. In the present report, we investigated the effects of licochalcone A (LA), a flavonoid extracted from licorice root, on the PI3K/AKT/mTOR and MAPK activation pathways in human gastric cancer BGC-823 cells. LA increased reactive oxygen species (ROS) levels, which is associated with the induction of apoptosis as characterized by positive Annexin V binding and activation of caspase-3, and cleavage of poly-ADP-ribose polymerase (PARP). Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented LA-induced apoptosis. Interestingly, we also observed that LA caused the activation of ERK, JNK, and p38 MAPK in BGC-823 cells. The antitumour activity of LA-treated BGC-823 cells was significantly distinct in KM mice in vivo. All the findings from our study suggest that LA can interfere with MAPK signaling cascades, initiate ROS generation, induce oxidative stress and consequently cause BGC cell apoptosis.

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Licochalcone A-induced human gastric cancer BGC-823 cells apoptosis by regulating ROS-mediated MAPKs and PI3K/AKT signaling pathways.

Influenza A virus is an important pathogenic virus known to induce host cell cycle arrest in G(0)/G(1) phase and create beneficial conditions for viral replication. However, how the virus achieves arrest remains unclear. We investigated the mechanisms underlying this process and found that the nonstructural protein 1 (NS1) is required. Based on this finding, we generated a viable influenza A virus (H1N1) lacking the entire NS1 gene to study the function of this protein in cell cycle regulation. In addition to some cell cycle regulators that were changed, the concentration and activity of RhoA protein, which is thought to be pivotal for G(1)/S phase transition, were also decreased with overexpressing NS1. And in the meantime, the phosphorylation level of cell cycle regulator pRb, downstream of RhoA kinase, was decreased in an NS1-dependent manner. These findings indicate that the NS1 protein induces G(0)/G(1) cell cycle arrest mainly through interfering with the RhoA/pRb signaling cascade, thus providing favorable conditions for viral protein accumulation and replication. We further investigated the NS1 protein of avian influenza virus (H5N1) and found that it can also decrease the expression and activity of RhoA, suggesting that the H5N1 virus may affect the cell cycle through the same mechanism. The NS1/RhoA/pRb cascade, which can induce the G(0)/G(1) cell cycle arrest identified here, provides a unified explanation for the seemingly different NS1 functions involved in viral replication events. Our findings shed light on the mechanism of influenza virus replication and open new avenues for understanding the interaction between pathogens and hosts.

Influenza A virus NS1 induces G0/G1 cell cycle arrest by inhibiting the expression and activity of RhoA protein

Background
Our previous research results showed that Type II cGMP dependent protein kinase (PKG II) could block the activation of epidermal growth factor receptor (EGFR) and consequently inhibit the proliferation and the related MAPK/ERK-mediated signal transduction of gastric cancer cell line BGC-823, suggesting that PKG II might inhibit other EGFR-triggered signal transduction pathways and related biological activities of gastric cancer cells. This paper was designed to investigate the potential inhibition of PKG II on EGF/EGFR-induced migration activity and the related signal transduction pathways.

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PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

Our previous study revealed that Type II cGMP-dependent protein kinase (PKG II) inhibits epidermal growth factor (EGF)-induced MAPK/ERK and MAPK/JNK-mediated signal transduction through the inhibition of the phosphorylation/activation of the EGF receptor (EGFR). As EGFR also mediates several other signal transduction pathways besides MAPK-mediated pathways, the present study was designed to investigate whether PKG II was able to inhibit EGF/EGFR-induced phosphatidylinositol-3-kinase (PI3K)/Akt-mediated signal transduction. The AGS human gastric cancer cell line was infected with adenoviral constructs encoding a cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II, and treated with 8-pCPT-cGMP to activate the enzyme. Western blotting was used to detect the phosphorylation/activation of the key components of the signal transduction pathway, including EGFR, PI3K, Akt, mTOR and NF-κB. The levels of apoptosis-related proteins, including Bax, Bcl-2, caspase 9 and DNA fragment factor (DFF), were also determined by western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining was used to detect the apoptosis of the AGS cells. The results revealed that EGF treatment increased the phosphorylation (activation) of EGFR, PI3K, Akt and mTOR, and increased the nuclear localization (activation) of NF-κB. EGF treatment also reduced the apoptosis of the AGS cells and increased the expression of the anti-apoptotic protein, Bcl-2, but had no effect on the expression of the pro-apoptotic protein, Bax, and did not alter the levels of caspase 9 and DFF. Increasing the PKG II activity of AGS cells by infecting them with Ad-PKG II and stimulating them with 8-pCPT-cGMP inhibited the EGF-induced activation of EGFR, PI3K, Akt, mTOR and NF-κB; caused an increase in caspase 9 breakdown (activation) and DFF levels; and reversed the anti-apoptotic effect of EGF. The results suggest that PKG II may also inhibit EGF-induced signal transduction of PI3K/Akt-mediated pathways, and further confirm that PKG II is able to block the activation of EGFR.

