Research paper Anti-ageing properties of Khelma Longevity TM : treatment of human fibroblasts increases proteasome levels and decreases the levels of oxidized proteins Konstantinos Voutetakis 1 , Vasiliki Delitsikou 1 , Michel Georges Magouritsas 2 , Efstathios S. Gonos 1, * 1 National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, 48 Vassileos Constantinou Ave., 11635 Athens, Greece 2 Khelma Hellas S.A., Fidiou 11, 10678 Athens, Greece A R T I C L E I N F O Article history: Available online xxx A B S T R A C T We have determined the putative anti-ageing properties of Khelma Longevity TM , a formula based on various natural compounds from the Mediterranean area. Human primary fibroblast cultures were treated with a wide range of concentrations of Khelma Longevity TM for 1 day or 3 consecutive days. Following these treatments, two major and complementary biomarkers of ageing were measured, namely, the proteasome and the amount of oxidized proteins. It was observed that 24 h of treatment with Khelma Longevity TM resulted in a maximum increase of about 41% of the total protein levels of 20S proteasome. Levels of oxidized proteins were reduced by almost 6.5-fold following longer treatments. Specifically we have observed a maximum decrease of protein carbonyls to 84.7% in comparison with nontreated control cells following 3 days of continuous treatment with Khelma Longevity TM . These results support the notion that formulas rich in natural compounds from the Mediterranean area possess anti-ageing properties. © 2017 Elsevier B.V. All rights reserved. Introduction Ageing is a natural and inevitable biological process accompa- nied by a progressive decline in cellular and organismal homeostasis. Cellular damage is caused mainly by reactive oxygen species (ROS) [1], which are continually generated as by-products of a number of cellular processes, as well as when cells are exposed to various environmental cytotoxic factors. In turn, these unstable and reactive derivatives can cause damage to biological macro- molecules such as lipids, proteins and nucleic acids [2]. In vitro studies, mainly on fibroblast cultures, have shown that these cells have a limited replicative capacity and, after serial passage, they enter a state of irreversible growth arrest termed replicative senescence [3]. Senescence is accompanied by various features including impaired function of the proteasome and increased levels of damaged proteins [4]. 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 [5]. The core particle, the 20S proteasome, is a barrel-shaped complex composed of 28 subunits, 7 different a- and 7 different b-subunits, arranged in an a1-7 b1-7 b1-7 a1-7 structure. The 20S proteasome is also central to the ATP/ubiquitin dependent intracellular protein degradation pathway where it represents the proteolytic core of the 26S complex [6]. Many studies have shown a general decline in proteasome activities in different aged tissues [7]. We have demonstrated proteasome activation by overexpressing the b5 subunit in different human cell lines. The proteasome-activated cell lines exhibited increased rates of proteolysis and cell survival following treatment with various cytotoxic agents [8,9]. Importantly, over- expression of the b5 subunit significantly extended lifespan in human primary cultures [9]. Following these genetic studies, we also showed that proteasome activation is possible through treatment with several compounds, either through promotion of conformational changes in proteasome structure [10] or through the activation of the transcription factor Nrf2 [11]. In accordance with the genetic activation of the proteasome, use of these compounds also results in cellular lifespan extension [11,12]. These in vitro studies indicated that proteasome activation is feasible and results in significant delay of senescence in human cells [13]. Apart from the above work on proteasome activation, several other natural compounds possess putative anti-ageing properties. Work on them was initiated thanks to the great efforts of the late Prof Brian Clark. The compounds include resveratrol [14], ginger [15], coumarin [16] and various other plant polyphenols [17]. Given these findings, we sought to investigate the anti-ageing properties of a potent, natural anti-oxidant supplement on human * Corresponding author. E-mail address: sgonos@eie.gr (E.S. Gonos). http://dx.doi.org/10.1016/j.nbt.2017.03.002 1871-6784/© 2017 Elsevier B.V. All rights reserved. New Biotechnology xxx (2016) xxx–xxx G Model NBT 960 No. of Pages 4 Please cite this article in press as: K. Voutetakis, et al., Anti-ageing properties of Khelma Longevity TM : treatment of human fibroblasts increases proteasome levels and decreases the levels of oxidized proteins, New Biotechnol. (2017), http://dx.doi.org/10.1016/j.nbt.2017.03.002 Contents lists available at ScienceDirect New Biotechnology journa l homepage: www.e lsevier.com/loca te/nbt