Tretinoin And Lycopene As Protectors Against Uv

Published Date: 02 Nov 2017

Abstract

Background: The search for new anti-skin cancer drugs is particularly focused on natural compounds. Tretinoin (acid form of vitamin A) is involved in the control of cell differentiation and proliferation in several tissues, particularly in the skin epithelium. Lycopene (carotenoid with non vitamin A activity) has been proposed to play a critical role on anticarcinogenic action at different levels in both in vitro and in vivo tumour models.

Scope of the review: This review focuses on the effect of UV-radiation on skin, and the photoprotection conferred by two different active molecules: tretinoin and lycopene against photoaging and photocarcinogenesis, respectively. Regarding this context, skin cancer (non- melanoma and melanoma cancer) is addressed as well as other current therapeutic strategies.

Major Conclusions: UV irradiation may induce a number of pathobiological cellular changes, including photoaging and photocarcinogenesis. Tretinoin can be used for photoaging treatment by different mechanisms, including the inhibition of the activated protein-1 and blocking dermal matrix degradation followed by sun exposure. The photoprotective properties of lycopene remain unclear. Some studies point out a positive and others a negative effect both in in vitro and in vivo models. Currently, researchers recognize that crucial gaps endure in our understanding of the role of carotenoids as effective modulators of apoptosis, cell cycle dynamics and/or of their in vivo behavior as cellular antioxidants. On other hand, it remains to confirm if these compounds, supplied through a suitable diet combining different natural antioxidants, contribute to basal protection and improve skin defense against UV light-mediated damage to skin.

General Significance: The development of novel preventive and therapeutic strategies for skin disorders depends on our understanding of the molecular mechanism of UV damage on skin cells. The use of several effective phytocompounds, working through different (preventive and/ or corrective) pathways in the cell, may be an effective approach for reducing UV-B generated damage mediated photoaging and photocarcinogenesis.

Keywords: Photoaging; Photocarcinogenesis; Lycopene; Tretinoin

Introduction

The effects of solar ultraviolet radiation (UVR) on most living organisms and, particularly, on human health are well known. Some of the beneficial and most detrimental effects of UVR on human health have been recently reviewed by Norval et al. (1). In order to cope with the consequences of UVR exposure, both plants and animals possess various protective small molecules and enzymatic defences such as the families of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). If the concentration of free radicals increases significantly and reaches a critical level, the generated oxidative stress can destroy cells and cell compartments and, consequently, origin premature skin aging and also skin cancer (2).

The search for new anti-cancer drugs is particularly focused on natural compounds, such as botanical antioxidants, available in the regular human diet. On one hand these compounds rarely exhibit severe side-effects, on other hand they efficiently regulate a wide range of cellular pathways involved in carcinogenesis (3). Chemically, botanical antioxidants can be polyphenols, phenols, flavonoids, isoflavonoids, phytoalexins, anthocyanidins and carotenoids. They can work in preventive ways (sunscreen and antioxidant effects) and/or in corrective ways (redox regulation of signal transduction pathway) (4, 5). The use of these several effective phytocompounds, working through different pathways in the cell, in both skin care products may be an effective approach for reducing UV-B generated reactive oxygen species (ROS) mediated photoaging and photocarcinogenesis (4).

Carotenoids are protective pigments at the light harvesting complex I and II in plants photosynthetic systems, and are in abundant quantities in several crops (6, 7). Carotenoids defence properties in human cells may involve their potent antioxidant activity (eg., they act as efficient scavengers of singlet species) and their ability to induce cellular protective responses (eg., DNA repair, phase 2 cytoprotective enzymes) (8). In fact, the influence of carotenoids and/or their metabolites on the expression of certain genes and on the inhibition of regulatory enzymes has been discussed in context of cancer preventive properties of these compounds (9).

In this review, a special focus will be given to the two carotenoid related compounds tretinoin and lycopene in the context of their protective effects of photoaging and photocarcinogenesis, respectively.

Tretinoin is a natural-occurring acid of retinol, an active metabolite of vitamin A, and is an agent involved in the control of cell differentiation and proliferations in several tissues, particularly in the skin epithelium (eg., binds to and activates retinoic acid receptors (RAR), inducing changes in gene expression that leads to cell differentiation, decreased cell proliferation, and inhibition of tumourigenesis). It has been demonstrated that photoaging resulting from UV-B radiation can be ameliorated by retinoid treatment. Pretreatment of human skin with tretinoin blocks dermal matrix degradation following sun exposure inhibiting the induction of the activated protein-1 (AP-1) transcription factor and AP-1 regulated matrix-degrading metalloproteinases (MMP).

Tretinoin is largely used in cosmetics, and is commercially available worldwide under several brand names.

Lycopene is a non-provitamin A carotenoid that is responsible for the red to pink colors seen in tomatoes, pink grapefruit, and other foods (10). Lycopene has been recognized as the most effective singlet oxygen quencher among carotenoids (11) and it is currently receiving considerable scientific attention (3). Epidemiological, tissue culture, and animal studies provide convincing evidence supporting the role of lycopene in the prevention of chronic diseases. Human intervention studies are now being conducted to validate epidemiological observations and to understand the mechanisms of action of lycopene in disease prevention (12).

The anticancer activity of lycopene has already been demonstrated in both in vitro and in vivo tumour models (13). The mechanisms underlying the inhibitory effects of lycopene on carcinogenesis could involve ROS scavenging, upregulation of detoxification systems, interference with cell proliferation, induction of gap-junctional communication, inhibition of cell cycle progression and modulation of signal transduction pathways (13). Some of these mechanisms will be discussed in this review within the photocarcinogenesis context.

UV-Radiation and its Effects on Skin

Although both the environment and genetics may play a role in the development of skin cancer, UVR is considered to be one of the most efficient skin carcinogens and mutagens as well as immunosuppressive agent. The UV spectrum is composed by: UV-C (200-280 nm), UV-B (280-320 nm) and UV-A (320-400 nm) (Table 1). The UV-C has the higher energy radiation, but it is (almost) blocked by the ozone layer and the UV-B and UV-A represent about 5 % and 90 % of UVR, respectively. Although there are beneficial effects of UVR on skin, such as the generation of vitamin D and therapy of some skin diseases (atopic eczema, psoriasis), there are also acute and chronic harmful effects especially if the UVR exposure is too prolonged. "Sunburn" or erythema, acanthosis (increase of epidermal thickness), desquamation, immunosuppression (increase of interleukins IL-10 and IL-12) and immediate pigmentation darkening are the main effects of acute exposure. The effects of chronic exposure include the solar elastosis, i.e. the accumulation of elastin in the superficial dermis, degradation of collagen and skin cancer (4, 11).

