The cytotoxic effects of brown Cuban propolis depend on the nemorosone content and may be mediated by mitochondrial uncoupling.

Three main types of Cuban propolis directly related to their secondary metabolite composition have been identified: brown, red and yellow propolis; the former is majoritarian and is characterized by the presence of nemorosone. In this study, brown Cuban propolis extracts were found cytotoxic against HepG2 cells and primary rat hepatocytes, in close association with the nemorosone contents. In mitochondria isolated from rat liver the extracts displayed uncoupling activity, which was demonstrated by the increase in succinate-supported state 4 respiration rates, dissipation of mitochondrial membrane potential, Ca(2+) release from Ca(2+)-loaded mitochondria, and a marked ATP depletion. As in cells, the degree of such mitotoxic events was closely correlated to the nemorosone content. The propolis extracts that do not contain nemorosone were neither cytotoxic nor mitotoxic, except R-29, whose detrimental effect upon cells and mitochondria could be mediated by its isoflavonoids and chalcones components, well known mitochondrial uncouplers. Our results at least partly unravel the cytotoxic mechanism of Cuban propolis, particularly regarding brown propolis, and raise concerns about the toxicological implication of Cuban propolis consumption.

Clusianone, a naturally occurring nemorosone regioisomer, uncouples rat liver mitochondria and induces HepG2 cell death.

Clusianone is a member of the polycyclic polyprenylated acylphloroglucinol family of natural products; its cytotoxic mechanism is unknown. Clusianone is a structural isomer of nemorosone, which is a mitochondrial uncoupler and a well-known cytotoxic anti-cancer agent; thus, we addressed clusianone action at the mitochondria and its potential cytotoxic effects on cancer cells. In the HepG2 hepatocarcinoma cell line, clusianone induced mitochondrial membrane potential dissipation, ATP depletion and phosphatidyl serine externalization; this later event is indicative of apoptosis induction. In isolated mitochondria from rat liver, clusianone promoted protonophoric mitochondrial uncoupling. This was evidenced by the dissipation of mitochondrial membrane potential, an increase in resting respiration, an inhibition of Ca(2+) influx, stimulation of Ca(2+) efflux in Ca(2+)-loaded mitochondria, a decrease in ATP and NAD(P)H levels, generation of ROS, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. The cytotoxic and uncoupling actions of clusianone were appreciably less than those of nemorosone, likely due to the presence of an intra-molecular hydrogen bond with the juxtaposed carbonyl group at the C15 position. Therefore, clusianone is capable of pharmacologically increasing the leakage of protons from the mitochondria and with favorable cytotoxicity in relation to nemorosone.

Administration of a murine diet supplemented with conjugated linoleic acid increases the expression and activity of hepatic uncoupling proteins.

Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (~2 % CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45 % of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.

Nanoassisted laser desorption-ionization-MS imaging of tumors.

The ability of nanoassisted laser desorption-ionization mass spectrometry (NALDI-MS) imaging to provide selective chemical monitoring with proper spatial distribution of lipid profiles from tumor tissues after plate imprinting has been tested. NALDI-MS imaging identified and mapped several potential lipid biomarkers in a murine model of melanoma tumor (inoculation of B16/F10 cells). It also confirmed that the in vivo treatment of tumor bearing mice with synthetic supplement containing phosphoethanolamine (PHO-S) promoted an accentuated decrease in relative abundance of the tumor biomarkers. NALDI-MS imaging is a matrix-free LDI protocol based on the selective imprinting of lipids in the NALDI plate followed by the removal of the tissue. It therefore provides good quality and selective chemical images with preservation of spatial distribution and less interference from tissue material. The test case described herein illustrates the potential of chemically selective NALDI-MS imaging for biomarker discovery.