Targeting mitochondria by α-tocopheryl succinate overcomes hypoxia-mediated tumor cell resistance to treatment

Rapidly proliferating tumor cells easily become hypoxic. This results in acquired stability towards treatment with anticancer drugs. Here, we show that cells grown at 0.1 % oxygen are more resistant towards treatment with the conventionally used anticancer drugs doxorubicin and cisplatin. The stimulation of apoptosis, as assessed by the number of cells in the SubG1 fraction of the cell cycle, release of cytochrome c into the cytosol, activation of caspase-3, and cleavage of PARP, was markedly suppressed under low oxygen content or when hypoxia was mimicked by deferoxamine. Hypoxia or deferoxamine treatment was accompanied by stabilization of the hypoxia-inducible factor (HIF-1). The downregulation of HIF-1 using siRNA technique restored cell sensitivity to treatment under hypoxic conditions to the levels detected under normoxic conditions. In contrast to cisplatin or doxorubicin, α-tocopheryl succinate (α-TOS), a compound that targets mitochondria, stimulated cell death irrespective of the oxygen concentration. Moreover, under hypoxic condition cell death induced by α-TOS was even enhanced. Thus, α-TOS can successfully overcome resistance to treatment caused by hypoxia, which makes α-TOS an attractive candidate for antitumor therapy via mitochondrial targeting.     

Role of calcium in the histamine release induced by D-galactosamine from rat mast cells

Summary Rat peritoneal mast cells were isolated and purified by differential centrifugation in Ficoll. Cells pooled from three to four rats were suspended at approximately 10⁶ cells/ml in a buffered salt solution and incubated for 1 h at 37°C in 300 l volumes in the absence or presence (9×10^–4 M) of calcium chloride. Addition of D-galactosamine hydrochloride (DGM; 2.8×10^–4 M) caused (in addition to basal release) a mean ±SEM percent histamine release of 15.7±5.2 in the presence of Ca⁺⁺ and 19±4.9 in the absence of Ca⁺⁺ (p>0.05). It is suggested that D-galactosamine does not require extracellular Ca⁺⁺ for the release of histamine from the rat mast cell.A preliminary analysis of these results was presented at the International Symposium on calcium entry blockers and tissue protection, Rome, 15–16 March 1984.     

Chronic sensory denervation reduces thrombin-stimulated endothelin release from aortic endothelial cells

The long-term (trophic) influence of perivascular nerves on the endothelium was investigated by measuring changes in thrombin-stimulated release of the potent vasoconstrictor, endothelin, after selective chronic denervation. Rat pups were treated with either guanethidine or capsaicin to destroy sympathetic or sensory nerves, respectively. The abdominal aortas from the rats at three months of age (5 pooled per experiment) were incubated with 4U thrombin/ml in medium for 24 h at 37°C, and the amount of endothelin released from the preparation determined by immunoassay. After neonatal sensory denervation there was a significant reduction in the thrombinstimulated release of endothelin compared to the controls (0.012±0.012 (4) compared to 0.063±0.012 (6), pmol/cm²/24 h, p<0.02). There was no change in endothelin release after sympathetic denervation. In summary, sensory nerves play a trophic role in the expression of endothelin in endothelial cells of the intima.     

Bombesin-like immunoreactivity in the pituitary gland

Summary Bombesin has been shown to stimulate release of anterior pituitary hormones both in vivo and in vitro. The aim of this study was to determine whether bombesin-like immunoreactivity could be detected in the human pituitary. Significant concentrations were found in the human anterior gland (4.6±1.5 pmoles/g), posterior gland (1.5±0.4 pmoles/g) and stalk (8.1±0.8 pmoles/g). Significant amounts were also observed in the guinea-pig pituitary. Gel permeation chromatography revealed the presence of 2 major molecular forms of bombesin-like immunoreactivity, one co-eluting with porcine gastrin-releasing peptide and the other with amphibian bombesin.     

Flow-induced release of adenosine 5′-triphosphate from endothelial cells of the rat mesenteric arterial bed

Adenosine 5-triphosphate (ATP) was released into the perfusate of rat isolated mesenteric arterial beds during each of two consecutive increases in flow. There was no significant difference between the amounts of ATP released on each occasion. Substance P was also released into the perfusate by increased flow, although its release was more variable. Removal of the endothelium of the mesenteric vessels with sodium deoxycholate led to a significant reduction (74%) in the amount of ATP released compared with the release before the endothelium had been removed. This suggests that the ATP released into the mesenteric arterial perfusate during increased flow arises from endothelial cells.     