Through lipid peroxidation, protein cross linking and DNA damage, the UV-A and UV-B radiation can cause photoaging and photocarcinogenesis. UV-A is considered the "aging ray" and UV-B the "burning ray". Whereas the principal site of action of UV-B is the epidermis, UV-A acts mainly in the dermis (14).

Table 1 - Global Solar Spectral Irradiance measured at sea level (15).

Wavelength

(nm)

Domain

Energy (W/m2)

%

280-320

UV-B

 4-2

 6.8

320-400

UV-A

 72-50

400-780

Visible

 580-660

 55.4

780-1400

IR A

 410-430

 37.8

1400-3000

IR B

The minimal erythema dose (MED) is the threshold dose required to cause a perceptible reddening of the skin 24 h after exposure. The estimated MED narrow band-UV-B radiation in north Indian patients varied from 500 to 1100 mJ/cm2  (16). However, MED values differ among individuals and depend on the actual endogenous protection provided by melanin and on the skin type. Melanin levels determine the skin colour and are related to some extent to the skin type, which is often categorized according to the Fitzpatrick scale ranging from type-I to IV. Skin type I is assigned to people with white or freckled skin, green or light –blue eyes, red hair and high sensitivity to sun light; skin type IV shows black skin, dark brown eyes and black hair, almost never experiencing sunburn. The repair of UVR skin damages occurs within 24 h only if the irradiation dose is ≤ 60 % of the MED (17, 18). The sunburn starts to develop few hours after irradiation, culminating about 18-24h post-irradiation. When a cell becomes irreversibly damaged by UVR exposure, cell death follows via apoptotic mechanisms, leading to the appearance of sunburn cells in the epidermis (18, 19).

The skin has natural defenses against the damaging effects of UVR, such as: (1) The SC that absorbs, reflects and scatters UV rays; (2) skin pigmentation: melanin absorbs and scatters UVR, and is a free radical/ROS scavenger; (3) naturally occurring antioxidants such as lipophilic carotenoids, alpha-tocopherol, glutathione and ascorbic acid; (4) antioxidant enzymes such as CAT, SOD and GPx; (5) DNA repair mechanisms and (6) epidermal hyperplasia and hyperkeratosis that increase the physical barrier to UVR (20, 21).

The importance of the skin’s natural defenses against the damaging effects of UVR is illustrated by several clinical syndromes, such as Xeroderma Pigmentosum and Oculocutaneous Albinism (20).

Photoaging and Photocarcinogenesis

Aging is a complex process in which several mechanisms operate simultaneously. These include accumulation of mutations in the genome, accumulation of toxic metabolites, hormonal deprivation, increased formation of ROS, and cross-linking of macromolecules by glycation (22). ‘Photoaging’ or ‘extrinsic aging’ is the process by which sunlight or artificial UVR gradually induces clinical and histological changes in the skin (20). On one hand, UV-A radiation is a main cause for premature skin aging (23) because represents the major percentage of total UVR and, compared to UV-B and UV-C, has a higher depth of penetration into the dermis (24). On the other hand, UV-B-induced DNA damage is an initiator of the events leading to MMP-1 induction, the main enzyme responsible for collagen 1 digestion (25). UVR-induced cutaneous alterations may be characterized by dryness, rough texture, irregular pigmentation (hyper or hypomelanosis), yellowish color, telangiectasia, plaque-like thickening, deep creases and wrinkles. These skin changes may vary considerably among skin types (20, 26).

Histological changes in the skin due to chronic UVR exposure involve the epidermis and the dermis (Table 2). Ultimately UVR-induced damage to the epidermal cells may lead to malignant transformation (20).

Table 2- UVR-induced Histological Changes in Epidermis and Dermis Layers (adapted from (20, 24, 26))

Epidermis

Dermis

epidermal hyperplasia or atrophy

disappearance of dermal papillae

thickening of the basement membrane

↑ and irregular distribution of melanocytes and melanosomes

atypical proliferation of keratinocytes (solar keratosis)

↓ naturally occuring antioxidants (↑ROS)

thickening of Stratum Corneum (SC)

elastosis

presence of deformed collagen fibers

↓collagen

dilated blood vessels

↑ Matrix Metalloproteinases (MMPs)

Solar Elastosis is clinically manifested as yellow discoloration and pebbly surface of the skin and is a prominent feature of photoaged skin. The dermis displays accumulation of elastotic material (degraded elastic fibres and disorganized tropoelastin and fibrillin) in the mid and upper dermis. In addition, glycosaminoglycans and proteoglycans increase in photodamaged skin, whereas the amount of collagen decreases. In UVR-induced collagen degradation is generally incomplete, leading to the accumulation of partially degraded collagen fragments in the dermis, which may interfere with skin integrity. Moreover, the large collagen degradation products inhibit new collagen synthesis. There is also a cellular accumulation of lipofuscin (a highly cross-linked, modified protein aggregate) associated with chronological aging that further inhibits proteasome function (24).

UVR also damages telomeres disproportionately due to their greater proportion of target TT and G bases compared with the chromosome overall. Such damage is postulated to disrupt the telomere loop, expose the TTAGGG overhang, and promote aging. Intrinsic aging is accompanied in most cases by repeated cell divisions that shorten telomeres (24).

Photodamaged skin frequently displays an increased number of hyperplastic fibroblasts as well as increased inflammatory cells. This chronic inflammation in photoaged skin is termed heliodermatitis. In severe photodamaged skin, perivascular veil cells may display dilations (telangiectases). UVR has also been implicated in local and systemic immunosuppression which may have implications in cutaneous tumour surveillance (24, 27). Rijken (20) believes that neutrophils may play an important role in the pathophysiology of photoaging.

