Replicative senescence in human fibroblasts is accompanied with alterations of various biological processes, including the impaired function of the proteasome. The proteasome is responsible for the removal of both normal and damaged proteins. Due to its latter function, proteasome is also considered a representative secondary antioxidant cellular mechanism. Nrf2 is a basic transcription factor responsible for the regulation of the cellular antioxidant response that has also been shown to regulate several proteasome subunits in mice. We have established in this study the proteasome-related function of Nrf2 in human fibroblasts undergoing replicative senescence. We demonstrate that Nrf2 has a declined function in senescence, whereas its silencing leads to premature senescence. However Normal human fibroblasts undergo a limited number of divisions in culture and progressively reach a state of irreversible growth arrest, a process termed replicative senescence. Senescence has been attributed to a combination of genetic and environmental factors (1). Senescent fibroblasts are viable and fully functional, however, they exhibit several morphologic and biochemical changes as compared with their young/proliferating counterparts (2). We and others (3) have reported loss of proteasome function upon aging of several human tissues as well as in senescent primary cultures. Our previous work in human embryonic fibroblast cultures undergoing replicative senescence has shown that the reduced levels of proteasome activities are accompanied by lower proteasome content due to the down-regulation of its ␤-catalytic subunits (4). The fundamental link between cellular senescence and proteasome function is further supported by our additional studies (5, 6) demonstrating that when the proteasome is partially inhibited in young fibroblasts cultures, an irreversible senescence-like phenotype is triggered. Importantly, reconstitution of the levels of the "down-regulated" ␤-subunits by genetic means in human embryonic fibroblasts, results in enhanced rates of assembled, and functional proteasome and delays senescence by ϳ15% (7).The proteasome is the major cellular non-lysosomal threonine protease, implicated in the removal of normal as well as abnormal, denatured, or otherwise damaged proteins (8). It is a well organized protein complex that contains a proteolytic active 20 S core, composed of 28 subunits, 7 different ␣-and 7 different ␤-subunits, arranged as ␣ 1-7 ␤ 1-7 ␤ 1-7 ␣ 1-7 structure (9). The ␤ 5, ␤ 1 , and ␤ 2 subunits are the catalytic centers of chymotrypsin-like (CT-L), 3 caspase-like/PGPH (peptidylglutamyl-peptide hydrolyzing), and trypsin-like activities, respectively, cleaving peptide bonds after hydrophobic, acidic, and basic residues, respectively (10). The 20 S proteasome is also central to the ATP/ubiquitin-dependent intracellular protein degradation pathway where it represents the proteolytic core of the 26 S complex (11).Although the regulation of human proteasome is not yet entirely elucidated, reports in mice indica...

BackgroundPollen is a bee-product known for its medical properties from ancient times. In our days is increasingly used as health food supplement and especially as a tonic primarily with appeal to the elderly to ameliorate the effects of ageing. In order to evaluate the chemical composition and the biological activity of Greek pollen which has never been studied before, one sample with identified botanical origin from sixteen different common plant taxa of Greece has been evaluated.ResultsThree different extracts of the studied sample of Greek pollen, have been tested, in whether could induce proteasome activities in human fibroblasts. The water extract was found to induce a highly proteasome activity, showing interesting antioxidant properties. Due to this activity the aqueous extract was further subjected to chemical analysis and seven flavonoids have been isolated and identified by modern spectral means. From the methanolic extract, sugars, lipid acids, phenolic acids and their esters have been also identified, which mainly participate to the biosynthetic pathway of pollen phenolics. The total phenolics were estimated with the Folin-Ciocalteau reagent and the total antioxidant activity was determined by the DPPH method while the extracts and the isolated compounds were also tested for their antimicrobial activity by the dilution technique.ConclusionsThe Greek pollen is rich in flavonoids and phenolic acids which indicate the observed free radical scavenging activity, the effects of pollen on human fibroblasts and the interesting antimicrobial profile.

Homeostasis is a key feature of the cellular lifespan. Its maintenance influences the rate of ageing and it is determined by several factors, including efficient proteolysis. The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Alterations of proteasome function have been recorded in various biological phenomena including ageing and replicative senescence. Proteasome activities and function are decreased upon replicative senescence, whereas proteasome activation confers enhanced survival against oxidative stress, lifespan extension and maintenance of the young morphology for longer in human primary fibroblasts. Several natural compounds possess anti-ageing/anti-oxidant properties. In this study, we have identified quercetin (QUER) and its derivative, namely quercetin caprylate (QU-CAP) as a proteasome activator with antioxidant properties that consequently influence cellular lifespan, survival and viability of HFL-1 primary human fibroblasts. Moreover, when these compounds are supplemented to already senescent fibroblasts, a rejuvenating effect is observed. Finally, we show that these compounds promote physiological alterations when applied to cells (i.e. whitening effect). In summary, these data demonstrate the existence of naturally occurring anti-ageing products that can be effectively used through topical application.

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