Patupilone-induced apoptosis is mediated by mitochondrial reactive oxygen species through Bim relocalization to mitochondria.

Among the new microtubule-targeted agents, the epothilone family of molecules has shown promising anticancer potential, and clinical trials are currently underway for patupilone (epothilone B) in various cancer indications. In this study, we characterized novel aspects of patupilone's cellular action that may underlie its potent cytotoxicity in human neuroblastoma cells. Patupilone induced mitochondrial membrane potential collapse, mitochondrial morphological changes, and cytochrome c release, leading to apoptosis. Within the first 2 h, patupilone increased the generation of reactive oxygen species (ROS; i.e., superoxides and hydrogen peroxide, 33+/-6 and 51+/-3% increase, respectively), specifically from mitochondria. ROS scavengers and mitochondrial DNA depletion [rho(-) cells] significantly protected cells against patupilone cytotoxicity, indicating that ROS generation is a key event in the initial phase of apoptosis. Although the Bim expression level was not modified by patupilone, this proapoptotic protein accumulated in the mitochondrial compartment (2.4-fold increase at IC70) after only a 6-h treatment. In contrast, Bax and Bcl-2 mitochondrial levels were not changed during treatment. It is noteworthy that ROS inhibition prevented Bim relocalization to mitochondria and mitochondrial membrane changes induced by patupilone. Altogether, our data reveal that patupilone-mediated ROS production by mitochondria initiates the intrinsic signaling cascade by inducing Bim accumulation in mitochondria. These results might explain the superior activity of patupilone in tumor cells compared with paclitaxel that is, until now, the clinical reference among microtubule-stabilizing agents. Furthermore, our data highlight the importance of mitochondria that simultaneously assume the role of activator and integrator of apoptotic signals triggered by patupilone.

Impairment of sodium pump and Na+/H+ antiport in erythrocytes isolated from cancer patients.

We sought to determine whether the impairment of sodium pump activity and Na+/H+ exchange reported in tumorigenic cells was specific to these cells or more general. Sodium pump activity and Na+/H+ exchange were measured in erythrocytes from 49 cancer patients and 51 healthy subjects. Cancer patients with a newly detected cancer or in relapse and without associated pathologies known to modify these sodium transporters were included in this study. Two sodium pump statuses reflecting its physiological modulation were evidenced for healthy subjects (10.3 +/- 0.2 and 19.4 +/- 0.8 mW/liter of cells). In cancer patients, only one basal status lower than those of controls was observed (8.3 +/- 0.5 mW/liter of cells; P < 0.001). Cooperativity of the Na+/H+ antiporter is the same in cancer patients and controls (2.58 +/- 0.27 versus 2.60 +/- 0.15). The intracellular pH (pHi) dependence curve of the antiporter was shifted toward more acidic values, and optimal pH1 was lower in cancer patients than in controls (5.80 +/- 0.03 versus 6.08 +/- 0.02; P < 0.0001). The mean maximal rate and the Km of H+ for the Na+/H+ antiporter were higher: 8.4 +/- 1.2 versus 4.6 +/- 0.4 mmol H+/liter of cells/h (P < 0.01) and 514 +/- 12 versus 322 +/- 16 nM (P < 0.05), respectively. Alterations of these Na+ transporters, therefore, were not restricted to cancerous cells. Among the alterations, the acidic shift in the pHi dependence of Na+/H+ exchange appears associated with cancer because this behavior has never been reported in other pathologies.

Inhibition of sodium pump by bepridil. An in vitro and microcalorimetric study.

The effects of diltiazem, verapamil, bepridil, nicardipine and nifedipine were studied in vitro on Na+,K(+)-ATPase from dog kidney (EC 3.6.1.37). Except diltiazem, all the drugs tested showed an inhibitory effect on Na+,K(+)-ATPase activity in a dose-dependent manner. Among these drugs bepridil is far more effective than the others (IC50 approximately 10(-4) M). Competition studies showed that bepridil acted in a non-competitive manner with the ATP-Mg2+ complex and in a partially competitive manner with K+. Since ouabain acted similarly under the same experimental conditions, we tested the interaction of bepridil and ouabain on Na+,K(+)-ATPase. With low doses of ouabain, the enzyme inhibition corresponded to a potentiated synergy of the two drugs. We then studied the action of bepridil on the sodium pump activity of intact red blood cells by an ex vivo microcalorimetric technique. At 10(-5) M bepridil caused a significant decrease in sodium pump activity (33 +/- 8%).

