Anti-ageing properties of Khelma Longevity™: treatment of human fibroblasts
increases proteasome levels and decreases the levels of oxidized proteins

Konstantinos Voutetakis1, Vasiliki Delitsikou1, Michel Georges Magouritsas2 & Efstathios S. Gonos1, 3

1: National Hellenic Research Foundation, Athens, Greece

2: Khelma Hellas S.A., Athens, Greece

3: corresponding author; sgonos@eie.gr

Abstract

We have determined the anti-aging properties of Khelma Longevity™ Caps Dietary Supplement, Khelma Hellas S.A. (Khelma Longevity™).To this end human primary fibroblast cultures where treated with a wide range of concentrations of Khelma Longevity™ (0.1 ug/ml, 1 ug/ml, 10 ug/ml and 50 ug/ml) for 1 and 3 consecutive days. Following these treatments, two major and complementary biomarkers of aging where measured, namely, the proteasome and the amount of oxidized proteins. Proteasome is the major cellular proteolytic machinery that degrades, among others, oxidized and other damaged proteins (Nobel Prize in 2004). The proteasome has an impaired function during aging; however, healthy centenarians have a functional proteasome, while proteasome activation by natural compounds delays aging significantly. We have found that 24 hours of treatment with Khelma Longevity™ Caps Dietary Supplement resulted in a maximum increase of 40.96 % of the total protein levels of 20S proteasome. Accordingly, 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 to controls non-treated cells following 3 days of continuous treatment with Khelma Longevity™ Caps Dietary Supplement. These results clearly support the anti-aging properties of Khelma Longevity™ Caps Dietary Supplement.

Introduction

Aging is a natural, inevitable phenomenon with a progressive decline in cellular and organismal homeostasis accompanied by an increased risk of degeneration and mortality over time. The free radical, or oxidative stress, theory of aging proposes that the cellular damage caused by reactive oxygen species (ROS) plays a key role in the aging process and a key determinant of species longevity (Finkel and Holbrook 2000; Harman 1956). ROS are continually generated as by-products of a number of cellular processes and are also produced when the cells are exposed to various external stimuli such as ultraviolet light, ionizing radiation, and environmental cytotoxic substances (Jiang and others 2011). These unstable and reactive derivatives can cause damage to biological macromolecules such as lipids, proteins, and nucleic acids (Kultz 2005). The most common products of protein oxidation are the protein carbonyl derivatives of lysine, arginine, proline, and threonine (Shringarpure and Davies 2002). These derivatives are chemically stable and serve as markers of oxidative stress for most types of ROS. A wide range of age-related diseases such as Alzheimer’s disease, Parkinson’s disease, dementia and diabetes are associated with protein carbonylation (Dalle-Donne and others 2003).

In vitro, aging process was usually represented by an irreversible growth arrest of normal human fibroblasts, where they undergo a limited number of divisions in culture and progressively they reach at a state termed as replicative senescence (Trougakos and others 2006). Several cellular mechanisms malfunction upon senescence with the main secondary anti-oxidant mechanism, namely the proteasome, included (Chondrogianni and Gonos 2005). 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 (Navon and Ciechanover 2009). Once cells are challenged with oxidative stress, the proteasome is activated. It degrades oxidatively and otherwise damaged proteins, thus preventing the formation and accumulation of protein aggregates that in turn have additional deleterious side effects (Farout and Friguet 2006; Friguet and others 2000) . The core particle, namely the 20S proteasome, is a barrel-shaped complex made of 28 subunits, 7 different α- and 7 different β- subunits, arranged as α1-7 β1-7 β1-7 α1-7 structure (Demartino and Gillette 2007). 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 (Glickman and Ciechanover 2002). From in vitro studies it is evident that the 20S proteasome complex mainly recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP is not very effective (Davies 2001; Grune and others 1997).

Many studies have already shown that proteasome function and cellular senescence are inextricably linked biological processes (Chondrogianni and Gonos 2005; Hohn and others 2013) and when the proteasome is partially inhibited in young fibroblasts cultures, an irreversible senescence-like phenotype is triggered (Chondrogianni and Gonos 2004; Chondrogianni and others 2008). On the contrary, restoration of normal level of proteasome subunits reduces aging markers in fibroblasts from elderly persons (Hwang and others 2007) while proteasome activation by genetic manipulation (Chondrogianni and others 2005; Hwang and others 2007; Liu and others 2007) or by natural compounds (Katsiki and others 2007), may delay the senescence process by ~15%.

In the last decade, there has been much interest in the potential health benefits of dietary natural anti-oxidants (Fusco and others 2007; Pandey and Rizvi 2009). Plant polyphenols (Pandey and Rizvi 2009), L-carnosine (McFarland and Holliday 1994; Shao and others 2004), Vitamin E (Packer and Smith 1974), are few examples of natural compounds that have been shown to exert a pro-longevity effect on human primary cultures. Given these findings, we sought to investigate the anti-aging properties of a potent, natural anti-oxidant supplement on human primary fibroblast cultures evaluating the 20S proteasome content and the amount of oxidized proteins.

