The Immune System and Pathogenesis of Melanoma and Non-melanoma Skin Cancer.

Tumor development is the result of genetic derangement and the inability to prevent unfettered proliferation. Genetic derangements leading to tumorigenesis are variable, but the immune system plays a critical role in tumor development, prevention, and production. In this chapter, we will discuss the importance of the immune system as it relates to the development of skin cancer-both melanoma and non-melanoma skin cancers (NMSC).

Topical application of ST266 reduces UV-induced skin damage.

Ultraviolet radiation (UVR) has a significant impact on human skin and is the major environmental factor for skin cancer formation. It is also believed that 80% of the signs of skin aging are attributed to UVR. UVR induces inflammatory changes in the skin via the increase in oxidative stress, DNA damage vascular permeability, and fluctuation in a myriad of cytokines. Acutely, UVR causes skin inflammation and DNA damage, which manifest as sunburn (erythema). ST266 is the secretome of proprietary amnion-derived cells that have been shown to reduce inflammation and accelerate healing of various wounds by promoting migration of keratinocytes and fibroblasts in preclinical animal studies. We hypothesized that ST266 has anti-inflammatory effects that can be used to reduce ultraviolet (UV) erythema and markers of inflammation. In this study, we examined the in vivo effects of ST266 on post UV-irradiated skin by measuring erythema, level of cyclobutane pyrimidine dimer (CPD), and expression level of xeroderma pigmentosum, complementation group A (XPA). We demonstrated that ST266 has the potential to reduce the acute effects of UV-induced skin damage when applied immediately after the initial exposure. In addition, ST266 is shown to reduce erythema, increase XPA DNA repair protein, and decrease damaged DNA.

PEGylated Dendrimers as Drug Delivery Vehicles for the Photosensitizer Silicon Phthalocyanine Pc 4 for Candidal Infections.

Fungi account for billions of infections worldwide. The second most prominent causative agent for fungal infections is Candida albicans (C. albicans). As strains of fungi become resistant to antifungal medications, new treatment modalities must be investigated to combat these infections. One approach is to employ photodynamic therapy (PDT). PDT utilizes a photosensitizer, light, and cellular O2 to produce reactive oxygen species (ROS), which then induce oxidative stress resulting in apoptosis. Silicon phthalocyanine Pc 4 is a photosensitizer that has exhibited success in clinical trials for a myriad of skin diseases. The hydrophobic nature of Pc 4, however, poses significant formulation and delivery challenges in the use of this therapy. To mitigate these concerns, a drug delivery vehicle was synthesized to better formulate Pc 4 into a viable PDT agent for treating fungal infections. Utilizing poly(amidoamine) dendrimers as the framework for the vehicle, ∼13% of the amine chain ends were PEGylated to promote water solubility and deter nonspecific adsorption. In vitro studies with C. albicans demonstrate that the potency of Pc 4 was not hindered by the dendrimer vehicle. Encapsulated Pc 4 was able to effectively generate ROS and obliterate fungal pathogens upon photoactivation. The results presented within describe a nanoparticulate delivery vehicle for Pc 4 that readily kills drug-resistant C. albicans and eliminates solvent toxicity, thus, improving formulation characteristics for the hydrophobic photosensitizer.

Activated T cells exhibit increased uptake of silicon phthalocyanine Pc 4 and increased susceptibility to Pc 4-photodynamic therapy-mediated cell death.

