11 March 2025
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1 Department of Health Sciences, School of Life and Health Sciences, University of Nicosia, 2417 Nicosia, Cyprus
2 Bioactive Molecules Research Center, School of Life and Health Sciences, University of Nicosia, 2417 Nicosia, Cyprus
* Author to whom correspondence should be addressed.
Objective: Dermo-cosmetics have significantly advanced, focusing on innovative and effective products such as cosmeceuticals—cosmetics infused with bioactive ingredients for skin benefits. Synthetic peptides are prominent among these bioactive molecules, noted for their enhanced effects in cellular processes related to skin physiology. Specifically, the glycoprotein E-cadherin plays a crucial role in cellular adhesion and has shown promise in wound healing studies, although its broader cellular functions remain underexplored. Despite their widespread use, many cosmetic peptides lack genetic validation of their effects. This study focuses on the synthetic, amphiphilic acetyl hexapeptide-1, aimed to possess wound healing and anti-aging properties, with a novel exploration of its molecular mechanisms, specifically its effect on the expression of the CDH-1 gene, which encodes E-cadherin—a key protein in cellular adhesion and wound healing. Methods: In this investigation, the acetyl hexapeptide-1 was synthesized in house, followed by cell culture assessment and molecular evaluation. Human hepatocytes HepG2 were exposed to the synthetic hexapeptide to assess cytotoxic effects and examine its impact on gene expression, specifically targeting the wound healing-associated gene CDH-1, as well as apoptosis-related genes BAX, Bcl-2, Caspase-9, and Cyclin D1. Results: No cytotoxic effects were observed in cell cultures. Gene expression analysis revealed a significant increase in E-cadherin expression, along with the NO modulation of apoptosis-related genes (BAX, Bcl-2, Caspase-9) and the cell cycle-related gene Cyclin D1. These findings suggest peptide’s role in enhancing cellular adhesion, without any cytotoxic effects. Conclusions: The findings of this study provide promising insights into the potential molecular properties of synthetic acetyl hexapeptide-1, implying its applicability in cosmeceuticals. These cosmetic peptides hold enormous potential and diverse applications not only within skincare. To fully understand their benefits and expand their scope, additional investigations are warranted to comprehensively explore their molecular mechanisms across a spectrum of applications.
The skin, the human body’s largest organ, comprises three main layers: the epidermis, dermis, and hypodermis. The outermost layer, the epidermis, directly interfaces with harmful elements in the environment. The dermis serves as the supporting and protective shield of the epidermis, is rich in extracellular matrix, and has high vascularity and mechanical receptors that contribute to the strength, nutrient supply, and immunity of the skin [1].
The skin functions as a physiological barrier, and its main role in the body is protection from various external factors, such as UV radiation and pathogenic microorganisms that can penetrate through the skin, while it is also an important factor of homeostasis, controlling the loss of water in the body through evaporation. The dysfunction of these mechanisms, which occur naturally over time due to a reduction in the integrity and thickness of the layers, leads to skin aging [2,3]. Also, its protective properties also vary in terms of protecting the internal organs and thus the survival of the organism. As an external organ, it is quite often subject to injury, and, therefore, its healing property is essential for survival [4].
Skin healing and repair is one of the most complex processes of the human body, involving multiple steps and the parallel action of various cell types from all layers [5]. The first response to an injury is the blockage of open blood vessels and the activation of platelets to form a fibrous clot to stop the bleeding and for the accumulation of anti-inflammatory cells at the site of the injury. Once the inflammation is dealt with, the process of angiogenesis follows, which involves the growth of endothelial cells, their metastasis, and the formation of branches so that new vessels can form. At the same time, the fibroblasts at the lesion site proliferate to form a clot and eventually a contractile fibrous tissue, while also expressing extracellular matrix (ECM) proteins, contributing to the growth phase [6]. Still, re-epithelialization, i.e., repair of the skin epithelium and thus the regeneration of skin appendages, is observed [7].