The oxidative stress generated by UVR in keratinocytes and fibroblasts can modify proteins forming carbonyl derivatives which accumulate in the papillary dermis of photodamaged skin. In addition to their oxidative effects on lipids and proteins, ROS also attack nucleic acids, in particular nuclear and mitochondrial DNA. The 8- hydroxyl-2´-deoxyguanosine (8-OHdG) is a representative DNA base –modified product generated by ROS and is considered as one of the most important biomarkers of carcinogenesis. It induces G-C  T-A transversions during DNA replication. Normally they are quickly removed from DNA. Therefore, their residual presence in DNA of normal and neoplastic tissues is the result of the imbalance between oxidative attack and DNA repair (28). Upon DNA repair, 8-OHdG is excreted in the urine. According to the European society of pigment research (29), a strong generation of 8-OHdG was related with the proliferation of melanocytes.

Overall, the increase of ROS levels may regulate a cascade of signal transduction, including the involvement of Mitogen-activated protein kinase (MAPK) pathways, and leading to the up-regulation of the activator protein 1 (AP-1) and of the Nuclear Factor (NF-kB), and to the down-regulation of the Transforming Growth Factor (TGF-β). While NF-kB regulation increases interleukin-1 family (IL-1) and Tumour Necrosis Factor (TNF-α) levels, the AP-1 up-regulation activates MMPs. On the other hand, the decrease of TGF-β expression decreases the synthesis of collagen. Ultimately, the combination of these changes lead to the increase of collagen breakdown and elastin production in extracellular matrix and, therefore, to matrix degradation (Fig.1) (30).

Figure%20x%20-%20photoaging

Fig.1- Effects of ROS increase on transcriptional regulation of some important proteins in the skin matrix, involving NF-kB, AP-1 and TGF-β pathways. Adapted from Chen et al. (30)

Photocarcinogenesis is a complex multistage phenomenon involving three distinct stages exemplified by initiation, promotion and progression. Each of these stages involves biochemical and molecular alterations in the cell (Fig.2).

DNA repair

Cell Cycle Arrest

Apoptosis

Fig.2- Brief overview of the main cell mechanisms involved in photocarcinogenesis. Adapted from Matsumura & Ananthaswamy (31).

The apoptosis of keratinocytes plays a critical role in regulating epidermal development and restraining carcinogenesis. The extrinsic pathway (via cytoplasm) is stimulated by UV-B or binding of Fas ligand, tumor necrosis factor, or other cytokines to death receptors that results in the activation of a caspase cascade. The intrinsic pathway (via mitochondria) is also stimulated by UV-B and involves mitochondrial depolarization and higher membrane permeability, leading to the release of multiple proapoptotic factors including cytochrome c, Smac and the apoptosis inducing factor (AIF). Cytochrome c promotes the activation of caspase-9 and cytochrome-c dependent activation is promoted by the mitochondrial protein Smac by eliminating the inhibition via the inhibitor of apoptosis (IAP). On other hand, AIF translocates to the nucleus and mediates caspase-independent apoptosis (31).

The extrinsic and intrinsic pathways are potentially linked by the Bid protein, which is cleaved by caspase-8, and the activated fragment causes mitochondrial content release. Activation of upstream caspases (caspase-8 or -9) leads to the activation of downstream caspases (caspase-3 or -7) resulting in the cleavage of intracellular substrates, cellular condensation and nuclear fragmentation. Apoptosis inhibitors include caspase inhibitors and the big complex Bcl-2 family proteins, most of which prevent mitochondrial membrane permeability. Caspase-14 is a keratinocyte-specific caspase only found in skin. It was demonstrated that caspase-14 was only expressed in HaCaT cells and normal human keratinocytes under culture conditions mimicking terminal differentiation, and could not be induced by death receptors signaling or UV-B irradiation. Although the targets of activated caspase-14 have not been defined yet, these are likely focal epidermal hyperplasia (32).

In general, the process of skin cancer development involves many different cellular mechanisms and events, as synthetically indicated (4, 33-35):

• abnormal stimulation of DNA synthesis and unability to repair DNA damages. A major reason of these abnormal events is the frequent mutation of the suppressor tumour p53 gene, which protects cells from DNA damage through repair mechanisms or apoptosis of damaged cells;

• formation of pyrimidine photodimers in nucleic acids and activation of the ataxia telangiectasia mutated family proteins of keratinocytes;

• deregulation of apoptosis leading to abnormal proliferation of keratinocytes, inflammation, epidermal hyperplasia and immunosuppression (increase of IL-10, IL-12);

• activation of proto-oncogenes (e.g. Ras);

cell cycle deregulation;

• increase of ROS levels and depletion of antioxidant enzymes activity;

• impairment of signal transduction pathways and induction of ornithine decarboxylase (ODC) activity (an important enzyme linked to the development of skin tumors) and of cyclooxygenase-2 (COX-2, an enzyme involved in inflammation process);

• photoisomerization of trans to cis-urocanic acid, which acts as an endogenous sunscreen against UV-B.

Exposure to UVR also causes alterations in the morphology and function of antigene-presenting Langerhans cells (36), release of immunosuppressive cytokines (37), and enhanced prostaglandin synthesis (38). In fact, Langerhans cells and dendritic cells of lymphatic system are the major cellular chromophores absorbing radiation in the UV-B wavelength range. Langerhans cells also contribute to abolish the immune response to antigens applied at the local irradiated site(25).

Excessive UV irradiation leads to an increase in intracellular free calcium in keratinocytes, resulting in the activation of the inflammasome (a multiprotein of the innate immune complex) and in the synthesis and release of IL-1, which can trigger the synthesis of other proinflammatory cytokines (8, 39). IL-10 appears to be a key mediator of UVR-induced immunosupression (8, 40), while IL-12 was found to accelerate the removal of UVR-induced DNA lesions in keratinocytes by inducing nucleotide-excision repair, strongly suggesting that it plays a protective role in photocarcinogenesis (8, 41). In addition, UVR induces the synthesis of granzyme B and perforin in keratinocytes, proteins present only in cytotoxic lymphocytes and natural killer cells, rendering keratinocytes cytotoxic to transformed cells. This behavior suggests that UV-irradiated keratinocytes participate in skin cancer surveillance (24).

