Clinical Applications of Polypodium leucotomos (Fernblock®): An Update.

Exposure to sun radiation leads to higher risk of sunburn, pigmentation, immunosuppression, photoaging and skin cancer. In addition to ultraviolet radiation (UVR), recent research indicates that infrared radiation (IR) and visible light (VIS) can play an important role in the pathogenesis of some of these processes. Detrimental effects associated with sun exposure are well known, but new studies have shown that DNA damage continues to occur long after exposure to solar radiation has ended. Regarding photoprotection strategies, natural substances are emerging for topical and oral photoprotection. In this sense, Fernblock®, a standardized aqueous extract of the fern Polypodium Leucotomos (PLE), has been widely administered both topically and orally with a strong safety profile. Thus, this extract has been used extensively in clinical practice, including as a complement to photodynamic therapy (PDT) for treating actinic keratoses (AKs) and field cancerization. It has also been used to treat skin diseases such as photodermatoses, photoaggravated inflammatory conditions and pigmentary disorders. This review examines the most recent developments in the clinical application of Fernblock® and assesses how newly investigated action mechanisms may influence its clinical use.

Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy.

Photodynamic Therapy (PDT) with methyl-aminolevulinate acid (MAL-PDT) is being used for the treatment of Basal cell carcinoma (BCC), but recurrences have been reported. In this work, we have evaluated resistance mechanisms to MAL-PDT developed by three BCC cell lines (ASZ, BSZ and CSZ), derived from mice on a ptch+/- background and with or without p53 expression, subjected to 10 cycles of PDT (10thG). The resistant populations showed mesenchymal-like structure and diminished proliferative capacity and size compared to the parental (P) cells. The resistance was dependent on the production of the endogenous photosensitiser protoporphyrin IX in the CSZ cell line and on its cellular localisation in ASZ and BSZ cells. Moreover, resistant cells expressing the p53 gene presented lower proliferation rate and increased expression levels of N-cadherin and Gsk3ß (a component of the Wnt/ß-catenin pathway) than P cells. In contrast, 10thG cells lacking the p53 gene showed lower levels of expression of Gsk3ß in the cytoplasm and of E-cadherin and ß-catenin in the membrane. In addition, resistant cells presented higher tumorigenic ability in immunosuppressed mice. Altogether, these results shed light on resistance mechanisms of BCC to PDT and may help to improve the use of this therapeutic approach.

Extract of Deschampsia antarctica (EDA) Prevents Dermal Cell Damage Induced by UV Radiation and 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Exposure to natural and artificial light and environmental pollutants are the main factors that challenge skin homeostasis, promoting aging or even different forms of skin cancer through a variety of mechanisms that include accumulation of reactive oxygen species (ROS), engagement of DNA damage responses, and extracellular matrix (ECM) remodeling upon release of metalloproteases (MMPs). Ultraviolet A radiation is the predominant component of sunlight causative of photoaging, while ultraviolet B light is considered a potentiator of photoaging. In addition, different chemicals contribute to skin aging upon penetration through skin barrier disruption or hair follicles, aryl hydrocarbon receptors (AhR) being a major effector mechanism through which toxicity is exerted. Deschampsia antarctica is a polyextremophile Gramineae capable of thriving under extreme environmental conditions. Its aqueous extract (EDA) exhibits anti- photoaging in human skin cells, such as inhibition of MMPs, directly associated with extrinsic aging. EDA prevents cellular damage, attenuating stress responses such as autophagy and reducing cellular death induced by UV. We demonstrate that EDA also protects from dioxin-induced nuclear translocation of AhR and increases the production of loricrin, a marker of homeostasis in differentiated keratinocytes. Thus, our observations suggest a potential use exploiting EDA's protective properties in skin health supplements.

Fernblock Prevents Dermal Cell Damage Induced by Visible and Infrared A Radiation.

