Antiproliferative and apoptosis-inducing activity of an oxidovanadium(IV) complex with the flavonoid silibinin against osteosarcoma cells.

Flavonoids are a large family of polyphenolic compounds synthesized by plants. They display interesting biological effects mainly related to their antioxidant properties. On the other hand, vanadium compounds also exhibit different biological and pharmacological effects in cell culture and in animal models. Since coordination of ligands to metals can improve or change the pharmacological properties, we report herein, for the first time, a detailed study of the mechanisms of action of an oxidovanadium(IV) complex with the flavonoid silibinin, Na2[VO(silibinin)2]·6H2O (VOsil), in a model of the human osteosarcoma derived cell line MG-63. The complex inhibited the viability of osteosarcoma cells in a dose-dependent manner with a greater potency than that of silibinin and oxidovanadium(IV) (p < 0.01), demonstrating the benefit of complexation. Cytotoxicity and genotoxicity studies also showed a concentration effect for VOsil. The increase in the levels of reactive oxygen species and the decrease of the ratio of the amount of reduced glutathione to the amount of oxidized glutathione were involved in the deleterious effects of the complex. Besides, the complex caused cell cycle arrest and activated caspase 3, triggering apoptosis as determined by flow cytometry. As a whole, these results show the main mechanisms of the deleterious effects of VOsil in the osteosarcoma cell line, demonstrating that this complex is a promising compound for cancer treatments.

Antitumor properties of a vanadyl(IV) complex with the flavonoid chrysin [VO(chrysin)2EtOH]2 in a human osteosarcoma model: the role of oxidative stress and apoptosis.

Flavonoids, a polyphenolic compound family, and the vanadium compounds have interesting biological, pharmacological, and medicinal properties. We report herein the antitumor actions of the complex [VO(chrysin)2EtOH]2 (VOchrys) on the MG-63 human osteosarcoma cell line. Oxovanadium(IV), chrysin and VOchrys caused a concentration-dependent inhibition of cell viability. The complex was the strongest antiproliferative agent (p < 0.05). Cytotoxicity and genotoxicity studies also showed a concentration effect. Reactive oxygen species (ROS) and the alterations in the GSH/GSSG ratio underlie the main mechanisms of action of VOchrys. Additions of ROS scavengers (vitamin C plus vitamin E) or GSH to the viability experiments demonstrated beneficial effects (p < 0.01). Besides, the complex triggered apoptosis, disruption of the mitochondria membrane potential (MMP), increased levels of caspase 3 and DNA fragmentation measured by the sub-G1 peak in cell cycle arrest experiments (p < 0.01). Collectively, VOchrys is a cell death modulator and a promissory complex to be used in cancer treatments.

Blood flow to the brown adipose tissue of conscious young rabbits during hypoxia in cold and warm conditions.

Brown adipose tissue (BAT) non-shivering thermogenesis is stimulated by cold temperature and depressed by hypoxia. We investigated the extent to which changes in metabolic rate during cold and hypoxia, singly or combined, were accompanied by changes in BAT perfusion. One-month-old rabbits were instrumented for measurements of regional blood flow by the coloured microsphere technique. One group of rabbits was tested in warm (24 degrees C, n=17), and the other in cold (13 degrees C, n=9) conditions, first in normoxia (inspired oxygen concentration FIO2 about 21%, arterial oxygen saturation SaO2 approximately 88%) followed by hypoxia (FIO2 approximately 10%, SaO2 approximately 54%). In warm conditions, oxygen consumption (VO2, measured by an open-flow method) averaged 22 ml.kg-1.min-1 (STPD), and BAT blood flow 98 ml.100g-1.min-1. In hypoxia, VO2 dropped on average to 87%, whereas BAT flow dropped to 43% of the normoxic values. In the cold during normoxia, VO2 averaged 31 ml.kg-1.min-1 (STPD), and BAT blood flow was 155 ml.100g-1.min-1. In cold and hypoxia VO2 dropped to 19 ml.kg-1.min-1 (STPD) (i.e. 60% of the normoxic value), whereas BAT blood flow was not altered significantly (148 ml.100g-1.min-1). Hence, BAT blood flow decreased in hypoxia in absence of cold stimuli, whereas it remained high when hypoxia occurred during cold, despite the major drop in VO2. We conclude that cold is more important than hypoxia in determining BAT perfusion, and that changes in BAT blood flow are not a mechanism for the hypoxic control of V.O2.

Thermogenesis in newborn rats after prenatal or postnatal hypoxia.