Abnormal DNA methylation is a hallmark of most cancers. It is a marker of epigenetic events, i.e. susceptible to change with environmental factors, and a fundamental process that not only modulates gene expression, but also regulates the chromosomal stability. Hyper or hypomethylation in G-C rich regions can contribute to carcinogenesis by silencing tumour suppressor genes (e.g., p53), upregulating oncongenes at dipyrimidine sites and by decreasing genomic stability. The maximum absorption of DNA purine and pyrimidine bases lies between 230 and 300 nm. Therefore, DNA is a major UV-B-absorbing cellular chromophore in the skin The major types of DNA photoproducts induced by carcinogenic UV-B are: cyclobutane pyrimidine dimmers (CPD) and pyrimidine (6-4) photoproducts (33). Repair of photolesions is the primary response to DNA photodamage in surviving cells. Depending upon the primary DNA lesion, one or more repair pathways become active such as direct repair, base excision repair, mismatch repair, double strand break repair, and nucleotide excision repair. The latter is considered to be the major line of defense against carcinogenesis. Phosphorylated p53-induced transcription of p21 causes G/S cell cycle arrest, allowing the cellular repair pathway to remove DNA lesions before DNA synthesis and mitosis (25). However, if the damage persists into the S-phase of the cell cycle, other repair mechanisms might lead to mutagenesis resulting mainly in characteristic cytosine to thymine substitutions. When such mutations occur in the p53 gene, keratinocytes lose their ability to undergo the apoptosis following high dose UVR exposure. The degenerative changes in keratinocytes include mitochondrial swelling and rupture, condensation of the cytoplasm and the appearance of pyknotic nuclei. Macrophages bind and phagocytise apoptotic keratinocytes and their number increase dramatically in the skin after UV-B exposure (5, 8, 33, 42, 43). Chemopreventive agents may be used to correct aberrant methylation patterns and restore growth control in tumour cells and/ or against the adverse effects of solar UVR (43).

Several strategies are applicable for photoprotection and subsequent impairment of molecular and cellular functions. Behavioral changes (avoidance of sun exposure and protective covering) and sunscreens with a high sun protection factor are highly recommended during times of intense exposure. In order to increase the barrier for UVR, the compound should absorb UVR over a broad range of wavelength with high efficacy and sufficient photostability is also required. Topically applied organic (previously called chemical) and inorganic (previously called physical) sunscreens protect by absorbing or reflecting radiation at the skin surface, respectively. Organic sunscreens are usually ‘invisible’ and hence cosmetically appealing. However, the first-generation of organic sunscreens were quite unstable and reactive because of UVR absorption which could lead to the interaction of the suncreen with cutaneous molecules, causing allergic sensitization. Inorganic sunscreens contain 10–100 nm particles such as zinc oxide or titanium, are chemically inert and hence do not cause allergic sensitization (24). Nanoencapsulation of organic UV filters is a more recent approach to improve skin retention, photostability and the UV blocking ability of the free molecules(25).

For the induction of repair systems dealing with UVR-induced damage, compounds which interfere with stress-dependent signaling are demanded. Suppression of cellular and tissue responses like inflammation also require the application of compounds which affect intra- and intercellular pathways (11). Furthermore, DNA damage repair enzymes (enzyme T4 endonuclease V; photolyase; oxoguanine glycosylase 1; thymidine nucleotides); antioxidants and dietary lipids; iron quelators (eg. Kojik acid); osmolytes; retinoids (tretinoin); fluorouracil, imiquimod, lipospondin; chemical peels (eg. α-hydroxy acids), dermabrasion, photodynamic, laser and radiofrequency therapies (44), injectables (eg. botulinum toxin and hyaluronic acid), and surgical procedures have also been proposed as strategies for the prevention and/ or treatment of photoaging (20, 24). Another approach is related to the induction of melanin production without sun exposure. Although the regulation of the tanning response is complex and only partially elucidated, evidence suggests that UVR-induced DNA damage or its repair is at least one of the initial signals that stimulates melanogenesis (24). Gilchrest et al. (45) showed that administration of the repair enzyme T4 endonuclease V to irradiated cultured pigmented S91 cells (melanoma cells) increased both their rate of repair and UVR-induced pigmentation. However, this enzyme was effective only in UV-irradiated cells. These data suggest that tanning can be stimulated through enhanced DNA repair.

Other approaches for increasing skin pigmentation rely on the introduction into the skin of telomere homologue oligonucleotides (that would mimic telomere loop disruption and stimulate DNA damage responses, including melanogenesis); Forskolin, a cell-permeable diterpenoid (that activates adenylate cyclase to induce melanogenesis) and α-melanocyte-stimulating hormones analogues (24). Finally, natural substances extracted from herbs (polyphenols, phenolic acids, flavonoids, carotenoids) can acts as other potential photoprotective resources owing to their UVR absorbing and antioxidant properties (46). However, the photoprotective efficacy of these agents, is not itself comparable to the use of a sunscreen (7).

Skin Cancer

Skin cancer remains one of the most common human cancers and, despite educational efforts, it shows a high incidence which has a tremendous impact on public health and healthcare expenditures (47). Although both the environment and genetic components play a role in the development of skin cancer, UVR is considered to be one of the most efficient skin carcinogenic and mutagenic agents, besides being an effective immunosuppressive agent (48).

The risk of skin cancer is expected to increase as the population ages and larger amounts of UVR reach the surface of the Earth because of depletion of the stratospheric ozone (43, 49). Although keratinocytes are resistant to UVR- induced damage, repeated exposure results in accumulated DNA mutations that can lead to skin malignancies. Therefore, it is desired to develop newer and effective chemopreventive agents and strategies, which can inhibit or slow down the UVR-induced risk of melanoma and non-melanoma skin cancers (Fig.3) especially among high-risk human populations (43). Preventive strategies including personal behavioral modifications and public educational initiatives are considered to be both life-changing and life-saving (50).

Fig.3 shows the fate of the different solar UVR components. The specific DNA lesions produced by UV-A and UV-B in this range of wavelengths include the production of various types of DNA photoproducts (33).

