The study of cellular cytotoxicity of argireline® — an anti-aging peptide*

Argireline® is well known, innovative anti-aging product used in the cosmetic market. This short chain peptide is used as active ingredient in dermal ointments and creams. Argireline® prevents formation of skin lines and wrinkles in a very similar way to the botulinum toxin (Botox), inhibiting neurotransmitter release at the neuromuscular junction. Argireline® does not require under skin muscle injections and it is believed to be relatively safe. However, despite the fact that some toxicity data has been provided by the product manufacturer, there is an evident lack of reliable information about cyto- toxicity of argireline® in the literature. The aim of the presented study was to estimate the antiproliferation effect of argireline® solution in several concentrations. The influence of argireline® on cellular proliferation was examined against: human embryonic kidney HEK-293 cell line, human neuroblastoma IMR-32 cell line, and human primary skin fibroblasts. Tests were performed using formazan-based cell proliferation assay: EZ4U, which allows to measure the efficiency of mitochondrial oxidative activity in living cells. The argireline® inhibi- tory concentration, IC 50 values were calculated and the results were compared to the IC 50 value of the reference compound: doxorubicin. In conclusion, the considered method resulted in dose-dependent argireline® antiproliferation effects. However, the significant cytotoxicity of argireline® solution was observed under 18 to 10 000 fold higher concentrations (depending on cells that were examined) in comparison to doxorubicin.


INTRODUCTION
Over the last years cosmetic industry has been extensively developed towards biologically active products with drug-like effects. Cosmetic applied topically, such as creams, lotions and ointments can contain small biological active chemical compounds like: carbohydrates, lipids, antioxidants, peptides, and proteins (Schroeder, 2012). They are used in anti-solar, anti-aging, anti-wrinkle and firming products. Those active compounds help to improve appearance of aging skin by bettering its hydration, firmness and texture (Lupo et al., 2007). Peptides are being incorporated in cosmetic formulas thanks to their wide biological effects. They play important role in aging, inflammation, cell proliferation and migration, angiogenesis, protein synthesis and regulation, pigmentation, and in extracellular matrix synthesis (Zhang et al., 2009). Cosmeceutical peptides are believed to be relatively safe, but the information about their cytotoxicity is still insufficient. Therefore the aim of presented study was to estimate the anti-proliferation effect of argireline ® 0.05% solution, as an example of peptide used in cosmetic market.
Argireline ® acts in similar way as botulinum neurotoxins (Botox ® , Allergan, Irvine, CA) causing muscle paralysis by inhibiting activity in the presynaptic neuronal exocytosis machinery (Fields et al., 2008). Active peptide inhibits skin muscle contraction, especially in the forehead, neck, nasal flare and around the eyes (Gorouhi et al., 2009). Argireline ® is blocking Ca 2+ dependent neurotransmitter release (acetylcholine) at neuromuscular junction. This peptide is reacting with SNARE complex, consisting of three synaptic proteins SNAP-25, VAMP and syntaxin. Argireline ® competes * e-mail: grosickim@gmail.com *Presented at 5th European Congress of Life Sciences, Eurobiotech 2013. Kraków, Poland Abbreviations: DX, doxorubicin; EZ4U, easy for you; HEK-293, human embryonic kidney; HSF, human skin fibroblast with the SNAP-25 protein in the SNARE complex, essential for neuronal exocytosis, (Chen et al., 2001). Due this complex is destabilized, preventing catecholamine and acetylcholine vesicle fusion with plasma membrane (Fields et al., 2008). This peptide can significantly limit neurosecreation at micromolar concentration (Blanes-Mira at al., 2002). The effectiveness of argireline ® has been confirmed in several anti-wrinkle