Sun overexposure leads to higher risk of photoaging and skin cancer. The contribution of infrared (IR) and visible light (VIS) radiation is currently being taken into account in their pathogenesis. Erythema, hyperpigmentation, genotoxicity or the increase of matrix metalloproteinases (MMPs) expression are some of the effects induced by these types of radiation. Extracts of various botanicals endowed with antioxidant activity are emerging as new photoprotective compounds. A natural extract from Polypodium leucotomos (Fernblock®, FB) has antioxidant and photoprotective properties and exhibits a strong anti-aging effect. In this study, we evaluated the protective capacity of FB against the detrimental effects of infrared A (IRA) and VIS radiation in human dermal fibroblasts. We analyzed the effects of FB on the morphology, viability, cell cycle and expression of extracellular matrix components of fibroblasts subjected to VIS and IRA. Our results indicate that FB prevents cell damage caused by VIS and IRA. Moreover, it reduces the increase in MMP-1 and cathepsin K expression induced by both VIS and IRA radiation, and curbs alterations in fibrillin 1, fibrillin 2 and elastin expression. All these findings support FB as a feasible approach to prevent or treat skin damage caused by IRA or VIS exposure.

Effects of photodynamic therapy on dermal fibroblasts from xeroderma pigmentosum and Gorlin-Goltz syndrome patients.

PDT is widely applied for the treatment of non-melanoma skin cancer pre-malignant and malignant lesions (actinic keratosis, basal cell carcinoma and in situ squamous cell carcinoma). In photodynamic therapy (PDT) the interaction of a photosensitizer (PS), light and oxygen leads to the formation of reactive oxygen species (ROS) and thus the selective tumor cells eradication. Xeroderma pigmentosum (XP) and Gorlin-Goltz Syndrome (GS) patients are at high risk of developing skin cancer in sun-exposed areas. Therefore, the use of PDT as a preventive treatment may constitute a very promising therapeutic modality for these syndromes. Given the demonstrated role of cancer associated fibroblasts (CAFs) in tumor progression and the putative CAFs features of some cancer-prone genodermatoses fibroblasts, in this study, we have further characterized the phenotype of XP and GS dermal fibroblasts and evaluated their response to methyl-δ-aminolevulinic acid (MAL)-PDT compared to that of dermal fibroblasts obtained from healthy donors. We show here that XP/GS fibroblasts display clear features of CAFs and present a significantly higher response to PDT, even after being stimulated with UV light, underscoring the value of this therapeutic approach for these rare skin conditions and likely to other forms of skin cancer were CAFs play a major role.

Glucose-functionalized amino-OPEs as biocompatible photosensitizers in PDT.

Photodynamic therapy (PDT) is a minimally invasive procedure that can provide a selective eradication of neoplastic diseases by the combined effect of a photosensitizer, light and oxygen. New amino oligo(phenylene-ethynylene)s (OPEs), bearing hydrophilic glucoside terminations, have been prepared, characterized and tested as photosensitizers in PDT. The effectiveness of these compounds in combination with UVA light has been checked on two tumor cell lines (HEp-2 and HeLa cells, derived from a larynx carcinoma and a cervical carcinoma, respectively). The compounds triggered a mitotic blockage that led to the cell death, being the effect active up to 3 µm concentration. The photophysical properties of OPEs, such as high quantum yield, stability, singlet oxygen production, biocompatibility, easy cell-internalization and very good response even at low concentration, make them promising photosensitizers in the application of PDT.

Combined Treatments with Photodynamic Therapy for Non-Melanoma Skin Cancer.