Photodynamic therapy (PDT) is an emerging treatment for malignant and inflammatory dermal disorders. Photoirradiation of the silicon phthalocyanine (Pc) 4 photosensitizer with red light generates singlet oxygen and other reactive oxygen species to induce cell death. We previously reported that Pc 4-PDT elicited cell death in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro, and furthermore that Jurkat cells were more sensitive than A431 cells to treatment. In this study, we examined the effectiveness of Pc 4-PDT on primary human CD3(+) T cells in vitro. Fluorometric analyses of lysed T cells confirmed the dose-dependent uptake of Pc 4 in non-stimulated and stimulated T cells. Flow cytometric analyses measuring annexin V and propidium iodide (PI) demonstrated a dose-dependent increase of T cell apoptosis (6.6-59.9%) at Pc 4 doses ranging from 0-300 nM. Following T cell stimulation through the T cell receptor using a combination of anti-CD3 and anti-CD28 antibodies, activated T cells exhibited increased susceptibility to Pc 4-PDT-induced apoptosis (10.6-81.2%) as determined by Pc 4 fluorescence in each cell, in both non-stimulated and stimulated T cells, Pc 4 uptake increased with Pc 4 dose up to 300 nM as assessed by flow cytometry. The mean fluorescence intensity (MFI) of Pc 4 uptake measured in stimulated T cells was significantly increased over the uptake of resting T cells at each dose of Pc 4 tested (50, 100, 150 and 300 nM, p < 0.001 between 50 and 150 nM, n = 8). Treg uptake was diminished relative to other T cells. Cutaneous T cell lymphoma (CTCL) T cells appeared to take up somewhat more Pc 4 than normal resting T cells at 100 and 150 nm Pc 4. Confocal imaging revealed that Pc 4 localized in cytoplasmic organelles, with approximately half of the Pc 4 co-localized with mitochondria in T cells. Thus, Pc 4-PDT exerts an enhanced apoptotic effect on activated CD3(+) T cells that may be exploited in targeting T cell-mediated skin diseases, such as cutaneous T cell lymphoma (CTCL) or psoriasis.

The Response of microRNAs to Solar UVR in Skin-Resident Melanocytes Differs between Melanoma Patients and Healthy Persons.

The conversion of melanocytes into cutaneous melanoma is largely dictated by the effects of solar ultraviolet radiation (UVR). Yet to be described, however, is exactly how these cells are affected by intense solar UVR while residing in their natural microenvironment, and whether their response differs in persons with a history of melanoma when compared to that of healthy individuals. By using laser capture microdissection (LCM) to isolate a pure population of melanocytes from a small area of skin that had been intermittingly exposed or un-exposed to physiological doses of solar UVR, we can now report for the first time that the majority of UV-responsive microRNAs (miRNAs) in the melanocytes of a group of women with a history of melanoma are down-regulated when compared to those in the melanocytes of healthy controls. Among the miRNAs that were commonly and significantly down-regulated in each of these women were miR-193b (P<0.003), miR-342-3p (P<0.003), miR186 (P<0.007), miR-130a (P<0.007), and miR-146a (P<0.007). To identify genes potentially released from inhibition by these repressed UV-miRNAs, we analyzed databases (e.g., DIANA-TarBase) containing experimentally validated microRNA-gene interactions. In the end, this enabled us to construct UV-miRNA-gene regulatory networks consisting of individual genes with a probable gain-of-function being intersected not by one, but by several down-regulated UV-miRNAs. Most striking, however, was that these networks typified well-known regulatory modules involved in controlling the epithelial-to-mesenchymal transition and processes associated with the regulation of immune-evasion. We speculate that these pathways become activated by UVR resulting in miRNA down regulation only in melanocytes susceptible to melanoma, and that these changes could be partially responsible for empowering these cells toward tumor progression.

The immune system and skin cancer.