tests performed in vitro using chromaffin cells and in vivo on healthy volunteers. A clinical study published in the International Journal of Cosmetic Science reported that acetyl hexapeptide-3 at a 10% concentration in O/W emulsion reduces the depth of wrinkles up to 30% after 30 days of use (Blanes-Mira at al., 2002). Although the anti-wrinkle effect of argireline ® is now commonly known and well described in the literature, the cytotoxicity data of this compound was only found in commercial data sheets*. According to those data no signs of cytotoxicity were observed on human dermal fibroblasts and human epidermal keratinocytes, when argireline was administrated in concentrations between 10 μg/ml and 1 mg/ml. Also no genotoxicity was observed testing argireline ® in concentrations ranging from 5 to 0.05 mg/plate in Ames test, performed on several S. typhymurium strains: TA97, TA98, TA100, TA 102 and TA 1537. The acute oral toxicity of argireline ® has been also investigated. According to authors this small peptide exhibits its insignificant acute toxicity (2000 mg/kg) as compared with BTX-A (20 ng/kg) (Blanes-Mira at al., 2002). Considering presented data we demonstrated independent study of antiproliferation effect of argireline ® on human embryonic kidney HEK-293 cell line, human neuroblastoma IMR-32 cell line, and human primary skin fibroblasts. Experiments have been performed using formazan-based antiproliferation assay: EZ4U, which allows to measure the efficiency of mitochondrial oxidative activity in living cells. This test takes advantage of enzymatic bioreduction of tetrazolium salt into formazan product which can be quantified using spectrofotometric methods (Malich et al., 1997;Krzysztoń-Russjan et al., 2009).

MATERIALS AND METHODS:
Reagents. Argireline ® (MW=889,1g/mol) was kindly provided by Przedsiębiorstwo Produkcyjno-Handlowe Ryszard Kaczmarek i Synowie Sp. z o. o., Spółka Komandytowa, the official distributor of Lipotec S.A. prod-ucts in Poland. Argireline was in form of 0.05% water solution. Concentration of pure active peptide in solution has been estimated around 0.56 mM, according to the information provided by manufacturer. Doxorubicin hydrochloride (2 mg/ml) was from Ebewe Pharma Poland. EZ4U Non-radioactive cell proliferation and cytotoxicity assay, was from Biomedica. For cell culture Dulbecco's Modified Eagle's Medium -DMEM (Gibco) was used, supplemented with fetal bovine serum (Gibco) and streptomycin/penicillin (Polfa Tarchomin).
Human skin fibroblasts (HSF) were kindly provided by Prof. Justyna Drukała (Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University). Human fibroblasts were isolated from skin graft, obtained from 38 years old male patient.
Cell culture. HEK-293 and IMR-32 cell lines, as well as human dermal fibroblasts were cultured in Dulbecco's Modified Eagle's complete growth Medium (DMEM); media contained 10% fetal bovine serum (FBS), 100 mg/mL streptomycin and 100 U/mL penicillin. Cells were cultured at 37°C in an atmosphere containing 5% CO 2 .
In vitro proliferation assay using EZ4U. Cells were seeded in 96-well plates at a concentration of 2 × 10 4 cells/well (IMR-32), 1.5 × 10 4 cells/well (HEK-293) and 1 × 10 4 cells/well (HSF) in 200 μl culture medium and incubated for 24 h at 37°C and 5% CO 2 to reach 60% confluence. Next, argireline ® was added into microplates at the final concentrations 0.01 μM-100 μM. The cells were incubated with argireline ® for 48 h at 37°C and 5% CO 2 . Then, 20 μl of EZ4U labeling mixture was added and the cells were incubated for 5 h under the same conditions. The absorbance of the samples was measured using a microplate reader (PerkinElmer) at 492 nm. The activity of the standard drug doxorubicin (DX) was estimated in the same way by using EZ4U at the concentrations 0.001-50 μM (against IMR-32) and at 0.005-100 μM (against HEK-293) and at 0.005-50 μM (against HSF). All experiments were conducted in four repetitions. GraphPad Prism 5.01 software was used to calculate the experimental IC 50 values.