Sodium pump and Na+/H+ activities in uremic erythrocytes. A microcalorimetric and pH-metric study.

The sodium pump and Na+/H+ antiport activities in red blood cells from uremic hemodialyzed patients were measured concomitantly. The patients selected (n = 35) were normotensive and free of intercurrent illness known to affect Na transport. The Na pump activity of intact red blood cells in suspension in their own plasma was measured by flow microcalorimetry. The Na+/H+ antiport activity of the erythrocytes from the same patients was determined by a titrimetric technique. The mean global value of the sodium pump was lower in uremics than in controls (13.3 +/- 0.6 vs. 11.3 +/- 0.8 mW/l cells, P < 0.05). The Na+/H+ antiport maximal activity was decreased in uremics (2.9 +/- 0.3 vs. 4.6 +/- 0.5 mmol H+/l cells/h, P < 0.05). Our results thus confirm that uremia per se can affect sodium transport. Moreover it has been shown that a decrease in Na+/H+ antiport activity is able to counteract an impairment of sodium pump. The decrease found in this study could thus explain, at least in part, the absence of hypertension in the patients studied despite their decreased sodium pump activity.

Modulation defect of sodium pump evidenced in diabetic patients by a microcalorimetric study.

Sodium pump activity of intact erythrocytes in their own plasma was measured by microcalorimetry in 41 healthy subjects and 35 insulin-dependent diabetic patients. Results show that modulation of the sodium pump is altered in diabetic patients. Addition of insulin increases functioning of the Na(+)-K+ pump in controls but has no effect in diabetic patients. These subjects show a slower response of the Na(+)-K+ pump to the inhibitory effect of ouabain. Cross-incubation experiments suggest that these findings may be explained by the existence of a plasmatic factor that impairs the modulation of the sodium pump in diabetic patients.

A non ouabain-like inhibitor of the sodium pump in uremic plasma ultrafiltrates and urine from healthy subjects.

A non ouabain-like inhibitor of the sodium pump was separated from uremic plasma ultrafiltrates and normal urine. Under the same chromatographic conditions (C18 column and a gradient of acetonitrile as eluant), ouabain was eluted in a fraction different from the inhibitor. Affinity chromatography based on the formation of a complex between Na,K-ATPase and the inhibitor achieved the differentiation ouabain. Without magnesium and sodium phosphate, ouabain could not bind to enzyme whereas the inhibitor did. A study of Na,K-ATPase enzyme kinetics showed the inhibitor was not competitive for K+, which further differentiates it from ouabain. It was uncompetitive for ATP and seemed competitive for Na+. These results indicate that the inhibitor acts inside the cell, unlike ouabain, and thus its action mechanism appears to be original.

Sodium pump and Na+/H+ antiport restoration in erythrocytes from cancer patients in remission.

We previously showed that in erythrocytes from cancer patients, the sodium pump is decreased and the optimal intracellular pH for Na+/H+ antiport activity is shifted toward an acidic value. We now have studied these sodium transporters in erythrocytes from patients in remission. Moreover, we intended to explain why the transporters were impaired in erythrocytes, which have no apparent bearing on cancer tissues. The sodium pump was studied through a microcalorimetric method, and the Na+/H+ antiport by a titrimetric method. In patients in remission the sodium pump activity returned to normal: 15.10 +/- 6.00 vs. 14.12 +/- 5.28 mW/l cells for remission and control, respectively. The optimal intracellular pH for Na+/H+ antiport activity was identical in remission and control: 6.09 +/- 0.23 vs. 6.10 +/- 0.10. Restoration of sodium pump activity and optimal intracellular pH for Na+/H+ antiport activity in erythrocytes were thus linked to remission. Moreover, we showed that the impairments of the sodium transporters were due to the presence of plasma-borne factors, the existence of which explained why the sodium transporters were impaired in erythrocytes.

Sodium pump activity in uremic erythrocytes: a microcalorimetric study.