Carcinogenesis involves multiple mechanisms that disturb genomic integrity and encourage abnormal proliferation. The immune system plays an integral role in maintaining homeostasis and these mechanisms may arrest or enhance dysplasia. There exists a large body of evidence from organ transplantation literature supporting the significance of the immune suppression in the development of skin cancer. Nonmelanoma skin cancers are the most frequent neoplasms after organ transplantation, with organ transplant recipients having a 65-fold increase in squamous cell carcinoma incidence and 10-fold increase in basal cell carcinoma incidence. Similarly, UV-radiation (UVR) induced immunosuppression is correlated with the development of cutaneous malignancies in a dose-dependent manner. This was first shown several decades ago by Margaret Kripke, when transplanted tumors were rejected in mice with competent immune systems, but grew unchecked in immunosuppressed specimens. After UV exposure, chromophores initiate a cascade that leads to immunosuppression via derangement of Langerhans cells' antigen-presenting capacity. UV-irradiated Langerhans cells present antigens to Th2 cells, but fail to stimulate Th1 cells. A subset of T regulatory cells, specific for the antigen encountered after UVR, is also stimulated to proliferate. In general UV irradiation leads to a greater number of T regulatory cells and fewer effector T cells in the skin, shiftingthe balance from T-cell-mediated immunity to immunosuppression. These regulatory cells have the phenotype CD4+, CD25+, Foxp3+, CTLA-4+. These and many other changes in local immunity lead to a suppressed immune state, which allow for skin cancer development.

Introduction to photobiology.

Photobiology is the study of the local and systemic effects of incident radiation on living organisms. Solar radiation is made up of ultraviolet, visible and infrared radiation. Ultraviolet radiation is made up of UV-C, UV-B, and UV-A. Sun exposure can lead to sunburn, tanning, vitamin D production, photoaging, and carcinogenesis. Phototherapy is the use of nonionizing radiation to treat cutaneous disease. Various types of artificial light sources are used for photo testing and phototherapy.

Effect of botanicals on inflammation and skin aging: analyzing the evidence.

The skin and its immune system manifest a decline in physiologic function as it undergoes aging. External insults such as ultraviolet light exposure cause inflammation, which may enhance skin aging even further leading to cancer and signs of photoaging. There is a potential role for botanicals as an adjunct modality in the prevention of skin aging. Numerous over-the-counter anti-aging products are commercially available, many of which boast unverified claims to reduce stress, inflammation and correct signs of aging. In this article we reviewed the scientific literature for data on frequently published "anti-inflammaging" additives such as vitamins A, C and E and green tea. We also analyzed the evidence available on five promising ingredients commonly found in anti-aging products, namely, argan oil, rosemary, pomegranate, Coenzyme Q10, and Coffeeberry. Though there may be an increasing amount of scientific data on a few of these novel botanicals, in general, there remains a lack of clinical data to support the anti-aging claims made.

Silicon phthalocyanine 4 phototoxicity in Trichophyton rubrum.

Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of terbinafine for the skin and ciclopirox for the nails, whereas systemic agents, such as oral terbinafine and itraconazole, are also prescribed. These systemic drugs have severe side effects, including liver toxicity. Topical therapies, however, are sometimes ineffective. This led us to investigate alternative treatment options, such as photodynamic therapy (PDT). Although PDT is traditionally recognized as a therapeutic option for treating a wide range of medical conditions, including age-related macular degeneration and malignant cancers, its antimicrobial properties have also received considerable attention. However, the mechanism(s) underlying the susceptibility of dermatophytic fungi to PDT is relatively unknown. As a noninvasive treatment, PDT uses a photosensitizing drug and light, which, in the presence of oxygen, results in cellular destruction. In this study, we investigated the mechanism of cytotoxicity of PDT in vitro using the silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)] in T. rubrum. Confocal microscopy revealed that Pc 4 binds to cytoplasmic organelles, and upon irradiation, reactive oxygen species (ROS) are generated. The impairment of fungal metabolic activities as measured by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay indicated that 1.0 µM Pc 4 followed by 670 to 675 nm light at 2.0 J/cm(2) reduced the overall cell survival rate, which was substantiated by a dry weight assay. In addition, we found that this therapeutic approach is effective against terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) strains. These data suggest that Pc 4-PDT may have utility as a treatment for dermatophytosis.

Acute effects of fractional laser on photo-aged skin.