Fig.3- Exposure to UV radiation and development of melanoma and non-melanoma skin cancers. UV-A radiation reaches the dermis and to some extent also the subcutis, whereas UV-B does not pass beyond the epidermal layer.

The importance of epigenetic events is that it represents a mechanism by which gene function is selectively activated or inactivated. Because epigenetic events are susceptible to change, they represent excellent targets to explain how environmental factors, including dietary constituents, supplements, chemopreventive agents may modify cancer risk and tumour behaviour (43, 51).

The formation of skin cancers is a biological process much more complex than narrow scientific aspects that are individually and, sometimes, independently investigated. Recent studies have focused the investigation on the regulation of apoptosis in the skin and its application for understanding skin carcinogenesis (32). Acute UV irradiation causes apoptosis involving p53 and Fas-FasL pathways (FasL, a member of TNF superfamily involved in the elimination of apoptotic cells and prevention of cell transformation). Chronic UV irradiation results in deregulation of apoptosis, leading to the abnormal proliferation of keratinocytes with DNA damage, acquisition of p53 mutations, loss of Fas-FasL interaction besides decreased death receptors, Stat3 activation and variation of Survivin, Bcl-2 and Bcl-XL expression, all of which contribute to the onset of skin cancer (25, 32). Dysfunctional (decreased) apoptosis occurring in skin cancer has been studied and gene silencing by RNA interference may become a new approach to reverse apoptosis resistance (52) as it has been successful in tissue cultures and tumor tissues, as well as, in a mouse model (53).

The current tools for measuring cumulative sun damage are biopsy histology and skin microtopography. However, skin biopsies are too invasive for population studies and skin replicas render only superficial skin architecture data. Reflectance confocal microscopy (RCM) is a noninvasive imaging tool that allows in vivo imaging of the skin (54). The Raman spectroscopic measurements are well suited for the screening of cancer patients prior to chemotherapy, to detect high-risk patients, who can be treated successfully with topically applied antioxidants, thus preventing the occurrence of palmar–plantar erythrodysesthesia (PPE), a dermal side effect of chemotherapy (2).

Non-Melanoma Cancers

The non-melanoma skin cancers (NMSC), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most frequent cutaneous malignancies and represent around 80% and 16% of all skin cancers, respectively, whereas malignant melanomas represent only about 4% of all skin cancers (55). Both BCC and SCC are derived from the basal layer of the epidermis. SCC especially affects people over 40 years of age who sunburn easily without tanning, and people who are immunosuppressed (55). BCC doesn’t metastasize but can be locally invasive and destructive. On the contrary, SCC is an invasive non-melanoma cancer and can metastasize. Dermoscopy improves the diagnosis of NMSC at different stages of progression (55).

The formation of these cancers is based on constitutional and/or inherited factors usually combined with environmental factors, mainly UV-irradiation through long term sun exposure. UVR can randomly induce DNA damage in keratinocytes, but it can also mutate genes essential for control and surveillance in the skin epidermis (56). Various repair and safety mechanisms exist to maintain the integrity of the skin epidermis. For example, and as already reported, UVR damaged DNA may be repaired and, if this is not possible, the DNA damaged cells are eliminated by apoptosis (57). It is currently considered that deregulated cell proliferation and apoptotic pathways lead to the development of cancer. Therefore, it appears that exploiting the apoptotic potential of cancer cells might lead to new therapies that could be less toxic to normal cells due to their physiologically controlled survival pathway.

Mutations in the Hedgehog pathway related genes, especially PTCH (a human homologue of the patched gene in Drosophila melanogaster), are well known to represent the most significant pathogenic event in BCC (25). In general, BCC have reduced expression of Bax and increased expression of Bcl-2, while Bcl-XL is overexpressed in SCC. However, a relatively higher apoptotic rate in BCC may account for the slow growth of clinical lesions. In SCC, expression of Survivin, Bcl-2 and Bcl-XL is associated with metastasis or poor prognosis. Progression of SCC is also associated with constitutive activation of keratinocytes survival signaling pathways (32).

Prevention of SCC is directly achievable through sunscreen protection (58) being dietary factors and nonsteroidal anti-inflammatory drugs potential second-line preventions (59). By targeting different pathways identified as important in the pathogenesis of NMSC, an approach combining multiple agents (with different targets in the cell) or the addition of chemopreventative agents to topical sunscreens may offer the potential for novel and synergistic therapies in preventing and/ or treating NMSC (60). Recently published data showed the failure of topical tretinoin in chemoprevention of NMSC (61). Photodynamic therapy (PDT) with topic 5- aminolaevulinic acid was used in NMSC therapy as a non-invasive therapy with possibility of treatment for multiple lesions in only one session (62). However, the recurrence rate increase after drug-PDT diode laser single session can be observed in a long-term follow-up, and the repetitive sessions is strongly recommended (62). Other potential therapies could include the introduction of p53, bortezomib (proteasome inhibitor) and stat3 decoy (32).

Melanoma Cancers

Cutaneous malignant melanoma arises through the interplay of both environmental and genetic factors and is highly invasive. This type of skin cancer is capable of metastasizing to distant sites. Melanoma is thought to develop starting with a benign nevus (63). The propensity to develop nevi is genetically determined. For the skin with low propensity, repeated and cumulative exposure to the sun is required for melanoma development. However, in people with high nevus propensity, even short exposure to the sun could lead to melanoma development (64). The role attributed to the intermittent sun exposure in the genesis of most melanoma and BCC in the case of sunbeds has been also discussed (65). Patients with advanced melanoma with dissemination to distant sites and visceral organs have a very poor prognosis. PDT for patients with skin metastases from melanoma only works in 20 −30 % of these patients (66). Melanoma research on the mechanisms by which UVR initiates this cancer are needed to improve prevention strategies and have been investigated in mouse models (67). Eggermont & Robert (68) reviewed the later 40 years of lack of progress on melanoma treatment. However, in the last decade, great advances have been made, both in terms of targeted drugs that kill melanoma cells and in terms of host immune system modulating drugs, which use however still remains under discussion (69, 70).