Oxygen consumption (VO2) was measured in normoxia as ambient temperature (Ta) was lowered from 40 to 15 degrees C, at the rate of 0.5 degrees C/min (thermoneutrality approximately 33 degrees C). In 2-day-old rats born in hypoxia after hypoxic gestation, the Ta-VO2 relationship was as in controls; their interscapular brown adipose tissue (IBAT) was hypoplastic (less proteins and DNA), with lower concentration of the mitochondrial uncoupling protein thermogenin. In 8-day-old rats exposed to hypoxia postnatally (day 2 to day 8), at any Ta below thermoneutrality VO2 was higher than in controls; also, in this group IBAT was hypoplastic with decreased thermogenin. Additional measurements under various experimental conditions indicated that the increased thermogenic capacity was not explained by the smaller body mass and increased blood oxygen content or by the eventuality of intermittent cold stimuli during the chronic hypoxia. On the other hand, chronic hypercapnia (3% CO2 in normoxia, from day 2 to day 8) also resulted in increased normoxic thermogenesis. We conclude that chronic hypoxia in the perinatal period 1) reduces IBAT mass and thermogenin concentration and 2) can increase the newborn's thermogenic capacity because of stress-related mechanisms not specific to hypoxia.

Brown adipose tissue and its uncoupling protein in chronically hypoxic rats.

1. Hypoxia is known to decrease thermogenesis. We set out to determine whether this is accompanied by alterations in the brown adipose tissue, which is a major source of non-shivering thermogenesis. 2. Measurements were performed on 25- and 64-day-old rats, after 4 days of hypoxia (10% inspired O2), and on approximately 3.5-month-old rats in hypobaric hypoxia since birth, at an ambient temperature of 25 degrees C. 3. All hypoxic rats had higher haematocrit and lower body mass than corresponding controls. 4. In the 25-day-old rats, hypoxia had minimal and non significant effects on brown adipose tissue mass, proteins and DNA concentration. The content of the mitochondrial uncoupling protein thermogenin, evaluated by immunoblot after electrophoretic separation, relative to the cytoskeleton actin (UCP/Act), was not significantly altered. 5. In 25-day-old rats exposed for 4 days to cold (ambient temperature = 7-9 degrees C), brown adipose tissue was hyperplastic, with increased UCP/Act; hypoxia did not appreciably alter the response to cold. 6. In the 2-month-old rats, after 4 days of hypoxia UCP/Act was reduced to about 40% of control. 7. In the 3.5-month-old rats maintained in hypoxia since birth, brown adipose tissue mass was reduced in proportion to body mass, with little effect on total proteins and DNA; UCP/Act was decreased to about 50% of control. 8. We conclude that chronic hypoxia had a minimal effect on brown adipose tissue total proteins and DNA content. However, the uncoupling protein content can be greatly reduced, depending upon age and duration of hypoxia.

Electrophoretic analysis of contractile proteins of the diaphragm in chronically hypoxic rats.

We questioned whether the relative composition of the diaphragm proteins [myosin heavy chain (MHC), actin, tropomyosin (TM)-alpha and TM-beta, and MHC isoforms] was altered by chronic hypoxia. Rats were exposed to hypobaric hypoxia (inspired O2 pressure approximately 95 mmHg) from birth for 60 days or 9-11 mo. From the muscle homogenate, relative protein content was computed by densitometric analysis after protein separation with polyacrylamide gel electrophoresis and identification by immunoblotting. The relative concentrations of MHC, actin, TM-alpha and TM-beta did not differ from those of control rats for either duration of exposure. The main MHC isoforms (I, IIa, IIb, IIx) remained unaltered after 60 days but changed significantly after 9 mo of hypoxia, mostly because of a decrease in the slow (I) component and an increase of II complex. We conclude that chronic hypoxia of long duration modifies the expression of MHC isoforms in the rat diaphragm. Qualitative similar changes also occurred in a muscle of the thigh, the rectus femoris; hence, the changes in diaphragm MHC probably reflected the systemic effects of hypoxia rather than the increased mechanical load imposed by the chronic hyperventilation.

Metabolism and ventilation in hypoxic rats: effect of body mass.

Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured by the flow-through method, and ventilation (VE) by the barometric technique in post-weaning age rats of 50, 100, 250 and 400 g, (5 males and 5 females in each group), at ambient temperature congruent to 24 degrees C. In normoxia, VO2, VCO2 and VE decreased with the increase in body weight (BW), whether normalization was by BW or by BW minus the weights of fat and skeleton; VE/VO2 and rectal temperature remained constant. In hypoxia (10% inspired O2), VE VO2 increased in all groups, to 2-2.5 times the normoxic values, because of a significant increase in VE (hyperpnea) and decrease in VO2 (hypometabolism); arterial PCO2, measured in some 100 g and 400 g rats, dropped similarly. However, the hyperpnea was about twice as large, and metabolism and body temperature decreased significantly less, in the 400 g than in the 50 g rats. The cost (ml O2) of breathing, computed in the paralysed animal artificially ventilated, averaged approximately 0.7% (normoxia) and 2% of VO2 (hypoxia), with no systematic differences with BW. The results agree with the concept that the metabolic response to hypoxia can be an important determinant of the magnitude of the hyperpnea.