BACKGROUND: Nonablative fractional photothermolysis (FP) laser treatment has shown clinical efficacy on photo-aged skin. Few studies have examined the molecular responses to FP. OBJECTIVE: To characterize the dynamic alterations involved in dermal matrix remodeling after FP laser treatment. METHODS: A single multipass FP treatment was performed. Baseline, day 1, and day 7 biopsies were obtained. Biopsies were sectioned and stained for histology and immunofluorescence confocal microscopic. Heat shock protein-70 (HSP-70) and matrix metalloproteinase-1 (MMP-1) expression and extracellular matrix (ECM) autofluorescence were examined. Quantitative real-time polymerase chain reaction (qRT-PCR) experiments were performed probing for collagen 1A1 (COL1A1) and COL3A1. RESULTS: All three patients were Caucasian women aged 49, 62, and 64 with Fitzpatrick skin types II, III, and IV. Transient neutrophilic infiltration found on day 1. Protein expression of HSP-70 and MMP-1 were up-regulated on day 1, reverting to baseline by day 7. ECM autofluorescence decreased from baseline to day 7. qRT-PCR showed a minor decrease in COL1A1 and COL3A1 messenger RNA 1 day after treatment. Variable results between patients receiving equal treatment were evident.

Photodynamic therapy: current evidence and applications in dermatology.

Photodynamic therapy (PDT) involves the activation of a photosensitizing drug, which preferentially localizes to diseased skin, by irradiation with light to cause selective cytotoxic damage. Since its discovery in the early 20th century and the development of topical photosensitizers 2 decades ago, PDT is increasingly being used in dermatology for a wide range of neoplastic, inflammatory, and infectious cutaneous conditions. Topical 5-aminolevulinic acid and methyl aminolevulinic acid, the most commonly used agents in PDT, have received Food and Drug Administration approval for the treatment of actinic keratoses, and many second-generation photosensitizers are under investigation. Compared with conventional therapies, PDT has the advantage of being noninvasive and capable of field treatment. It is also associated with quicker recovery periods and excellent cosmetic results. Because of these benefits, PDT is being evaluated as a potential treatment option for many dermatologic conditions and has been shown to be effective for certain nonmelanoma skin cancers. Although research is still limited, PDT might also have a therapeutic benefit for cutaneous T-cell lymphoma, acne, psoriasis, leishmaniasis, and warts, among others. This article is a review of the clinical applications of PDT in dermatology and summarizes the current evidence in literature describing its efficacy, safety, and cosmetic outcome.

Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans.

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 µM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis.

Preliminary clinical and pharmacologic investigation of photodynamic therapy with the silicon phthalocyanine photosensitizer pc 4 for primary or metastatic cutaneous cancers.

Photodynamic therapy (PDT) for cutaneous malignancies has been found to be an effective treatment with a range of photosensitizers. The phthalocyanine Pc 4 was developed initially for PDT of primary or metastatic cancers in the skin. A Phase I trial was initiated to evaluate the safety and pharmacokinetic profiles of systemically administered Pc 4 followed by red light (Pc 4-PDT) in cutaneous malignancies. A dose-escalation study of Pc 4 (starting dose 0.135 mg/m(2)) at a fixed light fluence (135 J/cm(2) of 675-nm light) was initiated in patients with primary or metastatic cutaneous malignancies with the aim of establishing the maximum tolerated dose (MTD). Blood samples were taken at intervals over the first 60 h post-PDT for pharmacokinetic analysis, and patients were evaluated for toxicity and tumor response. A total of three patients (two females with breast cancer and one male with cutaneous T-cell lymphoma) were enrolled and treated over the dose range of 0.135 mg/m(2) (first dose level) to 0.54 mg/m(2) (third dose level). Grade 3 erythema within the photoirradiated area was induced in patient 2, and transient tumor regression in patient 3, in spite of the low photosensitizer doses. Pharmacokinetic observations fit a three-compartment exponential elimination model with an initial rapid distribution phase (∼0.2 h) and relatively long terminal elimination phase (∼28 h), Because of restrictive exclusion criteria and resultant poor accrual, the trial was closed before MTD could be reached. While the limited accrual to this initial Phase I study did not establish the MTD nor establish a complete pharmacokinetic and safety profile of intravenous Pc 4-PDT, these preliminary data support further Phase I testing of this new photosensitizer.

