Human tumor cell sensitivity to oleandrin is dependent on relative expression of Na+, K+ -ATPase subunitst.

The membrane enzyme Na+, K+ -ATPase is known to help maintain ion homeostasis in mammalian cells. Newly identified functions of this enzyme suggest that inhibition of Na+, K+ -ATPase by cardiac glycosides may be useful to patients with cancer. Twelve human tumor cell lines were chosen to examine determinants of human tumor cell sensitivity to cardiac glycosides. In vitro cell culture models of human glioma HF U251 and U251 cells as well as human parental and modified melanoma BRO cells were also included in these studies. Data derived from both models and twelve tumor cell lines indicated that high expression of Na+, K+ -ATPase alpha 1 isoform in the presence of low alpha 3 expression correlated with increased resistance to inhibition of cell proliferation by cardiac glycosides such as oleandrin, ouabain and bufalin. Interestingly, increased expression of Na+, K+ -ATPase alpha 1 and therefore total Na+, K+ -ATPase activity is associated with increased cellular levels of glutathione. The altered enzyme activity and glutathione content were associated with a delayed and diminished release of cytochrome c and caspase activation. Additionally, an increased colony-forming ability was noted in cells with high levels of Na+, K+ -ATPase alpha 1 expression, suggesting that Na+, K+ -ATPase alpha 1 isoform may be actively involved in tumor growth and cell survival. Its inhibition by cardiac glycosides may provide a strategy for effective cancer therapy.

Determinants of human and mouse melanoma cell sensitivities to oleandrin.

Oleandrin, a cardiac glycoside component of Nerium oleander, has been shown to induce apoptosis in malignant cells. While human tumor cells are very sensitive to growth inhibition by oleandrin, murine tumor cells are extremely resistant. Using human BRO and mouse B16 melanoma cell lines, we explored several possible determinants of cell sensitivity to oleandrin and compared with ouabain. The studies include Na+, K(+)-ATPase activity and its isoforms as well as the cellular uptake of these cardiac glycosides. Oleandrin and ouabain induced apoptosis was detected in BRO cells while no evidence of cell death was observed in B16 cells even at concentrations 1000-fold higher than that used for BRO cells. Cellular uptake of oleandrin and ouabain was 3-4 fold greater in human BRO tumor cells than murine tumor cells. Partially purified Na+, K(+)-ATPase from human BRO cells was inhibited at a concentration that was 1000-fold less than that was required to inhibit mouse B16 enzyme to the same extent. Using Western blot analyses, human BRO cells were found to express both the sensitive alpha3 isoform and the less sensitive alpha1 isoform of Na+, K(+)-ATPase while mouse B16 cells expressed only the alpha1 isoform. These data suggest that differential expressions of Na+, K(+)-ATPase activities and its isoforms in BRO and B16 cells as well as cellular drug uptake may be important determinants of tumor cell sensitivity to cardiac glycosides.

The spectrum of Gram-positive bloodstream infections in patients with hematologic malignancies, and the in vitro activity of various quinolones against Gram-positive bacteria isolated from cancer patients.

OBJECTIVES: To determine the current spectrum of Gram-positive bloodstream infections (BSI) in patients with hematologic malignancies at our institution, and to determine the in vitro activity of various fluoroquinolones against clinical Gram-positive isolates collected from such patients. METHODS: Institutional microbiology records from 493 consecutive episodes of Gram-positive BSI were reviewed. The in vitro activity of six fluoroquinolones against 477 clinical isolates was determined using an NCCLS approved, broth-dilution method. RESULTS: The most common Gram-positive organisms isolated from the bloodstream of patients with hematological malignancies were coagulase-negative staphylococci (33%), Staphylococcus aureus (15%), viridans group streptococci (10%), and the enterococci (8%). Acute leukemias were the most common underlying malignancies, and 73% of patients were neutropenic when they developed their BSI. The newer generation quinolones--moxifloxacin and gatifloxacin--had the best overall in vitro activity against the Gram-positive isolates tested, and were at least 2 to 8-fold more potent than the early generation quinolones (ofloxacin and ciprofloxacin). Of the 477 isolates tested, 405 (85%) were from patients receiving quinolone (ciprofloxacin or levofloxacin) prophylaxis. CONCLUSIONS: In patients with hematologic malignancies, Gram-positive BSI are caused by a large number of bacterial species and many occur despite antimicrobial prophylaxis. The newer generation quinolones--moxifloxacin and gatifloxacin--have better in vitro activity against these organisms than early generation agents (ciprofloxacin and ofloxacin).