ECM proteins are a family of proteins, composed of a set of nuclear proteins to form the final ECM, where they play a catalytic role in providing the required structural support for cells and tissues. Some of these are collagen and elastin [8]. Specifically in the proliferation phase, the deposition of ECM by fibroblasts begins, forming the so-called temporary matrix, due to their migration to the site of injury. The temporary matrix allows cell separation and metastasis of the cells, to be eventually replaced by granular tissue, which is rich in fibronectin and provides the basis for the coating of collagen molecules, which is the main component of the ECM complex and is what will eventually form the wound scar. Over the years, there is deterioration and aging of this protein complex, which is directly related to the inability of adult skin to heal [9].
As the mechanisms of skin healing and the ability of cells to anchor together are analyzed, it is important to mention E-cadherin, a glycoprotein encoded by CDH-1 gene, which is found at elevated levels in regions where cell-to-cell contact occurs, the so-called adhesion junctions. These tight junctions of cells are essential for the formation of structured tissues and even allow the transmission and communication of cell membrane molecules and intracellular pathways. This glycoprotein has been shown to have a role in cell division, tissue development, organogenesis, and epithelial maintenance and to regulate other key cellular processes such as metastasis, proliferation, apoptosis, and cell differentiation, while its deficiency has been associated with malignancy and tumor development [10]. Several studies have been conducted on the expression of E-cadherin in cases of injury and how it seems to contribute to the healing process. However, its key role in healing processes such as mitosis, maturation, metastasis, and cell recruitment is undisputed [11,12].
Cosmeceuticals are hybrid formulations between cosmetics and pharmaceuticals that contain bioactive ingredients to achieve a more pronounced anti-aging, anti-wrinkle, and moisturizing effect or could even be classified as cosmetic products that are expected to exhibit a therapeutic effect and benefit the skin with continued use [2]. Despite the rapid increase in their use over the last four decades, they are not considered drugs and are not officially recognized by the U.S Food and Drug Administration (FDA) or the European Union. Their increased use is based on the theoretical benefits they provide, which, however, have only been confirmed by in vitro studies of the active ingredients. Dermo-cosmetics are now a separate subcategory between cosmetics and pharmaceuticals, and, in Europe and Japan, they fall under the subcategory of cosmetics, while, in the US, they are considered part of the pharmaceutical subcategory and are provided as over-the-counter (OTC) products [13].
A plethora of peptides have been utilized in cosmetic products due to the increased market demand for innovative and effective products that delay skin aging [14]. Initially, the incorporation of peptides in cosmetic formulations was only aimed at delivering larger molecules and thus increasing their permeability. A typical example is the synthesis of the copper tripeptide GHK-Cu, which had the potential to transport many copper molecules through the skin in order to increase skin healing and also to have an anti-inflammatory and anti-aging effect on the skin [15,16]. The scientific community has focused over the past decades on the synthesis, evaluation, and use of these peptides due to the multiple functions they exhibit such as the regulation of homeostasis molecules, growth, and reproduction, as well as antioxidant, anti-inflammatory, and anti-microbial activity [17]. In addition, factors such as their high safety in use, their hypoallergenicity, and the low-cost development of these molecules have been important parameters for their increased use, as have their proven properties through in vitro and in vivo studies, the main ones being anti-aging, moisturizing and healing, and collagen regulation [15,18].
Earlier reports emphasize that the size of cosmetic peptides must be less than 500 Da to be able to penetrate the skin, the lipophilicity coefficient must be between 1 and 3, and their melting point must be below 200 °C. Still, their aqueous solubility had to be >1 mg/mL, and their structure had to have little to no polar center. However, more recent studies have refuted the belief that only small peptides cross the skin barrier by modifying the amino acid side chains during peptide synthesis, resulting in improved characteristics such as transdermal permeability, binding to appropriate receptors, stability, and solubility [19]. Despite plenty of evidence of their beneficial effect by their addition to the formulation of cosmetic products, there are few reports on the conclusions from their use in cosmetic products. Indeed, there is a particular demand from consumers for validated studies on the safety and efficacy of products of natural and organic origin [20].
Acetyl hexapeptide is a synthetic peptide with the following amino acid sequence Ac-Ala-Arg-His-Leu-Leu-Phe-Trp-NH 2 ([Figure 1](https://www.frankenthalerfoundation.org and it is exploited in skin creams and treatments as an ingredient that can enhance the anti-wrinkle action of other substances, as well as smooth wrinkles and muscles.