Effects of pimecrolimus versus triamcinolone on Langerhans cells after UV exposure.

BACKGROUND/PURPOSE: Pimecrolimus is a topical immunomodulator for atopic dermatitis. Concerns regarding malignancy risk resulted in its black box warning in 2006. The purpose of this study is to determine the effects of pimecrolimus on Langerhans cells (LC), mediators of the cutaneous immunity UV-irradiated skin. METHODS: A RCT was conducted investigating pimecrolimus 1% cream vs triamcinolone 0.1% cream on UV-irradiated epidermal LC on 20 healthy volunteers. Punch biopsies were stained with antibodies to CD1a, HLADR and CD83. RESULTS: Triamcinolone caused more depletion in UV-irradiated CD1a(+) epidermis relative to pimecrolimus treatment. (P=0.030). Using HLA-DR as a pan-marker for APCs, pimecrolimus caused marginally less depletion than triamcinolone (P=0.013). Using anti-CD83 as a maturation marker, UV-irradiated skin treated with pimecrolimus showed more mature LC than skin treated with triamcinolone (P=0.00090). CONCLUSION: UV-induced changes in LC are minimally affected by pimecrolimus, compared with triamcinolone.

Anti-angiogenic effects of epigallocatechin-3-gallate in human skin.

Epigallocatechin-3-gallate (EGCG) is the main polyphenol component of green tea. This compound exhibits antioxidant, immunomodulatory, photoprotective, anti-angiogenic, and anti-inflammatory properties. We conducted a small randomized, double blind, split face trial using a cream containing 2.5% w/w of EGCG. Four healthy volunteers with significant erythema and telangiectasia on the face applied EGCG cream to one side of the face, and vehicle control cream to the other, twice daily for six weeks. After six weeks, biopsies were taken from EGCG and vehicle treated sites. Immunohistochemistry was used to measure VEGF and HIF-1 α. HIF-1 α expression was decreased in EGCG treated sites, such that 28.4% of the epidermis showed positive staining in vehicle treated vs. 13.8% in EGCG treated sites (p<0.001). A similar decrease in VEGF expression was found (6.7% in EGCG vs. 11.0%in in vehicle-treated skin (p<0.005). EGCG topical treatments influence HIF-1 α induction and VEGF expression and may serve as a potential agent in the prevention of telangiectasias.

Silicon phthalocyanine (Pc 4) photodynamic therapy is a safe modality for cutaneous neoplasms: results of a phase 1 clinical trial.

BACKGROUND: Photodynamic therapy (PDT) is a non-invasive treatment for non-melanoma skin cancer. However, PDT systems currently used clinically have limitations such as pain and superficial tissue penetration. The silicon phthalocyanine Pc 4 is a second-generation photosensitizer with peak absorption in the far red at 675 nm. OBJECTIVE: To assess the safety and tolerability of topically applied Pc 4 followed by red light (Pc 4-PDT) in treating cutaneous neoplasms. STUDY DESIGN/MATERIALS AND METHODS: Forty three adults with a diagnosis of neoplasms including actinic keratoses, Bowen's disease, squamous cell carcinoma, basal cell carcinoma, or mycosis fungoides were treated with a single administration of Pc 4-PDT and followed for 14 days. The study utilized a light and Pc 4 dose escalation design in sequential groups of three subjects each. RESULTS: Pc 4-PDT was well tolerated with no significant local toxicity or increased photosensitivity. It has promising biologic effects, particularly in mycosis fungoides where 14 of 35 subjects demonstrated a clinical response, which correlates with Pc 4-PDT-induced apoptosis, as measured by increased active caspase-3 in the treated skin lesions. CONCLUSIONS: Pc 4-PDT is a safe and tolerable treatment modality that effectively triggers apoptosis in cutaneous neoplasms such as mycosis fungoides.