A new development in 2011 in melanoma adjuvant therapy resulted with interferon IFN-alpha2b on the basis of the ECOG 1697 trial which found no difference in recurrence-free or overall survival (71). In March 2011, pegylated IFN-alpha2b (PegIntron®) was approved by the Food & Drug Administration (FDA) for the treatment of stage III melanoma based on the result of the EORTC 18991 trial, which found that PegIntron® induced a significant increase in recurrence-free survival in patients with stage III melanoma, with lymph-node-positive melanoma (72, 73). Two other adjuvant therapy trials, the EORTC 18071 and DERMA trial are completed. The importance of ulceration and IFN sensitivity in adjuvant trials have led to the EORTC 18081 trial, in course, in patients with ulcerated primary melanomas to prospectively investigate IFN sensitivity (68). A current trial, ECOG 1609, is comparing treatment with Yervoy™ (Ipilimumab) against standard high-dose IFN-2b. Ipilimumab, anti-Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) monoclonal antibodies treatment induces low response rates but durable responses (74). Vemurafenib (Zelboraf® from Roche) was the first FDA approved treatment for the B-Raf proto-oncogene (BRAF) mutation-positive metastatic melanoma and it has been associated with high response rates in up to 70 % of patients (55). The combination of the BRAF inhibitors with MAPK/ERK kinase protein inhibitors have fewer side effects and induced higher response rates. However, resistance to the drugs was developed and relatively short periods of response were achieved (55).

In conclusion, new drugs and probable combinations are under investigation and substantial improvement in survival from melanoma is expected from the trials in course.

Photodamage Skin Care Agents

Tretinoin

Physicochemical Properties

Tretinoin, also known as all-trans-retinoic acid (ATRA), is a naturally occurring derivative of vitamin A (retinol). The 13-cis isomer is called isotretinoin. Tretinoin belongs to the first generation of non-aromatic retinoids. The physico-chemical properties of tretinoin are represented in Table 3. There are two sources of dietary vitamin A. Active forms, retinaldehyde and retinol, are obtained from animal products. Precursors or provitamins, which are converted to active forms by the body, are obtained from fruits and vegetables containing yellow, orange and dark green pigments, known as carotenoids (75).

Table 3- Physicochemical properties of Tretinoin (75-78).

Molecule

Tretinoin

Molecular Structure

Tretinoin chemical structure

IUPAC Name

(2E,4Z,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenoic acid

MF

C20H28O2

MW (g/mol)

300,44

Physical state

yellow to light-orange crystalline powder

Solubility

Insoluble in water (20 °C); Soluble in chloroform; slightly soluble in ethanol

Log P

 4-5

Melting Point (ï‚°C)

180 – 181

Stability

Stable only under ordinary conditions. Sensitive to air / light and heat, especially in solution

Bioavailability and Metabolism

The human cells convert precursors to retinol, and then the de-esterified alcohol can be converted to metabolically active retinal (the aldehyde of retinol) and retinoic acid (the oxidized form of retinol) formed by carboxylation of retinal. The steps of metabolism, especially the inactivation of the retinoids, are not yet fully understood (77).

When applied on skin, all forms of trans-retinoic acid isomerize partly on the epidermis in 9-cis and 13-cis-retinoic acid, among other metabolites. Approximately 80 % of retinoic acid remains on the skin surface, and it penetrates depending on the type of vehicle formulation (79).

Safety

The collateral effects of topical administration of tretinoin include redness, desquamation, dryness, and burning tingle (80). The transdermal penetration and systemic bioavailability of topical retinoids are not yet completely clarified. Despite its short half-life, some authors defend that overexposure to tretinoin may cause teratogenesis and embryotoxicity (81, 82). However, there is not a consensual opinion about the use of topical retinoids during the pregnancy (83).

Biological Activity and Pharmacological Action

The retinoic acid receptors are α, β and γ -RAR (related orphan receptor) and retinoid X receptor (RXR), and the cytosolic skin binding proteins are the CRABP (cellular retinoic acid binding proteins). This connection promotes various action mechanisms, such as normalization of proliferation and differentiation of the epidermis, decrease of prostaglandins, leukotrienes and cytokeratins release and inhibition of neutrophil chemotaxis (84). An alternative pathway may be via aquaporin type 3 present in keratinocytes, facilitating the movement of water and glycerol through the skin (79, 85).

Retinoids, including tretinoin, are important regulators of cell proliferation and differentiation, and are mainly used to treat acne and photodamaged skin and to manage keratinization disorders such as ichthyosis and keratosis follicularis.

Retinoids have also been used as chemopreventive and anticancer agents because of their pleiotropic regulator function in cell differentiation, growth, proliferation, DNA repair and apoptosis. Although oral retinoids have not been effective for chemoprevention of skin cancer in the general population (86), the use of topical retinoids as chemopreventive agents has yielded variable results, some positive (87) others not (51, 60, 61, 88). However, according to Shimizu et al. (89) ATRA can suppress the process of carcinogenesis both in vitro and in vivo, especially in the treatment of various cancers, including SCC. Tretinoin represents the class of anticancer drugs called differentiating agents and is used in the treatment of acute promyelocytic leukemia (90). Roméro et al. (91) have demonstrated in vitro that retinoic acids are potent inhibitors of UV-B induced melanogenesis via tyrosinase related proteins (TRP-1 and TRP-2) expressions.

Acne

Retinoids are considered the first line treatment for acne, being also used in maintenance therapy. These drugs cause the desobstruction of the pores, preventing the formation of white spots, which lead to an anti–inflammatory and comedolytic action. Also, it is usual the topical combination of antibiotics with retinoids to treat comedogenesis, bacterial growth and inflammation. This combination also increases the effectiveness and tolerance to the treatment. On the other way, benzoyl peroxide can be used in combination with topical retinoids to reduce the dose of antibiotic. It is important to use daily solar protection during the treatment due to the skin sensitization to the solar exposure (92, 93).

The currently commercial available formulations include 0.025-0.1 % of tretinoin topical creams or gels. However, new sustaining release systems are emerging, in which the active substance is vehiculated in microsponges or polymers in order to remain at the SC, the outermost layer of the skin, thus promoting comedolisis and modulation of the keratinocytes proliferation (94). Recently we have developed a new formulation of tretinoin-loaded ultradeformable vesicles, which revealed to be a promising delivery system for tretinoin dermal delivery without promoting skin irritation that is usually observed in currently available commercial formulations (95).