Non-melanoma skin cancer (NMSC) is the most common form of cancer in the Caucasian population. Among NMSC types, basal cell carcinoma (BCC) has the highest incidence and squamous cell carcinoma (SCC) is less common although it can metastasize, accounting for the majority of NMSC-related deaths. Treatment options for NMSC include both surgical and non-surgical modalities. Even though surgical approaches are most commonly used to treat these lesions, Photodynamic Therapy (PDT) has the advantage of being a non-invasive option, and capable of field treatment, providing optimum cosmetic outcomes. Numerous clinical research studies have shown the efficacy of PDT for treating pre-malignant and malignant NMSC. However, resistant or recurrent tumors appear and sometimes become more aggressive. In this sense, the enhancement of PDT effectiveness by combining it with other therapeutic modalities has become an interesting field in NMSC research. Depending on the characteristics and the type of tumor, PDT can be applied in combination with immunomodulatory (Imiquimod) and chemotherapeutic (5-fluorouracil, methotrexate, diclofenac, or ingenol mebutate) agents, inhibitors of some molecules implicated in the carcinogenic process (COX2 or MAPK), surgical techniques, or even radiotherapy. These new strategies open the way to a wider improvement of the prevention and eradication of skin cancer.

Photoactivation of ROS Production In Situ Transiently Activates Cell Proliferation in Mouse Skin and in the Hair Follicle Stem Cell Niche Promoting Hair Growth and Wound Healing.

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.

Isolation and characterization of PDT-resistant cancer cells.

Even though the efficacy of photodynamic therapy (PDT) for treating premalignant and malignant lesions has been demonstrated, resistant tumor cells to this therapy occasionally appear. Here, we describe the published methods to isolate resistant cancer cells to PDT and propose new procedures that may be used, as laboratory models allow a better understanding of resistance mechanisms. For this purpose, the treatment conditions, the photosensitizer (PS) or pro-drug, the cell line and the final selection - clonal of total population - must be taken into account. In general, high and repeated treatment doses are used. The resistant cell population characterization may include cell morphology, response to PDT, expression of death proteins or survival related genes and cell proliferation analysis. In addition, in vivo models such as the resistant cell transplantation to mice, allow evaluating tumorigenicity and aggressiveness, leading to the determination of the in vivo resistance. Summarizing, in order to improve clinical results, cellular models can help understand PDT-resistance mechanisms in vivo and in vitro.

Photodynamic effects induced by meso-tris(pentafluorophenyl)corrole and its cyclodextrin conjugates on cytoskeletal components of HeLa cells.

The aim of this work was to synthesize new corrole ß-cyclodextrin conjugates ßCD1 (with one ß-cyclodextrin moiety) and ßCD2 (with two ß-cyclodextrin moieties) from 5,10,15-tris(pentafluorophenyl)corrole (TPFC) and to test in vitro the efficacy of these compounds towards tumoral HeLa cells. No dark cytotoxicity was observed for TPFC and ßCD1 at the concentration used for PDT cell treatment, even during long incubation periods (24 h). Fluorescence microscopy showed that TPFC and ßCD1 accumulate in HeLa cells at lysosomes and in the Golgi apparatus, respectively. The cell survival after the PDT treatment with visible light was dependent on light exposure level and compound concentration. ßCD1 was able to penetrate efficiently in the cytoplasm of the HeLa cells. In particular, we have analyzed the photodynamic effect of the corrole derivatives on the microtubules of HeLa cells and the morphological alterations on the mitotic spindle. TPFC and ßCD1 caused photocytotoxicity in tumoral HeLa cells and induced a rapid metaphase blockage of cells that also showed clearly altered configurations of the mitotic spindle. The results showed that TPFC has the highest photosensitizing efficiency on tumoral cells.

Glycophthalocyanines as photosensitizers for triggering mitotic catastrophe and apoptosis in cancer cells.

