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cope Exosomes/microvesicles are originated from multivesicular bodies that allow the secretion of endolysosome components out of the cell. In the present work, we investigated the effects of curcumin, a polyphenol, on exosomes/microvesicles secretion in different cells lines, using U18666A as a model of intracellular cholesterol trafficking impairment. Methods and results In both HepG2 hepatocarcinoma cells and THP-1 differentiated macrophages, treatment with curcumin affected the size and the localization of endosome/lysosomes accumulated by U18666A, and reduced the cholesterol cell content. To ascertain the mechanism, we analyzed the incubation medium. Curcumin stimulated the release of cholesterol and the lysosomal β-hexosaminidase enzyme, as well as the exosome markers, flotillin-2 and CD63. Electron microscopy studies demonstrated the presence of small vesicles similar to exosomes/microvesicles in the secretion fluid. These vesicles harbored CD63 on their surface, indicative of their endolysosomal origin. These effects of curcumin were particularly intense in cells treated with U18666A. Conclusion These findings indicate that curcumin ameliorates the U18666A-induced endolysosomal cholesterol accumulation by shuttling cholesterol and presumably other lipids out of the cell via exosomes/microvesicles secretion. This action may contribute to the potential of curcumin in the treatment of lysosomal storage diseases.

Antipsychotics are established drugs in schizophrenia treatment which, however, are not free of side effects. Lipid rafts are critical for normal brain function. Several G protein-coupled receptors, such as somatostatin (SRIF) receptors, have been shown to localize to lipid rafts. The aim of this study was to investigate whether haloperidol treatment affects the composition and functionality of lipid rafts in SH-SY5Y neuroblastoma cells. Haloperidol inhibited cholesterol biosynthesis, leading to a marked reduction in cell cholesterol content and to an accumulation of sterol intermediates, particularly cholesta-8,14-dien-3beta-ol. These changes were accompanied by a loss of flotillin-1 and Fyn from the lipid rafts. We next studied the functionality of the SRIF receptor. Treatment with haloperidol reduced the inhibitory effect of SRIF on adenylyl cyclase (AC) activity. On the other side, haloperidol decreased basal AC activity but increased forskolin-stimulated AC activity. Addition of free cholesterol to the culture medium abrogated the effects of haloperidol on lipid raft composition and SRIF signaling whereas the AC response to forskolin remained elevated. The results show that haloperidol, by affecting cholesterol homeostasis, ultimately alters SRIF signaling and AC activity, which might have physiological consequences.

A cytotoxic analogue of LH-RH, AN-207, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier [D-Lys6]LH-RH, was developed for targeted therapy of cancers expressing LH-RH-receptors. To determine its possible side-effects on the pituitary gland, we investigated the gene expression of pituitary LH-RH-receptors and LH secretion in ovariectomized female and normal male rats after treatment with the maximum tolerated dose of AN-207. The effect of AN-207 on the gene expression of the pituitary GH-RH-receptors and GH secretion was also assessed in male rats. Five hours after a single i.v. injection of AN-207 at 175 nmol/kg, there was a 39-51% decrease in mRNA expression for the pituitary LH-RH-receptors in male and female rats. The carrier, at an equimolar dose, caused a similar reduction (37-39%), whereas the cytotoxic radical AN-201, at an equitoxic dose (110 nmol/kg), produced only a 12-24% decrease (NS) in the mRNA expression of LH-RH-receptors. AN-207 and the carrier analogue induced a comparable 90-100-fold increase in serum LH concentrations in male rats, and the same 12-fold elevation in OVX rats at 5 h. Seven days after treatment with AN-207, the mRNA levels for the LH-RH receptors and the serum LH concentration were back to normal in both sexes. AN-207, the carrier, and AN-201 had no significant effect on the expression of mRNA for GH-RH-receptors in the pituitary. In vitro, a continuous perfusion of pituitary cells with 10 nM AN-207 did not affect the hormone-releasing function of the targeted LH cells or the nontargeted GH cells. Our results demonstrate that cytotoxic LH-RH analogue AN-207, at the maximum tolerated dose causes only a transient decrease in the gene expression of the pituitary LH-RH receptors, and the levels of mRNA for LH-RH receptor fully recover within 7 days. Moreover, the carrier hormone moiety, and not the cytotoxic radical in AN-207 is responsible for this transient suppression. Our findings suggest that the therapy with cytotoxic LH-RH analogues will not inflict permanent damage to pituitary function.