Low concentrations of zinc in gastric mucosa are associated with increased severity of Helicobacter pylori-induced inflammation.

BACKGROUND: Chronic Helicobacter pylori infection is the most common cause of gastric cancer. H. pylori induces oxidative stress while zinc deficiency results in increased sensitivity to it. In Ecuador, the prevalence of gastric cancer and zinc deficiency are high. We hypothesized that zinc deficiency in Ecuadorian people would cause increased H. pylori-induced inflammation in the gastric mucosa associated with lower tissue zinc concentrations. METHODS: Three hundred and fifty-two patients with dyspepsia underwent endoscopy to obtain gastric mucosa biopsies. Diagnosis of H. pylori infection and its severity, histopathology, mucosal zinc concentration, and inflammation intensity were determined. RESULTS: H. pylori-infected patients with non-atrophic chronic gastritis had lower concentrations of zinc in gastric mucosa than uninfected patients with the same type of gastritis (251.3 +/- 225.3 vs. 426.2 +/- 279.9 ng/mg of protein; p = .016). Considering all patients, the more severe the H. pylori infection, the higher the percentage of subjects with infiltration by polymorphonuclear (PMN) cells (p = .0001). Patients with high PMN infiltration had lower mucosal zinc concentrations than patients with low PMN infiltration (35.2 +/- 20.7 vs. 242.9 +/- 191.8 ng/mg of protein; p = .021). CONCLUSIONS: The degree of inflammation in H. pylori-induced gastritis appears to be modulated by gastric tissue zinc concentrations.

Molecular pathways leading to oxidative stress-induced phosphorylation of Akt.

Oxidative stress can activate a variety of intracellular signaling pathways. The authors previously reported the CaM-K inhibitor KN-93 inhibited hydrogen peroxide-induced phosphorylation of Akt on threonine 308 (T308). In this report they demonstrate that phosphorylation of T308 in response to hydrogen peroxide treatment is not inhibited by LY294002, suggesting that phosphorylation of this residue in response to oxidative stress is largely PI3K independent. In contrast, hydrogen peroxide-induced phosphorylation of Akt on serine 473 (S473) was downregulated by both PI3K and CaM-K inhibition, indicating that hydrogen peroxideinduced phosphorylation of Akt on S473 was largely dependent on both PI3K and a CaM-K activity. Further, it is reported that p56(Lck) had a substantial role in hydrogen peroxide-induced phosphorylation of S473, but only a minimal role in hydrogen peroxide-induced phosphorylation of T308. These data suggest that in response to hydrogen peroxide, two pathways are activated in Jurkat T lymphocytes that converge to result in the phosphorylation of Akt on S473 and T308. One pathway involves the CaM-Ks that may directly phosphorylate Akt on T308. In this pathway, neither the Src kinases nor PI3K are required. The other pathway mediated by hydrogen peroxide results in the phosphorylation of Akt on S473 and requires CaM-K, PI3K, and Src activity.

Activation of the calcium/calmodulin-dependent protein kinases as a consequence of oxidative stress.

Oxygen radicals have diverse effects on cells. In many cases, exposure to reactive oxygen intermediates (ROI) can induce cell death. Conversely, there is also evidence that suggests oxygen radicals can activate signaling pathways that are thought to prevent cell death. In this review, the authors discuss the finding that hydrogen peroxide and ROI-generating treatments trigger the activation of the calcium/calmodulin-dependent kinases (CaM-kinases), and the potential role this activation has in preventing apoptosis. Evidence is presented that CaM-kinase activation occurs by both calcium dependent- and independent-pathways in response to ROIs. In addition, the idea is discussed that ROIs have the potential to lead to the phosphorylation of calmodulin and through this mechanism potentiate the activation of the CaM-kinases. The concept that inhibition of the CaM-kinases as a mechanism to sensitize cells to the damaging effects of ROIs is also presented. Contrasting these studies, evidence is presented that exposure of the CaM-kinases directly to hydrogen peroxide also has the apparent ability to inhibit their activity.

Inhibition of the CaM-kinases augments cell death in response to oxygen radicals and oxygen radical inducing cancer therapies in MCF-7 human breast cancer cells.

Many cancer treatments induce cell death through lethal oxidative stress. Oxidative stress also induces the activation of the calcium/calmodulin-dependent kinases (CaM-Ks), CaM-KII and CaM-KIV. In turn, the CaM-Ks are known to induce the activation of antiapoptotic signaling pathways, such as Akt, ERK, and NF-kappaB in many different cell types. The aim of this study was to determine the role of CaM-Kinases in resistance to hydrogen peroxide and three oxidative stress-inducing cancer therapies in MCF-7 breast cancer cells. We found that oxidative stress induced CaM-Kinase activity in MCF-7 breast cancer cells and that CaM-K inhibition increased hydrogen peroxide-induced cell death in MCF-7 human breast cancer cells. When MCF-7 cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy in the presence of a CaM-K inhibitor a greater level of cell killing was observed than when cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy alone. In support of this finding, CaM-K inhibition increased hydrogen peroxide-induced apoptosis in MCF-7 cells, as determined by increased number of apoptotic cells, DNA fragmentation, and PARP cleavage. Pharmacological and molecular inhibition indicated that CaM-KII was participating in hydrogen peroxide-induced ERK phosphorylation in breast cancer cells indicating a potential mechanism by which this sensitization occurs. This is the first time that CaM-K inhibition is reported to sensitize cancer cells to reactive oxygen intermediate inducing cancer treatments.

Calcium/calmodulin-dependent protein kinases as potential targets in cancer therapy.