RESULTS AND DISCUSSION
Cytotoxicity tests, including formazan-based in vitro proliferation assays, are widely used in drug discovery research for testing safety of therapeutic agents before they are progressed to the clinic. The acceptable toxicity as well as the other parameters such as: absorption, distribution, metabolism, elimination, describe the ability of the compound to be an ideal drug candidate (Kerns et al., 2008). Additionally, in-vitro toxicity testing is a reliable alternative to the animals toxicity testing by reduction, replacement and refinement (3Rs) approach (Smith 2001). Argireline ® is the peptide with very high biological activity and therefore should be considered not only as an anti-wrinkle ingredient used in cosmetology but also as a potential toxic agent in case of long-term skin exposure or overdose. That justify the necessity of its safety testing just as any other drug. The incubation of argireline ® solution in different concentrations with human embryonic kidney HEK-293 and neuroblastoma IMR-32 cell lines for 48 h had dose dependent antiproliferative effect (Fig. 2). In lower concentration no difference between argireline ® and the control without active peptide was observed on proliferative activity of the examined cells. However, the significant antiproliferative activity was observed above 10 µM argireline ® concentration. The calculated IC 50 values were 34.862 μM against HEK-293 and 68.458 μM against IMR-32 respectively.
The antiproliferative effect of the commonly used drug in cancer chemotherapy doxorubicin (DX) on HEK-293 and IMR-32 was also examined. This compound is used in anticancer treatment and possesses well examined antiproliferative effects. Therefore, it is commonly used as a standard compound in comparison of cytotoxic properties of unknown substances. The obtained data showed very strong antiproliferative effect of DX against neuroblastoma cell line IMR-32 (IC 50 = 0.0051 μM) and human embryonic kidney HEK-293 cell line (IC 50 = 0.455 μM) (Fig. 3). Comparing the results of antiproliferative effect of argireline ® and doxorubicin (standard compound), it has to be pointed out that the IC 50 value of argireline ® is almost 75 fold higher than IC 50 of DX against HEK-293 cell line and more than 10 000 fold higher against IMR-32 cell line.
Argireline ® is a synthetic anti-aging peptide, applied topically, therefore we also examined its anti-proliferative effect on human skin cells. Argireline ® solution incubated in different concentrations with HSF had a dose-dependent antiproliferative effect on those cells (Fig. 4).
Cellular proliferation was not effected in argireline ® low concentration. The evident antiproliferative effect (67% inhibition) has been observed at 100 µM. The antiproliferative effect of DX against HSF was also examined. The results are shown in Fig. 5.
Contrary to HEK-293 and IMR-32, doxorubicin was less cytotoxic against HSF, effecting these cells above 1 µM (IC 50 = 5.628 µM). In the comparison with other examined cells, the antiproliferative effect of DX against HSF was more than 700 fold lower for IMR-32 (IC 50 = 0.0051 μM) and 8 fold lower for HEK-293 (IC 50 = 0.455 μM). Comparing the results of the antiproliferative effect of argireline ® and doxorubicin against HSF, the significant effect of argireline ® was observed at 100 µM, whereas for doxorubicin at 18 fold lower concentration   5.628 µM. However, argireline ® producer states that no signs of peptide cytotoxicity were observed on human dermal fibroblasts at concentrations between 10 μg/ml and 1 mg/ml*. This was not confirmed in our experiments, where argireline exhibited 67% antiproliferative effect after 48 h of incubation at 100 µM (90 μg/ml ). Unfortunately, the detailed comparison study is impossible due to unpublished data about the experiments which were undertaken by manufacturer. Therefore, further antiproliferation study should be also performed to verify the manufacturer results for human epidermal keratinocytes. In conclusion, this comparative study reveals that argireline ® seems to be a safe compound in compare to the reference compound doxorubicin. Notwithstanding this fact, taking to the account the cytotoxic activity against human skin fibroblasts the safety profile of this product is of concern and the use of argireline ® in very high doses or for a very long period of time must be considered as potentially dangerous for patients.