Photodynamic therapy (PDT) is a treatment modality for different forms of cancer based on the combination of light, molecular oxygen, and a photosensitizer (PS) compound. When activated by light, the PS generates reactive oxygen species leading to tumor destruction. Phthalocyanines are compounds that have already shown to be efficient PSs for PDT. Several examples of carbohydrate substituted phthalocyanines have been reported, assuming that the presence of carbohydrate moieties could improve their tumor selectivity. This work describes the photoeffects of symmetric and asymmetric phthalocyanines with D-galactose (so-called GPh1, GPh2, and GPh3) on HeLa carcinoma cells and their involvement in cell death. Photophysical properties and in vitro photodynamic activities for the compounds considered revealed that the asymmetric glycophthalocyanine GPh3 is very efficient and selective, producing higher photocytotoxicity on cancer cells than in nonmalignat HaCaT. The cell toxiticy after PDT treatment was dependent upon light exposure level and GPh3 concentration. GPh3 causes cell cycle arrest at the metaphase stage leading to multiple spindle poles, mitotic catastrophe, followed by apoptosis in cancer cells. These effects were partially negated by the pancaspase inhibitor Z-VAD-FMK. Together, these results indicate that GPh3 is an excellent candidate drug for PDT, able to induce selective tumor cell death.

Isolation and characterization of squamous carcinoma cells resistant to photodynamic therapy.

Photodynamic therapy (PDT) employing methyl δ-aminolevulinic acid (Me-ALA), as a precursor of the photosensitizer protoporphyrin IX (PpIX), is used for the treatment of non melanoma cutaneous cancer (NMCC). However, one of the problems of PDT is the apparition of resistant cell populations. The aim of this study was to isolate and characterize squamous carcinoma cells SCC-13 resistant to PDT with Me-ALA. The SCC-13 parental population was submitted to successive cycles of Me-ALA-PDT and 10 resistant populations were finally obtained. In parental and resistant cells there were analyzed the cell morphology (toluidine blue), the intracellular PpIX content (flow cytometry) and its localization (fluorescence microscopy), the capacity of closing wounds (scratch wound assay), the expression of cell-cell adhesion proteins (E-cadherin and ß-catenin), cell-substrate adhesion proteins (ß1-integrin, vinculin and phospho-FAK), cytoskeleton proteins (α-tubulin and F-actin) and the inhibitor of apoptosis protein survivin, in the activated form as phospho-survivin (indirect immunofluorescence and Western blot). The results obtained indicate that resistant cells showed a more fibroblastic morphology, few differences in intracellular content of the photosensitizer, higher capacity of closing wounds, higher number of stress fibers, more expression of cell-substrate adhesion proteins and higher expression of phospho-survivin than parental cells. These distinctive features of the resistant cells can provide decisive information to enhance the efficacy of Me-ALA applications in clinic dermatology.

Temperature sensing using fluorescent nanothermometers.

Acquiring the temperature of a single living cell is not a trivial task. In this paper, we devise a novel nanothermometer, capable of accurately determining the temperature of solutions as well as biological systems such as HeLa cancer cells. The nanothermometer is based on the temperature-sensitive fluorescence of NaYF(4):Er(3+),Yb(3+) nanoparticles, where the intensity ratio of the green fluorescence bands of the Er(3+) dopant ions ((2)H(11/2) --> (4)I(15/2) and (4)S(3/2) --> (4)I(15/2)) changes with temperature. The nanothermometers were first used to obtain thermal profiles created when heating a colloidal solution of NaYF(4):Er(3+),Yb(3+) nanoparticles in water using a pump-probe experiment. Following incubation of the nanoparticles with HeLa cervical cancer cells and their subsequent uptake, the fluorescent nanothermometers measured the internal temperature of the living cell from 25 degrees C to its thermally induced death at 45 degrees C.

Differential photodynamic response of cultured cells to methylene blue and toluidine blue: role of dark redox processes.

Cultured cells treated with equal concentrations of thiazine photosensitizers methylene blue (MB) or toluidine blue (TB) showed a distinct photodynamic lethality, with TB being much more effective, when exposed to red light from a LED source. This effect is accounted for because of the differences in the chemical reduction of MB and TB in the intracellular environment. While TB accumulates as blue granular structures, MB does not give such a localization pattern. However, upon exposure of MB-treated cells to oxidant agents, the dye becomes clearly localized in the cytoplasm as blue granules. We propose that massive reduction of MB to its leuco form inside the cell inhibits most of the photodynamic damage, while no such reduction occurs with TB.