In this review the authors discuss the expression and activation of a family of protein kinases known as the calcium/calmodulin-dependent kinases (CaM-kinase) and the role that these kinases have in the activation of antiapoptotic signalling pathways. In addition, the authors outline a novel mechanism of activation of these kinases by oxidative stress. Founded on this novel mechanism of activation and the role that these kinases have in activating antiapoptotic signalling pathways, the authors propose that the CaM-kinases would make very good targets for sensitising cancer cells to certain therapeutic treatments. Furthermore, the authors discuss the role that these kinases have in cell transformation and in the regulation of the cell cycle. Based on these roles the authors suggest that inhibition of the CaM-kinases not only has the potential to sensitise cancer cells, but also has the potential to induce cytostasis in these cells.

Calcium/calmodulin-dependent kinase I and calcium/calmodulin-dependent kinase kinase participate in the control of cell cycle progression in MCF-7 human breast cancer cells.

Calcium is universally required for cell growth and proliferation. Calmodulin is the main intracellular receptor for calcium. Although calcium and calmodulin are well known to be required for cell cycle regulation, the target pathways for their action remain poorly defined. Potential targets include the calcium/calmodulin-dependent kinases (CaM-K). The aim of this study was to determine the role of the CaM-Ks on cell proliferation and progress through the cell cycle in breast cancer cells. CaM-KI inhibition with either KN-93 or specific interfering RNA (siRNA) caused an arrest in the cell cycle in the human breast cancer cell line, MCF-7. This arrest occurred in the G(1) phase of the cell cycle. Supporting this finding, CaM-K inhibition using KN-93 also resulted in a reduction of cyclin D1 protein and pRb phosphorylation when cells were compared with control cultures. Furthermore, inhibition of the upstream activator of CaM-KI, CaM-KK, using siRNA also resulted in cell cycle arrest. In summary, CaM-KK and CaM-KI participate in the control of the G(0)-G(1) restriction check point of the cell cycle in human breast cancer cells. This arrest seems due to an inhibition in cyclin D1 synthesis and a reduction in pRb phosphorylation. To the best of our knowledge, this is the first time that CaM-KK has been reported to be involved in mammalian cell cycle regulation and that CaM-Ks are regulating breast cancer cell cycle.

Inhibition of CREB transcriptional activity in human T lymphocytes by oxidative stress.

Hydrogen peroxide (HP) induced the phosphorylation of cAMP response element binding protein (CREB) on Ser133 in Jurkat T lymphocytes via p38 and MSK1. Although CREB Ser133 was phosphorylated, increases in HP-stimulated CREB-mediated transcription were absent. T lymphocyte stimulation with anti-CD3 and anti-CD28 induced CREB Ser133 phosphorylation, as well as CREB-mediated transcriptional activity. When CD3/CD28-stimulated lymphocytes were treated with HP, Ser133 was phosphorylated, but TCR-induced CREB-mediated transcriptional activity was reduced. These data provide insight into a potential mechanism by which oxidative stress can alter T cell receptor-induced CREB activation and responsiveness.

Models of anergy in the human Jurkat T cell line.

We investigated two model systems to study anergy in a human T cell line. OKT3 or calcium ionophore stimulation of Jurkat cells, in the absence of costimulation, resulted in a steep reduction in the transcription and secretion of IL-2 in response to subsequent stimulation via CD3 and CD28. Treatment of anergic Jurkat cells with the combination of the phorbol ester, PMA, and ionomycin restored IL-2 production in cells rendered anergic by both mechanisms. However, hydrogen peroxide, which also stimulates kinases downstream of the proposed block that occurs in anergic murine cells, did not reverse the anergic state of these cells induced by either stimulus. The cause of unresponsiveness in these two models was found to differ. OKT3-induced anergy resulted in a substantial down-regulation of the CD3 on these cells. In contrast, anergy induced by treatment with a calcium ionophore did not result in CD3 down-regulation. These data indicate that the Jurkat cell line may serve as a suitable model for studying anergy in human T cells; however, the mechanism by which anergy is induced may vary dramatically in response to these two commonly used anergy-inducing strategies. Understanding the similarities and differences between these two models of anergy may lead to a better overall understanding of the anergic state of the T cell.

Participation of the calcium/calmodulin-dependent kinases in hydrogen peroxide-induced Ikappa B phosphorylation in human T lymphocytes.

NF-kappaB is an important transcription factor that has a role in a variety of responses such as inflammation, oncogenesis, apoptosis, and viral replication. Oxidative stress is well known to induce the activation of NF-kappaB. Cells can be exposed to either endogenously produced oxidants or oxidants produced by surrounding cells. In addition, ischemia reperfusion and certain cancer therapies such as chemotherapy and photodynamic therapy are thought to result in oxygen radical production. Because of the important role that NF-kappaB has in multiple responses, it is critical to determine the mechanisms by which oxidative stress induces NF-kappaB activity. We report that the calmodulin antagonist W-7 and the calcium/calmodulin-dependent (CaM) kinase inhibitors KN-93 and K252a, can block oxidative stress-induced IkappaB phosphorylation in Jurkat T lymphocytes. Furthermore, KN-93 but not KN-92 can block hydrogen peroxide-induced Akt and IKK phosphorylation. In addition, we found that expression of a kinase-dead CaM-KIV construct in two cell lines inhibits IkappaB phosphorylation or degradation and that expression of CaM-KIV augments hydrogen peroxide-induced IkappaB phosphorylation and degradation. Although the CaM kinases appear to be required for this response, increases in intracellular calcium do not appear to be required. These results identify the CaM kinases as potential targets that can be used to minimize NF-kappaB activation in response to oxidative stress.