Pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 human leukemia cells.

Pterostilbene, a naturally occurring structural analog of resveratrol, has been reported to exert antiproliferative and proapoptotic effects in various cancer types. Recently, it has been demonstrated to induce both autophagy and apoptosis in human bladder and breast cancer cell lines. The aim of this study was to evaluate the effects of pterostilbene on HL60 human leukemia cells. Cell morphology was examined using confocal and electron microscopy. Cell viability was determined by MTT, neutral red uptake and trypan blue exclusion assays. LC3 processing was studied based on Western blotting and immunofluorescence analyses. Flow cytometry was used to study cell cycle distribution, phosphatidylserine externalization, caspase activation, disruption of mitochondrial membrane potential and intracellular production of reactive oxygen species. DNA degradation was examined by gel electrophoresis. We found that treatment of HL60 cells with pterostilbene at the IC90 concentration resulted in the G0/G1 cell cycle arrest. Pterostilbene induced conversion of cytosolic LC3-I to membrane-bound LC3-II and accumulation of large LC3-positive vacuolar structures. Pterostilbene also led to phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase activation and disruption of mitochondrial membrane potential. Moreover, it did not induce oxidative stress. Our results suggest that pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 cells.

Increased rate of cholesterologenesis--a possible cause of hypercholesterolemia in experimental chronic renal failure in rats.

Hypercholesterolemia plays an important role in the lipid abnormalities in chronic renal failure (CRF). It is thought to contribute to both a progression of renal failure and atherosclerosis. Despite intensive research, the etiopathogenesis of hypercholesterolemia in CRF patients is still obscure. The present study was designed to evaluate the possible role of cholesterol overproduction in the development of hypercholesterolemia associated with experimental CRF. We found that plasma total cholesterol and cholesterol distributed in VLDL, LDL and HDL concentrations were significantly enhanced in CRF rats. Simultaneously, the rate of liver cholesterol biosynthesis in vivo (measured by determining the incorporation of tritium from tritiated water intraperitoneally injected into cholesterol ), liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and liver HMG-CoA reductase mRNA presence were elevated. Significant increases in activity of liver malic enzyme, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, NADPH-producing enzyme (required for cholesterol synthesis) have also been observed in CRF rats. In conclusion, the increased rate of liver cholesterol biosynthesis due to increase of HMG-CoA reductase and NADPH-producing enzyme gene expression could be one of the possible causes of hypercholesterolemia in CRF animals.

Diurnal rhythm of cholesterol biosynthesis in experimental chronic renal failure.

Changes in lipid metabolism are an important risk factor for vascular complications during chronic renal failure (CRF). In experimental CRF hypercholesterolemia has been found to be the main lipid disorder. It is probably due to enhanced cholesterologenesis. Mechanisms of these changes remain poorly understood. It is well known that activity of cholesterologenesis undergoes a significant diurnal rhythm. However, there was no evidence that this rhythm is still present in the course of experimental CRF. Results of our studies indicate that in contrast to puromycin induced nephrotic syndrome, diurnal rhythm of cholesterologenesis in CRF rats is preserved both in liver and in the intestine tissue. Significant higher incorporation of tritiated water into cholesterol fraction was found in vivo both in liver as well as in intestine of CRF rats, as compared to control animals. Increased (with comparison to the controls) incorporation of 14C-acetate, and 3H-mevalonate into CRF rat liver sterols indicate that mechanism of enhanced cholesterologenesis is more complex than simply due to the elevated level of mevalonate (potential substrate for cholesterologenesis) which has been reported in plasma of CRF animals.

[Metabolism of mevalonate in human placenta]. / Metabolizm mewalonianu w lozysku czlowieka.

Prenylation of protein rather than cholesterol synthesis, seems to be main "metabolic target" of mevalonate metabolism in the human placenta. It seems very probable that cytological transformation of cytotrophoblast cells to syncytiotrophoblast which occur during placental development, "switched off" activity of mevalonate biosynthesis in term placenta.