Photoaging

Tretinoin is also used on fine wrinkling, mottled hyperpigmentation, roughness associated with photodamaged skin (90). The described (long term) effects of topical retinoids on the skin are: (1) reduction and redistribution of epidermal melanin, (2) improved ultrastructural characteristics of the epidermis, (3) increased anchoring fibrils, (4) increased deposition of papillary dermal collagen, and (5) increased vascularity in the papillary dermis (21, 70).

UV irradiation results in a functional deficiency of vitamin A as a consequence of the lower expression of the two predominant retinoid receptors in human skin, RAR- and RXR-. The same happens in UV-B irradiated cultured keratinocytes and melanocytes. Contrarily to keratinocytes, irradiated melanocytes return to normal values 2 to 3 days after irradiation (27, 96, 97). Pretreatment of human skin with tretinoin blocks dermal matrix degradation following sun exposure inhibiting the induction of the AP-1 transcription factor and AP-1 regulated matrix-degrading MMPs. Tretinoin does not interfere with UVR-induced upregulation of tissue inhibitors of metalloproteinases (TIMP) thus favoring collagen preservation (24, 98). In fact, Singh et al. (26) demonstrated by photomicrography the restoration of procollagen-1 in the papillary dermis after treatment with 0.1 % tretinoin during 40 weeks.

Clinically modest but highly statistically signficant improvements in global appearance of photoaged skin treated with tretinoin were shown in several double-blind vehicle-controlled trials involving more than 700 subjects. These data led to the first U.S. FDA product (Renova®) approval for the indication of improving photoaged skin (24, 99). The beneficial effects are dose-dependent and increase with duration of therapy for at least 10–12 months (24, 99).

Kossard et al. (100) reported that 0.05 % tretinoin cream appeared to be effective in reversing epidermal atrophy and diminishing fine wrinkling, mottled hyperpigmentation and skin roughness in a randomized double-blind vehicle controlled trial. However, the solar elastosis was not treated at least during this period (6 months).

Fisher et al. (98) demonstrated that a single topical application of 0.1 % retinoic acid for 4 days promoted the proliferation of keratinocytes (increasing the number of cells and therefore the thickness of the epidermis), compression of the barrier and opening spaces between keratinocytes.

Lycopene

Physicochemical Properties

Lycopene is a naturally occurring carotenoid found in a number of fruits and vegetables such as the tomato, watermelon, pink grapefruit, guava, apricots, papaya and rosehip (101). It is an acyclic hydrocarbon made up of eleven conjugated double bonds and two unconjugated double bonds that can easily be attacked by electrophilic reagents, resulting in an extreme reactivity toward oxygen and free radicals. This reactivity of lycopene is the basis for its anti-oxidant activity (102).

Lycopene chemical structure has many conjugated carbon double bonds in the all-trans form, which account for lycopene´s stability and also for its attractive colour. Synthetic lycopene is extremely expensive (103), and the resulting compound is equivalent to natural lycopene, including the isomer content (104-106). While in the plant matrix or in solid form, lycopene is relatively stable, but after extraction from the matrix and dissolution in a non-polar organic solvent, lycopene is quite unstable (107). Therefore, the stability of lycopene must be a consideration during experiments, especially in in vitro studies. A summary of the main physicochemical properties of lycopene is represented in Table 4.

Table 4- Physicochemical properties of Lycopene (105, 106, 108, 109).

Molecule

Lycopene

Molecular Structure

chem_structure

IUPAC Name

(6E,8E,10Z,12Z,14E,16E,18E,20Z,22Z,24E,26E)-2,6,10,14,19,23,27,31-octamethyldotriaconta-2,6,8,10,12,14,16,18,20,22,24,26,30-tridecaene

MF

C40H56

MW (g/mol)

536.87264

Physical state

Orange Powder

Solubility

Insoluble in water, ethanol and methanol

Soluble in chloroform, hexane, benzene, carbon disulfide, acetone, petroleum ether and oil

Log P

15

Melting Point (ï‚°C)

172-175

Abs.

444, 470 and 502 nm

Stability

Sensitive to light, oxygen, high temperature, acids, catalyst, metal ions.

Store at -70ï‚°C. Combustible. Incompatible with strong oxidizing agents.

Bioavailability and Metabolism

Although lycopene represents as much as 50 % of carotenoids found in human serum, it cannot be synthesized in human organism (9). Lycopene has high bioavailability and, in most cases, its bioavailability from dietary sources is increased by coingestion of dietary lipids and thermal processing (for example, in tomato paste) due to cis-isomerization of the molecule because of the heat chemical reaction during processing. In addition, the physical disruption of the cell structure in processed tomato products compared to fresh tomatoes partially explains the difference in the bioavailability of lycopene. Although about 90% of the lycopene in dietary sources is found in the linear, all-trans conformation, human tissues contain mainly cis-isomers. Several research groups have suggested that cis-isomers of lycopene are better absorbed than the all-trans form because of the shorter length of the cis-isomer, the greater solubility of cis-isomers in mixed micelles, and/or as a result of the lower tendency of cis-isomers to aggregate (9, 47, 104, 106, 110, 111).

The lycopene distribution in skin tissue is about 0.2-0.6 nmol/g wet weight, with higher concentration in the upper skin layers because of the transportation of carotenoids to the skin surface via eccrine sweat glands and/or sebaceous glands and migration of epidermal keratinocytes in which carotenoids have been suggested to be loaded. Through Raman spectroscopy studies, significant differences regarding the distribution and level of carotenoids were observed within different skin areas (2, 7, 18).

Lycopene is cleaved in vitro to acycloretinal, acycloretinoic acid and apolycopenals (112) in a nonenzymatic manner. Kim et al. (113) suggested the susceptibility of carbonyl group of carotenoids, with a long chain of conjugated double bonds, to cleave by autooxidation, radical-mediated oxidation and by singlet oxygen. These reactions may also occur in in vivo conditions if the tissues are exposed to oxidative stress (114).