Results

Khelma Longevity™ promotes increased formation of 20S proteasome 

Initially, we aimed to reveal potential anti-ageing properties of Khelma Longevity™, investigating changes in a major biomarker of aging, the proteasome. Therefore, middle-aged HFL-1 cells were continuously treated with 0.1 ug/ml, 1 ug/ml, 10 ug/ml and 50 ug/ml Khelma Longevity™ or the diluents (0.1% DMSO) for 24 h and 75 h and their 20S proteasome content was measured. As shown in Fig.1, the total protein levels of 20S proteasome increased in a maximum level of 1.41 folds (40.96 %) and 1.25 folds (24.86%) after 24 h and 72 h, respectively, in the presence of 0.1 ug/ml Khelma Longevity™ as compared to the control cultures. Regarding the 1 ug/ml and 10 ug/ml Khelma Longevity™ concentrations promoted also 20S Proteasome increase with a rate gradually decreased, independently of treatment time. Neutralization of this phenomenon was observed in the concentration of 50 ug/ml Khelma Longevity™ after 24 h and 75 h of treatment.

Khelma Longevity™ decreases the content of protein carbonyls

As the proteasome is the major cellular non-lysosomal threonine protease, implicated in the removal of normal as well as oxidized proteins, we addressed the question whether and to what extent the Khelma Longevity™ ingredients reduce the oxidative load in HFL-1 cells in accordance with proteasome increase. Therefore, middle-aged HFL-1 cells were continuously treated with 0.1 ug/ml, 1 ug/ml, 10 ug/ml and 50 ug/ml Khelma Longevity™ or the diluents (0.1% DMSO) for 24 h and 75 h and protein carbonyls were measured. As shown in Fig. 2, the protein carbonyls were significantly decreased by 28.3% and 26.4% in comparison to controls not-treated cells, after 24 h treatment with 0.1 ug/ml and 1 ug/ml Khelma Longevity™, respectively. In higher concentrations of supplement the content of protein carbonyls gradually returned to control levels. On the other hand, the levels of protein carbonyls in HFL-1 cells were dramatically decreased after 72 h treatment with Khelma Longevity™ at almost all the concentrations studied. At the concentration of 50 ug/ml Khelma Longevity™ a decrease by almost 6.5-fold in protein carbonyls was observed.

Discussion

In this short study, we have identified Khelma Longevity™ as a potent proteasome activator with anti-oxidant properties on middle-aged human fibroblasts. Although the anti-oxidant ingredients of Khelma Longevity™ are already known, this is the first demonstration of its anti-ageing effects in in vitro studies that link it with the proteasome and its increase content. Khelma Longevity™ Caps Dietary Supplement contains a variety of flavonoids (or bioflavonoids) such as polyphenols, punicosides and proanthocyanidins which are a group of anti-oxidants with a wide consumption worldwide as remedies for anti-oxidant therapy. Flavonoids constitute a class of naturally occurring polyphenolic compounds, ubiquitously present in photosynthesizing cells (Saito 1974). The best-described property of almost every group of flavonoids is their capacity to act as anti-oxidants because of their scavenging activity (Cazarolli and others 2008; Croft 1998; Pietta 2000). Furthermore, flavonoids are used as nutraceutical ingredients with a wide range of  beneficial properties including anti-inflammatory (Cazarolli and others 2008; Mansuri and others 2014; Yamamoto and Gaynor 2001), antimicrobial (Cushnie and Lamb 2011; Manner and others 2013) as well as anti-cancer (Cazarolli and others 2008; Ravishankar and others 2013) actions. All this spectrum of activities may justify their consumption as therapeutics to several physiological disorders such as cardiovascular diseases (Hu and others 2014; Manach and others 2005) and cancer (Ravishankar and others 2013).

Our in vitro results show for the first time the direct anti-ageing effects of an anti-oxidant supplement on human cells. Our preliminary in vivo studies that include administration of Khelma Longevity™ to human volunteers exhibit positive results as regards to endurance, blood pressure, cardiovascular function but a larger population study is needed for more solid conclusions (data not shown).

Given that we have already shown that the proteasome can be activated through natural compounds (Chondrogianni and others 2010; Katsiki and others 2007) fortifying the cells against oxidative stress and giving them an extended lifespan, we have examined whether Khelma Longevity™ as a potent anti-oxidant supplement may stimulate the proteasome function. We show that 20S proteasome content is induced upon Khelma Longevity™ treatment in HFL-1 human primary cells, accompanied by a deleterious effect of proteasome on protein carbonyl levels. This proteasome action seems to be diminished for Khelma Longevity™ concentrations greater than 1ug/ml under 24 h treatment while the increase of 20S proteasome content at concentrations above 0.1ug/ml for longer treatment did not appear to affect the protein carbonyl levels in a dominant manner even if there was a maximum decrease of protein carbonyls up to 84.7% in comparison to controls non-treated cells. This may be due to the synergistic activation of more than one anti-oxidant mechanism in the cell under longer treatment because of the strong antioxidant effect of Khelma Longevity™ supplement.

Acknowledgments

The described work was supported by Khelma Hellas S.A. to ESG.

Original Scientific Paper