Purpose Activation of the c-Met and epidermal growth factor receptors (EGFR) promotes growth and survival of non-small cell lung cancer (NSCLC). Specific receptor antagonists have demonstrated efficacy in the clinic; however, tumors often become resistant to these therapies. We have investigated the ability of (-)-epigallocatechin-3-gallate (EGCG) to inhibit cell proliferation, and c-Met receptor and EGFR kinase activation in several NSCLC cell lines. Experimental Design NSCLC cell lines with variable sensitivity to the EGFR antagonist erlotinib were studied. Cell growth was evaluated using MTS and colony formation assays. Kinase activation was assessed via western blot analysis. Experiments were conducted with EGCG, the EGFR antagonist erlotinib and the c-Met inhibitor SU11274. The antagonists were also tested in a xenograft model using SCID mice. Results EGCG inhibited cell proliferation in erlotinib sensitive and resistant cell lines, including those with c-Met overexpression and acquired resistance to erlotinib. The combination of erlotinib/EGCG resulted in greater inhibition of cell proliferation and colony formation than either agent alone. EGCG also completely inhibited ligand-induced c-Met phosphorylation and partially inhibited EGFR phosphorylation. The triple combination of EGCG/erlotinib/SU11274 resulted in a greater inhibition of proliferation than EGCG with erlotinib. Finally, the combination of EGCG and erlotinib significantly slowed the growth rate of H460 xenografts. Conclusion EGCG is a potent inhibitor of cell proliferation, independent of EGFR inhibition, in several NSCLC cell lines, including those resistant to both EGFR kinase inhibitors and those overexpressing c-Met. Therefore, EGCG might be a useful agent to study as an adjunct to other anti-cancer agents.

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide ([Formula: see text]) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing[Formula: see text] could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce [Formula: see text] were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that [Formula: see text]released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

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It is becoming increasingly apparent that certain forms of acute and chronic inflammation are associated with enhanced production of nitric oxide (NO). Although substantial information has been obtained describing the regulation of NO synthase (NOS) in macrophages, little information is available regarding the biochemistry and molecular biology of NOS in circulating vs. extravasated polymorphonuclear leukocytes (PMNs). The objective of this study was to characterize the molecular and biochemical properties of the inducible NO synthase (iNOS) in circulating vs. extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicited in rats by intraperitoneal injection of 1% oyster glycogen or in humans by peritoneal dialysis of patients with peritonitis. Inducible NOS mRNA from circulating and elicited PMNs was quantified using slot blot hybridization analysis with a cDNA probe specific for iNOS. iNOS protein was identified using Western immunoblot analysis, and NOS activity was quantified by measuring the NG-monomethyl-L-arginine (L-NMMA)-inhibitable conversion of 14C-labeled L-arginine to L-[14C]citrulline. In a separate series of experiments, circulating or extravasated PMNs were cultured for 4 h and the accumulation of L-NMMA-inhibitable nitrite (NO2-) in the supernatant was determined and used as a measure of NO production in vitro. We found that circulating PMNs (rat or human) contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, circulating rat or human PMNs (2 x 10(6) cells/well) were unable to generate significant amounts of NO2- when cultured for 4 h in vitro. In contrast, iNOS mRNA levels in 4- and 6-h elicited rat PMNs increased 21- and 42-fold, respectively, when compared with circulating cells. Western blot analysis revealed the presence of iNOS protein in the elicited rat PMNs and iNOS enzymatic activity increased from normally undetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg for the 4- and 6-h elicited rat PMNs, respectively. Approximately 20-30% of the total iNOS activity was Ca(2+)-dependent. Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased from normally undetectable amounts for circulating PMNs to approximately 8 and 11 microM/10(6) cells for the 4- and 6-h elicited PMNs, respectively. Highly enriched preparations of extravasated human PMNs contained neither message, protein nor iNOS enzymatic activity. Taken together our data demonstrate that inflammation-induced extravasation of rat PMNs upregulates the transcription and translation of iNOS in a time-dependent fashion and that 20-30% of the total inducible NOS is Ca(2+)-dependent. In contrast, neither circulating nor extravasated human PMNs contained iNOS message, protein, or enzymatic activity. These data suggest that the human PMN iNOS gene is under very different regulation than is the rat gene.