Photodynamic therapy with the silicon phthalocyanine pc 4 induces apoptosis in mycosis fungoides and sezary syndrome.

Our current focus on the effects of Photodynamic Therapy (PDT) using silicon phthalocyanine Pc 4 photosensitizer on malignant T lymphocytes arose due to preclinical observations that Jurkat cells, common surrogate for human T cell lymphoma, were more sensitive to Pc 4-PDT-induced killing than epidermoid carcinoma A431 cells. Mycosis fungoides (MF) as well as Sezary syndrome (SS) are variants of cutaneous T-cell lymphoma (CTCL) in which malignant T-cells invade the epidermis. In this study, we investigated the cytotoxicity of Pc 4-PDT in peripheral blood cells obtained from patients with SS and in skin biopsies of patients with MF. Our data suggest that Pc 4-PDT preferentially induces apoptosis of CD4(+)CD7(-) malignant T-lymphocytes in the blood relative to CD11b(+) monocytes and nonmalignant T-cells. In vivo Pc 4-PDT of MF skin also photodamages the antiapoptotic protein Bcl-2.

Factors that affect skin aging: a cohort-based survey on twins.

OBJECTIVE: To identify environmental factors that correlate with skin photoaging, controlling for genetic susceptibility by using a questionnaire administered to twins. DESIGN: The survey collected information about each participant's Fitzpatrick type, history of skin cancer, smoking and drinking habits, and weight from a cohort of twins. Clinicians then assigned a clinical photodamage score to each participant. SETTING: The annual Twins Days Festival in Twinsburg, Ohio. PARTICIPANTS: A voluntary cohort of twins from the general community, mostly from Ohio, Pennsylvania, and the northeastern United States. The survey was completed on a voluntary basis by sets of monozygotic (MZ) and dizygotic (DZ) twins. A total of 130 surveys taken by 65 complete twin pairs were analyzed. MAIN OUTCOME MEASURE: Skin aging was assessed using a validated photographic scale of photodamage, graded by such characteristics as wrinkling and pigmentation change. RESULTS: Photodamage scores among twins of a pair, whether MZ or DZ, were highly correlated (P = .92). Factors found to predict higher photodamage include history of skin cancer (P < .001), zygosity status (MZ vs DZ) (P = .001), weight (P = .02), and cigarette smoking (P = .046). Alcohol consumption was significantly associated with lower photodamage scores (P = .003). CONCLUSIONS: The study of twins provides a unique opportunity to control for genetic susceptibility in order to elucidate environmental influences on skin aging. The relationships found between smoking, weight, sunscreen use, skin cancer, and photodamage in these twin pairs may help to motivate the reduction of risky behaviors.

Topical application of green and white tea extracts provides protection from solar-simulated ultraviolet light in human skin.

BACKGROUND: Tea polyphenols have been found to exert beneficial effects on the skin via their antioxidant properties. AIMS: We sought to determine whether topical application of green tea or white tea extracts would prevent simulated solar radiation-induced oxidative damages to DNA and Langerhans cells that may lead to immune suppression and carcinogenesis. METHODS: Skin samples were analysed from volunteers or skin explants treated with white tea or green tea after UV irradiation. In another group of patients, the in vivo immune protective effects of green and white tea were evaluated using contact hypersensitivity to dinitrochlorobenzene. RESULTS: Topical application of green and white tea offered protection against detrimental effects of UV on cutaneous immunity. Such protection is not because of direct UV absorption or sunscreen effects as both products showed a sun protection factor of 1. There was no significant difference in the levels of protection afforded by the two agents. Hence, both green tea and white tea are potential photoprotective agents that may be used in conjunction with established methods of sun protection.

Advances in photoprotection.

Major advancements in the realm of photoprotection have occurred over the last decade providing potential means to reduce the prevalence of ultraviolet radiation-related skin problems. This review elucidates current photoprotective methods and recent developments that may hold future promise.