Erythrocyte thermogenesis was studied by flow microcalorimetry in 25 healthy subjects and 27 uremic patients. The heat production (HP) from cells in plasma, decrease in HP induced by ouabain (a specific sodium pump inhibitor) and index of rate response to ouabain action were measured. HP was higher in uremic patients than controls. Sodium pump inhibition with ouabain induced the same decrease in HP in the two groups. The index of rate response to ouabain action was lower in uremic patients than in controls. The difference in total HP may be due to a different age distribution of erythrocytes. Mean sodium pump activity was identical in the two groups, but some patients had lower activity than controls. Ouabain seems to act more slowly in many patients than in controls, perhaps because of hindered binding of the inhibitor.

Small terrestrial mammals as reservoirs of leptospires in the Sava Valley (Croatia).

Small mammals trapped in seven sites along the river Sava in Croatia were examined for leptospiral infection by renoculture and serological methods. Of the 1749 animals caught 702 were examined by renoculture method and 626 were tested by microagglutination-lysis. The dominant animals species caught were Microtus arvalis (672 animals) and Apodemus agrarius (552 animals). Of the 115 strains of leptospires isolated the dominant serotypes were L. grippotyphosa (40) and L. pomona (35). Five strains of L. sejroe, 9 strains of L. australis, 1 strain of L. bataviae and 25 undetermined strains were isolated, the infection rate ranging from 3 to 32%. Serologic evidence of infection was demonstrated in 91 animals, the prevalence ranging from 0 to 43% of animals depending on the trapping site. Specific agglutinins most frequently encountered were those against L. pomona (50) and L. grippotyphosa (26), though agglutinins against L. jalna (5), L. bataviae (4), L. bratislava (3) etc., were also found. A high degree if association between L. grippotyphosa and the common vole as well as between L. pomona and the field mouse was found indicating that as far as these two types of leptospires are concerned the Sava valley may be considered as their vast natural focus. It was also concluded that wild small mammals in this region of Croatia may be a source of leptospiral infection for the local population as well as for outdoor-reared domestic animals (swine, bovine).

Chloride dependent intracellular pH increase induced by bepridil in human red blood cells: a possible involvement in correction of ischemic acidosis.

Inhibitors of Na+/H+ exchanger are reported to exert an anti-ischemic effect. Some calcium antagonists and particularly bepridil are commonly used as anti-ischemic agents. Therefore, in this study, we test the hypothesis that protective effect against ischemia may occur at least in part through an action on Na+/H+ exchanger. The effect of some calcium antagonists on Na+/H+ exchanger from acid-loaded and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt (DIDS)-treated human red cells (RBC) has been studied with a pHstat technique. Doses above those required to affect calcium channel (10(-5) and 10(-4) M) of nifedipine, nicardipine, verapamil and diltiazem had no effect on Na+/H+ exchanger activity. Ethyl isoproply amiloride (10(-4) M) completely inhibited the exchanger. Among the calcium antagonists tested, bepridil exhibited a particular effect, dissipating the pH gradient independently from the Na+/H+ exchanger activity. Bepridil's effect on DIDS-treated RBC was compared with that of a well known protonophore, carbamyl cyanide p-(trifluoromethoxy)phenyl hydrazone, and with that of tributyltin, which mediates a Cl-/OH- exchange across the cell membrane. Bepridil (> 2 x 10(-6) M) acts like tributyltin by dissipating the pH gradient whatever the external cation (Na+ or K+) or the membrane potential, and its action depends on the ratio intracellular [Cl-]/extracellular [Cl-]. The dissipation seems to occur through an OH-/Cl- exchange but other mechanisms may intervene. Moreover, intraerythrocytic pH measurement by 31P nuclear magnetic resonance clearly showed that bepridil permits the cell to recover normal pH faster than control.(ABSTRACT TRUNCATED AT 250 WORDS)

Redox regulation of human Rac1 stability by the proteasome in human aortic endothelial cells.