The regulation of matrix metalloproteinase activity is crucial for maintaining the proper balance of tissue remodeling vs. injury. Metalloproteinase proenzymes are activated when the active site zinc is exposed via a cysteine switch mechanism. Peroxynitrite, the product generated from the interaction between nitric oxide and superoxide, has been shown to release zinc from zinc-thiolate groups, suggesting that it might alter metalloproteinase activity. This study examined the effects of nitric oxide and superoxide generators on gelatinase A activity. Results showed that nitric oxide alone had no effect on gelatinase A activity relative to control, whereas superoxide-derived metabolites increased activity. The simultaneous generation of both nitric oxide and superoxide caused an inhibition of gelatinase A activity. This inhibition was reversed by the addition of hemoglobin, superoxide dismutase, or sodium urate, suggesting that peroxynitrite and/or peroxynitrous acid caused the inhibition. Authentic peroxynitrite also inhibited gelatinase A activity. We postulate that the relative fluxes of nitric oxide and superoxide at sites of inflammation may modulate metalloproteinase activity and thus affect matrix protein metabolism.

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Restless legs syndrome (RLS) is common in the elderly, with an estimated prevalence of 10 to 35% in individuals over 65 years of age. RLS is characterised by paraesthesias and dysaesthesias of the legs, typically occurring in the evening. The symptoms occur at rest and result in motor restlessness; movement often temporarily relieves the symptoms. Patients with poorly controlled RLS may develop related problems including insomnia (due to sleep-onset restlessness or periodic limb movements or related sleep fragmentation) and depression. RLS can be a primary disorder that develops in the young and includes familial cases. Secondary RLS occurs in association with iron-deficiency anaemia, uraemia and polyneuropathies. Typically, RLS is misdiagnosed or undiagnosed for years. In the elderly, both primary and secondary types of the disorder are common. It is thought that RLS represents lower CNS levels of, or reduced responsiveness to, dopamine. The symptoms improve with dopaminergic therapy. Ergotamine dopamine-receptor agonists such as pergolide, and the non-ergotamine dopamine-receptor agonists pramipexole and ropinirole, are becoming more commonly used to treat RLS. The dopamine precursor levodopa, in combination with carbidopa, is another effective therapeutic agent. An advantage of levodopa is lower cost than non-ergotamine and ergotamine dopamine-receptor agonists. However, the adverse effect of symptom augmentation appears to develop more frequently with levodopa than dopamine-receptor agonists; therefore, levodopa may currently be used somewhat less often as first-line therapy. Patients with painful symptoms may respond favourably to the anticonvulsants gabapentin and carbamazepine. Opioids and hypnosedatives are helpful in selected patients; however, these agents may have troubling adverse effects in the elderly. Correction of iron deficiency improves symptoms in patients with low ferritin levels. Lifestyle modification may also be helpful. Therapy is directed at symptoms, and most symptomatic patients benefit from treatment. It is important to consider RLS in the differential diagnosis of any patient with paraesthesias of the limbs.

Administration of endotoxin intravenously to unanesthetized sheep causes an acute lung injury characterized by increased microvascular barrier permeability and subsequent pulmonary edema. Endotoxin-induced sheep lung injury can be attenuated by leukocyte depletion, and may be mediated by toxic metabolites of oxygen. We studied effects of administering catalase, which catalyzes conversion of hydrogen peroxide to oxygen and water, to sheep subsequently infused with endotoxin to test the hypothesis that hydrogen peroxide plays a role in the pathogenesis of lung injury. We found that infusions of endotoxin (1 microgram/kg) into untreated sheep caused the expected biphasic response, a transient, early, marked pulmonary arterial hypertension followed by a prolonged increase in protein-rich lung lymph flow characteristic of increased microvascular permeability filtration in the lungs. Intraperitoneal injections of catalase (50 mg/kg) prior to infusing endotoxin in these same sheep resulted in substantial catalase activity in plasma and in lung lymph, and attenuated the expected changes in pulmonary arterial pressure, lung lymph flow, and arterial leukocyte counts and oxygen tension after endotoxin infusions. Furthermore, mechanical elevation of hydrostatic pressure in the lungs of a catalase-treated sheep infused with endotoxin resulted in increased lung lymph flow with a decreased protein concentration, indicating that the microvascular barrier to fluid and protein was functionally intact. Administration of catalase that was inactivated by reaction with hydrogen peroxide in the presence of aminotriazole or administration of the catalase vehicle, thymol, had no effects on the sheep responses to endotoxin. We conclude that hydrogen peroxide plays a role in the pathogenesis of endotoxin-induced acute lung injury in sheep.