Adenosine-5′-triphosphate levels in experimental CaNT and Fib/t tumours of varying volume and degree of hypoxia

Summary The variation of adenosine-5-triphosphate (ATP) content per unit mass of tumour, versus tumour volume was measured in vivo under normoxic conditions, using CaNT and Fib/t murine tumours grown in CBA and WHT mice respectively. A monotonically decreasing relation was found. Artificially induced tumour hypoxia resulting from 15 min of clamping was accompanied by reduced ATP levels.     

Adenosine-5'-triphosphate levels in experimental CaNT and Fib/t tumours of varying volume and degree of hypoxia

The variation of adenosine-5'-triphosphate (ATP) content per unit mass of tumour, versus tumour volume was measured in vivo under normoxic conditions, using CaNT and Fib/t murine tumours grown in CBA and WHT mice respectively. A monotonically decreasing relation was found. Artificially induced tumour hypoxia resulting from 15 min of clamping was accompanied by reduced ATP levels.     

Net ATP synthesis by running the red cell calcium pump backwards

Summary Ca²⁺ loaded inside-out vesicles from human red blood cells, yielding Ca²⁺ into a Ca²⁺ free medium with 4 mM EGTA, 2 mM ADP and 10 mM phosphate, produced an excess of 14.9 pmoles · min^–1 · (mg protein)^–1 of ATP compared to controls in which the transmembrane Ca²⁺ gradient was abolished by the ionophore A 23 187.We are obliged to Dr H. Fey and Miss H. Pfister (Veterinarybacteriological Institute Bern) and Dr H. Porzig (Pharmacological Institute Bern) for help and advice.     

Influence of hypoxia on contractility and calcium uptake in rabbit aorta

Summary The influence of hypoxia on noradrenaline (NA)-induced contractions and⁴⁵Ca uptake has been studied on isolated rabbit aortae. Hypoxia significantly decreased the contractility of aortic strips. NA stimulation resulted in increased or decreased⁴⁵Ca uptake by normoxic or hypoxic specimens, respectively. Relating⁴⁵Ca movement with mechanical activity, the results suggest that decrease in Ca⁺⁺ uptake may be mechanism for hypoxic relaxation of aortic smooth muscle.The author acknowledges facilities at the Wellcome Surgical Research Institute, University of Glasgow, U.K. Much of this work was in collaboration with Drs Sheila Jennett and J.D. Pickard.     

Parameters affecting the maximum cell concentration ofTetrahymena

Summary In cultures with efficient aeration a maximum cell concentration (MCC) of 6×10⁵ cells/ml (defined medium) and 5.5×10⁶ cells/ml (broth) can be reached. By culturing within Millicells with excess supply of medium and efficient removal of waste products a physical limit for MCC of about 13×10⁶ cells/ml is reached.     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

Summary The contraction induced by a Ca²⁺-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (F_o), immediate elastic recoil (SE), unloaded shortening velocity (V_us), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca²⁺-induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. F_o developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. V_us in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3±8.9% less than V_us induced by Ca²⁺ (1.6x10^–6M) in the control fibers. Addition of Ca²⁺ (1.6x10^–6M) to a contraction induced by MLCK-resulted in small increases in both F_o and V_us. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca²⁺. We conclude that with respect to actin-myosin interaction, MLCK- and Ca²⁺-induced contractions are similar.     

The effect of temperature on Ca2+ uptake and Ca2+-activated ATP hydrolysis by cardiac sarcoplasmic reticulum

Zusammenfassung Der Ca²⁺-Transport und die Ca²⁺-aktivierte ATP Hydrolyse (ATP extra Spaltung) durch Membranen des cardialen sarkoplasmatischen Retikulums zeigen die gleiche Temperaturabhängigkeit. Die Aktivierungsenergie der Ca²⁺-Aufnahme und der ATP extra Spaltung, gemessen bei Anwesenheit von Oxalat, beträgt 16.65±0.87 und 17.93±0.49 Kcal/Mol^–1.     

Inhibition of TSH-stimulated thyroid hormone release and potentiation of TRH-stimulated TSH release by indomethacin in perifusion systems of rat thyroids and pituitaries

Summary Using indomethacin (Ind), a prostaglandin, synthesis inhibitor, in vivo experiments in rats and in vitro experiments with perifusion systems of rat thyroids and pituitaries were conducted. After 35 days of intragastric infusion of Ind, serum TSH levels were markedly increased, the thyroid was swollen and, as a consequence, T₃ and T₄ levels were normal. The T₃ release from perifused rat thyroids under continuous stimulation with 10 mU/ml TSH was inhibited significantly (p<0.01) by 1.0×10^–6 M Ind. On the other hand, the TSH release from perifused rat pituitaries under TRH stimulation was enhanced conspicuously by Ind. It was concluded that Ind decelerated thyroid hormone release from the thyroid and accelerated TSH release from the pituitary in perifusion systems.     

Flow-induced release of adenosine 5'-triphosphate from endothelial cells of the rat mesenteric arterial bed.