[Structure and function of LDL receptors in familial hypercholesterolemia]. / Struktura i funkcja receptora LDL w hipercholesterolemii rodzinnej.

Structure and function of the LDL-receptor in genetic disorder--familial hypercholesterolemia is described as well as the structure of receptor coding gene. General outline of therapy in FH is also described.

Cytochrome P-450 in the mitochondria from human placenta in early pregnancy.

The early mitochondria show a lower cytochrome P-450 specific concentration and cholesterol side-chain cleavage activity as compared to the term placental mitochondria. These differences occur mainly in the heavy fraction of placental mitochondria.

Intramitochondrial compartmentation of progesterone biosynthesis from cholesterol in the human placenta.

Intramitochondrial distribution of the cholesterol side-chain cleavage reaction was investigated in human placental mitochondria. The matrix side of the inner mitochondrial membrane has been found to be most probably side of this reaction.

HMG-CoA reductase activity in the microsomal fraction from human placenta in early and term pregnancy.

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) mevalonate: NADP oxidoreductase (CoA acylating; EC 1.1.1.34) in microsomes from early- and term-pregnancy placenta has been found to be 24 +/- 2 and 6 +/- 3 pmol/min per mg protein, respectively. Inactivation of the enzyme required the addition of ATP and Mg2+ and was dependent on the time of preincubation. Reactivation of the enzyme was also dependent on the incubation time and prevented by the presence of fluoride--a phosphoprotein phosphatase inhibitor. These data suggest that (despite a low activity) placental HMG-CoA reductase is covalently modulated via the phosphorylation-dephosphorylation system. The conversion of [14C]acetate and [3H]mevalonate into digitonin precipitable placental sterols indicates that the lower reductase activity in term, than in early, placental microsomes is accompanied by a less active conversion of [14C]acetate in this tissue.

Cytochrome P-450 and steroidogenic activities of the human placental mitochondria.

The relationship between cytochrome P-450 concentration, cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase/isomerase activity, in different density mitochondrial preparations from human term placenta has been studied. The heavy mitochondrial fraction shows a higher cytochrome P-450 concentration and cholesterol side-chain cleavage activity as compared to the light mitochondrial fraction, it has however lower cytochrome P-450AROM level and 3 beta-hydroxysteroid dehydrogenase/isomerase activity.

The relationship between energy generation and cholesterol side-chain cleavage reaction in the mitochondria from human term placenta.

1. The interrelationship between progesterone (from cholesterol) biosynthesis and oxidative phosphorylation in human placental mitochondria was examined. 2. ADP and ATP stimulated the malate, succinate and alpha-ketoglutarate-supported progesterone biosynthesis probably via the energy-dependent pyridine nucleotide transhydrogenase activation. The effect of ADP was abolished by rotenone and antimycin in the presence of malate or alpha-ketoglutarate. 3. In the non-energized state of mitochondria malate may supported progesterone biosynthesis by the malic enzyme-dependent pathway. 4. The inhibitory effects of antimycin or cyanide, and the stimulatory effect of rotenone on the succinate-supported progesterone biosynthesis indicate that the succinate to malate conversion is a necessary condition for the stimulation of progesterone biosynthesis from cholesterol. 5. alpha-Ketoglutarate plus malonate did support progesterone biosynthesis also in the presence of ADP or ATP and to a lesser degree in the presence of DNP and rotenone. Arsenate in the presence of alpha-ketoglutarate, malonate, dinitrophenol and rotenone did not affect significantly progesterone biosynthesis. These results indicate that NADPH may be generated also by a non-energy-dependent transhydrogenation in placental mitochondria.

Regulation of progesterone biosynthesis in human placental mitochondria by Krebs cycle metabolites.

1. 2-Oxoglutarate, succinate, fumarate, malate and citrate, cis-aconitate and isocitrate stimulate conversion of cholesterol to progesterone in human placental mitochondria. 2. The stimulatory effect of dicarboxylic and tricarboxylic acids depends on the activity of malate dehydrogenase (decarboxylating) (NADP+) (EC 1.1.1.40) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42), respectively.