Figure 1. Structure of synthetic acetyl hexapeptide-1 (Ac-ARHLFW-NH 2).
A few studies have been conducted to confirm its properties, where differences were observed between the groups under study and the control group in terms of wrinkles, hair follicles, and skin hydration rate [21]. Another study conducted in 2023 by Ying Ye et al. examined a dermo-cosmetic product with retinol as a key ingredient. The results showed improvement in the photo-aging phenomena of women, reduction in wrinkle formation, even a reduction in pores, increase in skin elasticity, and smoothness and glow were observed [22]. Still, the effect of acetyl hexapeptide and palmitoyl pentapeptide on the formation of wrinkles and fine lines was examined and showed an improvement in wrinkle formation by 10% [23].
The above clinical studies seem to demonstrate the action of synthetic hexapeptide as an anti-wrinkle substance; however, the common thread in these studies is the lack of evaluation of its effect in genetic expression, i.e., how the peptide acts at the genetic level and whether it affects the expression of genes that help in anti-wrinkle action and wound healing. In fact, most peptides used in dermo-cosmetic formulations, although they have been tested in some clinical studies for altering skin physiology, are lacking in studies that genetically prove their activity. This fact should stimulate the scientific community for further studies so that new peptides can still be exploited in similar products [18]. The aim of the present study is to synthesize, characterize, and analyze the synthetic acetyl hexapeptide-1, followed by the evaluation of its effect on the growth rate of epithelial cells and determination of the safety of usage on a concentration of 0.6 mg/mL. Finally, this study investigates the effect of the synthetic acetyl hexapeptide-1 on the expression of the CDH-1 gene, which encodes E-cadherin—a protein integral to cellular adhesion, wound healing, and epithelial maintenance. By linking the peptide to CDH-1 expression, this research aims to elucidate the molecular basis of its purported healing and anti-wrinkle effects.
For peptide synthesis, microwave-assisted solid-phase peptide synthesis (SPPS) was performed using a Biotage Initiator+ SP Wave peptide synthesizer (Biotage AB, Uppsala, Sweden). The PS-PEG 2000 Fmoc Rink Amide resin (Agilent Technologies, Shropshire, UK), with a loading capacity of 0.46 mmol/g, was used as the solid support. Fmoc-protected amino acids (CBL Patras, Patras, Greece), Oxyma Pure (Merck KGaA, Darmstadt, Germany), and diisopropylcarbodiimide (DIC) (TCI Europe, Zwijndrecht, Belgium) were utilized as coupling reagents. Dichloromethane (DCM) (Merck, Germany), trifluoroacetic acid (TFA) (Merck, Germany), and acetic acid (Merck, Germany) were used for peptide cleavage and purification. The final lyophilization of the synthesized peptide was conducted using a Zirbus Vaco2 freeze dryer (Zirbus Technology GmbH, Bad Grund, Germany).
For peptide characterization, high-performance liquid chromatography (HPLC) was performed using an Alliance HPLC e2695 system (Waters Corporation, Milford, MA, USA), equipped with a PDA detector (2998, Waters, USA) controlled via Empower 3.0 software. The molecular mass of the peptide was confirmed using a QDa Aquity Mass Spectrometer (Waters, USA). Dynamic Light Scattering (DLS) analysis was conducted using a Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) to determine particle size distribution and stability.
For the cell culture experiments, HepG2 human hepatocellular carcinoma cells were kindly provided by Dr. Lefteris Zacharia, Associate Professor of Pharmacology at the University of Nicosia. All culture media and reagents were obtained from established suppliers. Dulbecco’s Modified Eagle Medium (DMEM) and Phosphate-Buffered Saline (PBS, pH 7.2) were sourced from GIBCO Laboratories (Thermo Fisher Scientific, Waltham, MA, USA). The cell culture medium was supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 1% Antibiotic-Antimycotic Solution, containing 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin B, all supplied by Sigma-Aldrich (St. Louis, MO, USA). For cell viability assessment, 0.05% Trypsin-EDTA solution (Gibco, Thermo Fisher Scientific, USA) was used for cell detachment, and Tr