Recently it was demonstrated that oxidation/cleavage products formed by chemical transformation impact the proliferation of certain cancer cells. In this process, the dialdehydes seem to be the most active metabolites (3). These studies strongly suggest that oxidation products as well as intact carotenoids have biological effects on human health, whether beneficial or harmful (112). 

Safety

Regarding the lycopene safety, no adverse effects from lycopene ingestion and topical administration have been described up to moment. Both pure crystalline lycopene and formulated lycopene (in stable conditions) are not genotoxic as demonstrated by a comprehensive battery of tests (106).

Biological Activity

In humans, carotenoids function primarily as dietary sources of provitamin A. When converted to vitamin A, these molecules play an important role mainly in vision and skin. However, lycopene lacks the b-ionone ring structure required to form vitamin A and has no provitamin A activity. The first reported in vivo activities of lycopene were the protection against bacterial infection, when lycopene was injected intraperitoneally to mice (115). Furthermore, protection against radiation and development of certain types of ascites tumours was also described (116, 117).

Several studies have demonstrated potential benefits of lycopene related to its biological functions, as it is described above for antioxidant, gap-junction communication, retinoid activity, cell proliferation and apoptosis properties and the consequent implications for prevention of photocarcinogenesis. Some of these biological functions may be mediated by lycopene metabolites with more or less activity or with an entirely independent function (114).

Epidemiological studies indicate that lycopene may be helpful in cardiovascular disease (118), diabetes (119) and cancer prevention (oral, esophageal, pancreatic, rectal, colon, cervical, breast cancer) (120) and, particularly, in prostate cancer prevention, which is reported in many clinical trials (47, 121). Being fat soluble, lycopene appears to be particularly effective in tissues with high lipid content, such as prostate. The skin, also a lipid-rich organ is likely to benefit from lycopene biological activity. However, the studies regarding the role of lycopene against some of these diseases are not completely conclusive and are still ongoing.

Although some published Phase I and II studies have established the safety of lycopene supplementation, these studies do not clearly address the potential efficacy of lycopene as a chemopreventive agent. It has been proposed that the most important studies that are expected to be reported during the next several years will be Phase II clinical trials that are placebo-controlled, randomized and double blind. Well designed and adequately powered clinical studies of lycopene efficacy are still needed (116).

Antioxidant Properties

The pharmacological properties of lycopene are mainly due to its antioxidant activity. In fact, antioxidant properties of many carotenoids have been long believed to play critical roles in their anticarcinogenic actions. Using tomatoes or tomato products, numerous studies have demonstrated decreased DNA damage (28), decreased susceptibility to oxidative stress in lymphocytes (122), and decreased low density lipoprotein (LDL) oxidation or lipid peroxidation (123). Oxidative stress is one of the major factors of chronic diseases and cancer. In vitro, ex vivo, and in vivo studies have been carried out to demonstrate the effects of lycopene against oxidative stress (105). However, data regarding the antioxidant effects of lycopene alone in biological systems are limited (114). The investigation into the interaction of free radicals with antioxidants has been strongly stimulated, as it has become possible to measure the carotenoids in human skin in vivo, online and non-invasively by resonance Raman spectroscopy (124). In the past, these investigations were only possible by means of chemical analysis of biopsies, i.e. invasive measurements (2, 125).

As mentioned above, lycopene is a powerful antioxidant both in vitro and in vivo against the oxidation of proteins, lipids and DNA. The reactivity of lycopene in biological systems depends on molecular and physical structure, location or site of action within the cells, ability to interact with other antioxidants, concentration and the partial pressure of oxygen (126).

Due to its highly lipophilic nature, lycopene exerts its maximal antioxidant activity at the level of cellular membranes and interacts with lipid components (127). Through protecting membranes from lipid peroxidation, it counteracts tumour initiation. It was demonstrated that lycopene is more efficient than β –carotene in preventing nitrogen dioxide induced oxidation of lipid membranes and subsequent cell death (3, 128).

Lycopene has been identified as one of the most potent scavengers of singlet species of oxygen free radicals – the highest among the carotenoids (twice as potent as -carotene and, approximately, 100 times more powerful than vitamin E). This effect is probably due to the greater number of conjugated double bonds in the lycopene structure (8, 114). At low oxygen tension, it can also scavenge peroxyl radicals, inhibiting the process of lipid peroxidation (18).

It is predicted that three possible mechanisms are involved in the action for lycopene towards ROS: radical addition, electron transfer to the radical and hydrogen abstraction (Fig.4). Lycopene is the best antioxidant based on electron transfer reactions (129).

1. Radical addition: Lycopene + R• → R-Lycopene•

2. Electron transfer: Lycopene + R• → Lycopene•+ + R–

3. Hydrogen abstraction: Lycopene + R• → Lycopene• + RHL

Fig.4- Possible reactions of lycopene with radical species (R•) (102, 105).

Regarding the interaction of lycopene with other antioxidants, it is already known that lycopene in combination with other antioxidants such as vitamins E and C, polyphenols and other carotenoids have wide potential for human health (Table 5) (126, 130). Bast et al. (131) suggested that lycopene might enhance the cellular antioxidant defense system by regenerating the non-enzymatic antioxidants vitamins E and C from their radicals. β-carotene not only quenches oxy radicals but could also enhance the radical-protective properties of both vitamins E and C, as well (Fig.5). In addition, the superior protection of these mixtures may be related to the specific location of different antioxidants in cell membranes and, also to different absorption wavelengths regarding the skin photodamage context. Recent formulations of antioxidant mixtures in the development of nutritional products have been in favor for their health benefits (105, 132).

Fig.5. Squeme of the mechanism proposed by Black & Lambert (133) by which β-carotene participates in quenching oxy radicals and interacts to enhance the antioxidant properties of vitamin E: vitamin E (TOH) firstly intercepts an oxy radical and the tocopherol radical cation (TOH.+) formed is repaired by β-carotene (CAR), which in turn forms the carotenoid radical cation (CAR.+) latter repaired by ascorbic acid.

Table 5- Examples of some lycopene interactions with other antioxidants (adapted from Kong et al. (105)).

Examples