Adenosine 5'-triphosphate (ATP) was released into the perfusate of rat isolated mesenteric arterial beds during each of two consecutive increases in flow. There was no significant difference between the amounts of ATP released on each occasion. Substance P was also released into the perfusate by increased flow, although its release was more variable. Removal of the endothelium of the mesenteric vessels with sodium deoxycholate led to a significant reduction (74%) in the amount of ATP released compared with the release before the endothelium had been removed. This suggests that the ATP released into the mesenteric arterial perfusate during increased flow arises from endothelial cells.     

Sodium concentration and the effect of angiotensin II on iieal smooth muscle

Zusammenfassung Die kontraktile Reaktion des isolierten Meerschweinchen-Ileums auf Angiotensin II variierte direkt mit der Natriumkonzentration der Nährflüssigkeit, nicht aber bei ACh; Atropin (10^–6 g/ml) reduzierte die Wirkung auf Angiotensin um 60–70% und hob die Wirkung auf äquivalente Mengen von ACh auf. In atropinhaltiger Ringerlösung potenzierte ein hoher Natriumgehalt (+ 30 meq./l) die Reaktion auf Angiotensin zehnfach.     

High K+-induced release of somatostatin from the cortical preparation of rat brain

Summary Release of endogenous somatostatin (SRIF) from the rat cerebral cortical slices incubated in Krebs-bicarbonate buffer was increased from the basal rate of 3.4±0.6% of the total SRIF content in 15 min at [K⁺]_o=5.6 mM, to 13.1±1.6% upon raising the [K⁺]_o to 56.6 mM. The high-K⁺ evoked SRIF release was absent when Ca⁺⁺ in the medium was replaced by Mn⁺⁺. The isolated synaptosomes from rat cerebral cortex contain 13.2±3.1 ng SRIF/mg protein compared to 0.33±0.01 ng/mg protein in the cortical tissue as a whole, suggesting that nerve terminals are the main source of the peptide released upon membrane depolarization.The study was supported by a grant from the Medical Research Council of Canada. Results of this work have been published in part as abstracts: Can. Physiol.9, 45 (1978), and Fedn Proc.37, 665 (1978).The authors are greatly indebted to Dr M. Gotz and the Ayerst Research Laboratories for the most generous supply of the synthetic somatostatin.     

The regulatory peptide system of the large bowel in equine grass sickness

Summary In recent years, distinct changes in regulatory peptides have been found in a number of gastrointestinal diseases. Grass sickness is a fatal disease of horses for which the etiology has yet to be fully ascertained. In this study, the peptide-containing nerves and ganglionic and mucosal endocrine cells of the ileum, colon and rectum were investigated in horses with sub-acute or chronic grass sickness and compared with normal controls using immunocytochemistry, at both the light and electron microscopical levels, and radioimmunoassay. A substantial loss of both peptide-containing cells and nerves was found in all of the sick horses, particularly in the ileum. Electron microscopy revealed marked degeneration of nerves in the gut wall. fibers containing granules immunostained for substance P or VIP, using the immunogold staining technique, underwent extensive degranulation in grass sickness, with the formation of multiple vacuoles.Radioimmunoassay of peptide content also showed that the most drastic changes occurred in the ileum. For example, VIP content was significantly reduced from 109±19.8 (mean±SEM) pmoles/g in controls to 6.8±1.4 pmoles/g in grass sickness (p<0.001) and substance P from 65.9±8.1 to 31.3±9.5 (p<0.02). These results may have applications in the diagnosis and treatment of grass sickness.The authors gratefully acknowledge the financial support of the Wellcome Trust and the Grass Sickness Fund.     

Denaturation map of the ribosomal DNA ofLytechinus variegatus sperm

Summary Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6±0.2 m, corresponding to a mol.wt of 7.2±0.4×10⁶. Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47±0.11 m (mol.wt 4.9±0.22×10⁶) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16±0.09 m (mol.wt 2.3±0.18×10⁶) is presumed to contain non-transcribed spacer sequences.Acknowledgments. This paper is dedicated to the memory of Dr D. P. Costello whose insprration made this work possible. I gratefully acknowledge the valuable advice of Drs D.W. Stafford and M.A. Bleyman.     

Total synthesis of derivatives of (±)-6-oxa and aza-estrone

Zusammenfassung Es wird über die Totalsynthese von verschiedenen Derivaten des (±)-6-Oxa- und Aza-Östrons und die Umwandlung von (±)-17-Oxy-3-methoxy-6-oxa-östratrien-1, 3, 5¹⁰ zu Verbindungen der (±)-19-Nor-6-oxa-testosteron-Klasse berichtet. Die biologische Wirksamkeit der Glieder in der 6-Oxa-Reihe wird beschrieben.