Preventive cosmetic actives for skin protection

This disruptive condition takes place in response to daily faced common stressors such as UV radiation, extreme weather conditions, pollution and contaminants exposure. However, there are also other stressful situations such as emotional stress, jet-lag, poor sleep quality, unhealthy habits, daily stress, illnesses, etc. Skin is the first protective barrier of our body, which implies that it is constantly challenged by a number of physical and chemical stressors that may impair its function and physiology. If stress conditions are maintained, the harmful effects on skin may cause undesirable long-term effects such as premature ageing leading to fragile, tired, dull skin. Protection response of all organisms to lethal cell aggressions is known as the heat shock response and is characterised by an increase in the synthesis of evolutionary highly conserved proteins: the Heat Shock Proteins (HSP) or stress proteins. Increased expression enables cells to resist damage from further stress exposure. This phenomenon is called tolerance, which is transient and also unspecific for the triggering stimulus, implying that heat shock may induce resistance against other stress conditions.1 Heat Shock Proteins were first reported to be induced by heat shock (temperatures above 42°C) and are believed to represent a mechanism of cellular stress response that protects intracellular proteins from damaging events and subsequent challenges.1,2,3 The HSP group of proteins is a conserved set of molecular chaperones involved in folding and protein transport. HSPs are expressed intracellularly in all cells and organisms from prokaryotes to humans, showing high evolutionary conservation, which implies that HSPs are necessary for survival under hostile environmental conditions.1,5 HSPs are expressed both constitutively (cognate proteins) and under stressful conditions (inducible forms). Consequently, they present both constitutive and inducible chaperone functions (Fig. 1): binding to several proteins and taking part in proteins conformational changes during folding, activation/inactivation, disaggregation, renaturation and intracellular transport. They are also known to directly participate in different aspects of protein maturation, especially in the early stages where the protein is still unfolded. Moreover, they might allow translocation, folding or assembly of new proteins.2,4 These abilities let them take part in normal cell life and proteotoxic stress response, and to protect skin cells from further stress exposure (tolerance). HSPs are classified into families according to their molecular weights. HSP70 family includes two major HSPs that are found both in the cytosol and the nucleus of the cells: Hsp73, which is constitutively expressed in all cells and tissues, and Hsp72 (also named Hsp70), which is the inducible form of HSP70 family. Hsp72 has been reported to help skin cells recover from cellular stress and prevent further damage when exposed to different stressful situations.4 Under stress conditions, Hsp72 expression is greatly enhanced, and despite being HSP70 inducible form, its role in keratinocytes is so important that it also presents basal expression. Hsp72 is expressed throughout all layers of the epidermis including other structures like hair follicles and sweat glands and is considered the major HSP responsible for the protective function in human skin.1,6 However, there is an agerelated reduction in Hsp72 expression after heat stress: protection afforded by Hsp72 induction may be impaired with ageing rendering the cell more vulnerable to environmental attacks. Daily, skin faces challenging situations such as UV exposure, high temperatures, desiccation, cold and pollution and heavy metals exposure. Low humidity levels result in osmotic stress and, consequently, loss of cellular water and denaturation of intracellular proteins. It has been suggested that Hsp72 could take part in protein stabilisation in epidermal keratinocytes after massive water loss.1,7 Stress proteins have recently become of interest in the investigation of ageing molecular mechanisms due to their role in protein refolding, stabilisation and degradation. These proteins are presumed to increase the ability of cells to recover from toxic effects of physiological stress.1,4,6 A boost in Hsp72 skin levels may create a protective shield providing stress tolerance against everyday challenges and stressful situations. A new molecular cosmetic peptide has been developed to confer stress tolerance to the skin. Molecular cosmetics is the result of bringing techniques from the pharmaceutical world into the cosmetic field to create new actives with a welldefined structure, higher purity, better efficacy, lower dosage and complete safety. This methodology allowed finding the best candidate capable of inducing HSP70 synthesis in a keratinocytes cell model. Thermostressine (INCI name: Acetyl Tetrapeptide-22) is a molecular cosmetic peptide identified by a combinatorial chemistry approach from a library of 331,776 tetrapeptides, which was screened by monitoring fluorescence of luciferase-based reporter gene assay to identify potential Hsp72 boosters in a keratinocytes cell model. The luminescence released by cells that express the luciferase-Hsp72 promoter construct when in contact with luciferase substrate was quantified using a luminometer. Activation of Hsp72 promoter was determined from the luciferase activity values and normalised regarding negative controls (non-treated cells). Proteasome inhibitor MG-132 was used as an Hsp72 promoter positive control. Acetyl Tetrapeptide-22 was selected as a human Hsp72 promoter inducer, with potential efficacy in conferring stress tolerance to the skin. After the screening, Acetyl Tetrapeptide- 22 efficacy was validated at translational level by quantification of HSP70 expression in human keratinocytes, proving to boost HSP70 expression and being able to protect skin cells from stressors. Other in vitro tests conducted with the peptide confirmed its protective efficacy against UVB radiation, heat, cold and dehydration of skin cells. By increasing skin Hsp72 levels, the proteotoxic stress that causes cellular damage can be modulated and its side-effects prevented, especially when the skin is repeatedly challenged or when ageing impairs skin stress response.

Materials and methods

Determination of expressed HSP70 in human keratinocytes