Rac1 has been shown to activate a NADPH oxidase complex producing superoxide anions in a variety of mammalian cell types. We evaluated the impact of Rac1-induced reactive oxygen species production on the turnover of Rac1 itself in human aortic endothelial cells. The concentration of a constitutively active mutant of Rac1 (Rac1(V12)) was increased by treatment of the cells with diphenylene iodinium (DPI), an inhibitor of the NADPH oxidase. Such an effect was not observed for the dominant negative form of Rac1 (Rac1(N17)). We showed a decrease in proteolytic degradation of Rac1(V12) in the presence of DPI, and showed that short term treatment with H(2)O(2) reverses the effect of DPI. We found that proteasome inhibitors (lactacystin and MG132) increased Rac1(V12) protein level. In support of this finding, we have identified in the primary sequence of Rac1 a potential destruction box domain, which is known to be a signal for protein degradation mediated by the ubiquitin/proteasome system. We show that Rac1(V12) is ubiquitinated before degradation. By contrast Rac1(N17) induces an accumulation of the ubiquitinated form of Rac1. These results suggest that Rac1 activation of NADPH oxidase is necessary for the proteolytic degradation of Rac1 itself.

Adhesion, actin cytoskeleton organisation and the spreading of colon adenocarcinoma cells induced by EGF are mediated by alpha2beta1 integrin low clustering through focal adhesion kinase.

Both epidermal growth factor (EGF) and the extracellular matrix components have been implicated in the pathobiology of adenocarcinomas by somewhat poorly understood mechanisms. We have addressed this problem using an in vitro model comprising the colon adenocarcinoma cell line HT29-D4, wherein the role of EGF and type IV collagen on cell adhesion was examined. We demonstrated that the effect of EGF on HT29-D4 cell adhesion was regulated by type IV collagen in a time- and dose-dependent manner. The incorporation of a panel of monoclonal antibodies to integrins alpha1beta1, alpha2beta1 and alpha3beta1 in adhesion medium revealed that EGF-mediated increase in the cell adhesion was mediated essentially by alpha2beta1, and the use of flow cytometry led us to conclude that this EGF effect was mediated by an increase in alpha2beta1 activation and not by an increase in cell surface expression of integrin. An indirect immunofluorescence technique was employed to demonstrate that focal adhesion kinase (FAK) and alpha2beta1 integrin were present in focal complexes in large EGF-induced lamellipodia whereas actin cytoskeleton was organised in small tips that colocalised with FAK. This pattern was observed at early time points (15 min) with a strong FAK tyrosine phosphorylation and with an increase in mitogen-activated protein kinase activity (5-15 min) as measured by immunoprecipitation and immunoblotting. We conclude that at early time points of cell adhesion and spreading, EGF exerted an inside-out regulation of alpha2beta1 integrin in HT29-D4 cells. This regulation seemed to be mediated by EGF-dependent FAK phosphorylation entailing an increase in integrin activation and their recruitment in numerous focal complexes. Furthermore after activation, FAK induced aggregation of actin-associated proteins (paxillin, vinculin and other tyrosine phosphorylated proteins) in focal complexes, leading to organisation of actin cytoskeleton that is involved in lamellipodia formation. Finally, activated alpha2beta1 integrins intervened in all these processes clustered in small focal complexes but not in focal adhesions.

Increase of frequency of post-traumatic stress disorder in disabled war veterans during prolonged stay in a rehabilitation hospital.

AIM: To explore possible causative factors in the development of post-traumatic stress disorder (PTSD) in disabled Croatian war veterans. METHOD: The sample comprised 42 disabled Croatian war veterans, aged 19 to 44 years, accommodated in the VaraZdinske Toplice Rehabilitation Hospital for the purpose of long-term physical rehabilitation. Manifestation of PTSD symptoms (Mississippi Scale for Combat-Related Post-Traumatic Stress Disorder) and anxiety levels (Spilberger's State Trait Anxiety Inventory) were tested in 1994 and 1999. RESULTS: Patients with PTSD symptoms had significantly higher anxiety levels then patients without PTSD symptoms. The percentage of patients manifesting PTSD increased from 19% in 1994 to 41% in 1999. Over the same period, the anxiety levels decreased in the patients with PTSD. CONCLUSION: Anxiety and PTSD seem to share common etiologic grounds. Nevertheless, staying in the same homogenous group for a substantial period of time, in combination with inadequate social support and deficient psychological care, may contribute to the development of the PTSD symptomatology.