Metabolism of homoharringtonine, a cytotoxic component of the evergreen plant Cephalotaxus harringtonia.

Homoharringtonine (HHT), first isolated from the Chinese evergreen Cephalotaxus harringtonia, has been demonstrated to have a broad antitumor activity in rodents and antileukemic effects in humans. We found that HHT was metabolized to an acid product [HHT-acid; 2'-hydroxy-2'-(alpha-acetic acid)-6'-hydroxy-6'-methylheptanoyl cephalotaxine] when incubated with either human plasma or mouse plasma in vitro. The conversion was faster, however, in mouse plasma, and was both time- and temperature-dependent. Boiled plasma prevented the conversion of HHT to HHT-acid, suggesting that the conversion was enzymatically mediated. When mice were given an intravenous (i.v.) injection of HHT (4 mg/kg), the HHT-acid metabolite was found in both plasma and urine. In mice, HHT-acid was detected in the plasma within 5 min of the i.v. injection of HHT and declined rapidly thereafter. The initial half-lives (t 1/2 alpha) of HHT and HHT-acid were 9 and 17 min, respectively. Twenty-four hours after HHT dosing in mice, approximately 29% of the dose was excreted in the urine as HHT and 20% as HHT-acid. High-pressure liquid chromatography and mass spectrometry were used to confirm the identity and quantify HHT and its metabolite, HHT-acid. The HHT concentration inhibiting 50% of the growth of human leukemic HL-60 cells was 20 ng/ml, while for HHT-acid it was 14,500 ng/ml, indicating that the acid form was more than 700 times less cytotoxic than HHT. The lethal dose of HHT affecting 50% (LD50) of mice was 6.7 mg/kg, but HHT-acid produced no apparent toxic effects at doses up to 280 mg/kg.

Murine pharmacokinetics and metabolism of oleandrin, a cytotoxic component of Nerium oleander.

Pharmacokinetic studies of [3H]oleandrin, a cardiac glycoside component of Anvirzel, were conducted in mice after either an i.v. dose (40 micrograms/kg) or a p.o. dose (80 micrograms/kg). Oleandrin was rapidly absorbed after oral dosing (Cmax at 20 min) although the elimination half-life was longer (2.3 +/- 0.5 h) than that after i.v. dosing (0.4 +/- 0.1 h). The AUC0-infinity values obtained after i.v. and p.o. dosing were 24.6 +/- 11.1 and 14.4 +/- 4.3 (ng.h/ml), respectively, resulting in an oral bioavailability of approximately 30%. After i.v. administration, oleandrin concentration in liver was approximately twice that measured in heart or kidney tissue. Oleandrigenin, the aglycone of oleandrin, was also found in these tissues. At 5 min, > 60% of the total radioactivity in liver was due to oleandrin while 28% of the given dose was present as oleandrigenin. Twenty-four hours following injection, 8% of total radioactivity was excreted in urine and contained both oleandrigenin (4.4% of the injected dose) and oleandrin (1.9%). Sixty-six percent of injected radioactivity was found in feces and consisted of oleandrin and oleandrigenin in equal amounts. Uptake of oleandrin in brain after i.p. injection of oleandrin (3 mg/kg) or oleander extract (700 mg/kg) was examined. Measured by LC/MS/MS, oleandrin content in brain was higher following injection of extract than it was with an equivalent dose of oleandrin. The data suggest that components within oleander extract may enhance transport of oleandrin across the blood brain barrier.

Antimicrobial activity of a novel des-fluoro (6) quinolone, garenoxacin (BMS-284756), compared to other quinolones, against clinical isolates from cancer patients.

The in vitro spectrum of a novel des-fluoro (6) quinolone, garenoxacin (BMS-284756), was compared with that of ciprofloxacin, levofloxacin, and trovafloxacin against 736 clinical isolates from cancer patients. Garenoxacin was the most active agent overall against Gram-positive organisms, with potent activity against Aerococcus spp., Micrococcus spp., Rhodococcus equi, Stomatococcus mucilaginous, Bacillus spp., Enterococcus faecalis, Listeria monocytogenes, methicillin-susceptible Staphylococcus spp., and all beta-hemolytic and viridans streptococci. Although ciprofloxacin was the most active agent tested against the Enterobacteriaceae garenoxacin inhibited the majority of these isolates at