The resveratrol analogue 4,4'-dihydroxy-trans-stilbene inhibits cell proliferation with higher efficiency but different mechanism from resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antiproliferative activity. We previously found that 4-hydroxy group in the trans conformation was absolutely required for the inhibition of cell proliferation. In the present work we have synthesized the resveratrol analogue 4,4'-dihydroxy-trans-stilbene, which contains two OH in 4' and 4 positions, with the aim of developing a compound with an antiproliferative potential higher than that of resveratrol, on the basis of the correlation between structure and activity previously observed. In comparison with resveratrol, 4,4'-dihydroxy-trans-stilbene inhibited cell clonogenic efficiency of fibroblasts nine times more although with a different mechanism. First, 4,4'-dihydroxy-trans-stilbene induced predominantly an accumulation of cells in G1 phase, whereas resveratrol perturbed the G1/S phase transition. Second, although both compounds were able to inhibit DNA polymerase (pol) delta in an in vitro assay, 4, 4'-dihydroxy-trans-stilbene did not affect pol alpha activity. Finally, 4,4'-dihydroxy-trans-stilbene increased p21(CDKN1A) and p53 protein levels, whereas resveratrol led to phosphorylation of the S-phase checkpoint protein Chk1. Taken together, our results demonstrated for the first time that the two hydroxyl groups on 4- and 4'- positions of the stilbenic backbone enhance the antiproliferative effect and introduce additional targets in the mechanism of action of resveratrol. In conclusion, 4,4'-dihydroxy-trans-stilbene has potent antiproliferative activities that differ from the effect of resveratrol shown in this system, suggesting that it warrants further development as a potential chemopreventive or therapeutic agent.

Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells.

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.

Poly(ADP-ribose) polymerase cleavage during apoptosis: when and where?

Poly(ADP-ribose) polymerase-1 (PARP-1) plays the active role of "nick sensor" during DNA repair and apoptosis, when it synthesizes ADP-ribose from NAD(+) in the presence of DNA strand breaks. Moreover, PARP-1 becomes a target of apoptotic caspases, which originate two proteolytic fragments of 89 and 24 kDa. The precise relationship between PARP-1 activation and degradation during apoptosis is still a matter of debate. In human Hep-2 cells driven to apoptosis by actinomycin D, we have monitored PARP-1 activity by the mAb 10H, which is specific for the ADP-ribose polymers, and we have observed that poly(ADP-ribose) synthesis is a very early response to the apoptotic stimulus. The analysis of the presence and fate of the p89 proteolytic fragment revealed that PARP-1 proteolysis by caspases is concomitant with poly(ADP-ribose) synthesis and that p89 migrates from the nucleus into the cytoplasm in late apoptotic cells with advanced nuclear fragmentation.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes.

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.