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Type II cGMP-dependent protein kinase inhibits epidermal growth factor-induced phosphatidylinositol-3-kinase/Akt signal transduction in gastric cancer cells.

The aim of this study was to elucidate the localization of RhoA in human cancer tissues and cancer cell lines. Immunohistochemistry and immunofluorescence microscopy were used to determine the localization of RhoA in cancer tissues and cell lines, respectively. Western blotting was used to determine the quantity of RhoA in cell nucleus, cytosol and membrane. Immunofluorescence microscopy revealed that in all cell lines examined in this experiment (including SGC-7901, Hela, A549 and SW480), RhoA was localized not only on the membrane, in the cytosol, but also in the nucleus. Within the nucleus its precise localization was in the nucleolus. In the transformed gastric epithelial cell line GES, RhoA was also found to be localized on the membrane, in the cytosol and the nucleus, but the nuclear location was less than that of cancer cell lines. Immunohistochemistry microscopy revealed that in cancer tissues the nuclear RhoA was obviously more than that of para-cancer tissue; in the cancer tissues undergoing inflammation and necrosis, nuclear RhoA was even higher. RhoA is generally distributed in the nucleus of cells and the distribution increases when the cells undergo tumorigenesis, suggesting a new role for RhoA.

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RhoA protein is generally distributed in the nuclei of cancer cells.

Receptor tyrosine kinases (RTKs) are key to the regulation of biological cell activities, particularly of tumor cells. In a previous study, we demonstated that type II cGMP-dependent protein kinase (PKG II) may inhibit the activation of epidermal growth factor receptor (EGFR), a key member of the RTK family, and inhibit the consequent signal transduction in gastric cancer cells. Since RTKs exhibit a high level of conservation in their molecular structure, we hypothesized that PKG II may exert a similar inhibitory effect on the activation of other members of the RTK family. The aim of the present study was to investigate the potential inhibitory effect of PKG II on the activation of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and insulin-like growth factor-1 receptor (IGF-1R). The AGS gastric cancer cell line was transfected with adenoviral vector encoding PKG II cDNA (Ad-PKG II) to increase the expression of PKG II and incubated with 8-pCPT-cGMP to activate the kinase. The cells were then stimulated with VEGF, PDGF and IGF-1, and the phosphorylation/activation of relative RTKs was detected by western blot analysis. The results demonstrated that stimulating cells with VEGF-C (100 ng/ml), PDGF-BB (100 ng/ml) and IGF-1 (100 ng/ml) for 5 min led to a clear increase of phosphorylation of VEGFR2 (Tyr 951), PDGFRβ (Tyr 751) and IGF-1R (Tyr 1161), respectively. Pre-infection of the cells with Ad-PKG II with a multiplicity of infection (MOI) of 100% overnight and pre-incubation of cells with 8-pCPT-cGMP (100 and 250 μM) for 1 h efficiently inhibited the ligand-binding-induced phosphorylation/activation of the RTKs. PKG II also inhibited the MAPK/ERK- and PI3K-mediated signal transductions induced by VEGF-C, PDGF-BB and IGF-1. The results demonstrated that PKG II may exert a wide range of inhibitory effects on the activation of RTKs and provided further evidence to confirm PKG II as a tumor suppressor.

https://www.spandidos-publications.com/10.3892/br.2013.85/download

Yueying Li

Papers

PKG II Inhibits EGF/EGFR-Induced Migration of Gastric Cancer Cells

Type II cGMP-dependent protein kinase inhibits epidermal growth factor-induced phosphatidylinositol-3-kinase/Akt signal transduction in gastric cancer cells.

RhoA protein is generally distributed in the nuclei of cancer cells.

Type II cGMP-dependent protein kinase inhibits activation of key members of the RTK family in gastric cancer cells

Ginkgol C17:1 inhibits tumor growth by blunting the EGF- PI3K/Akt signaling pathway.