Alicia Zamarrón

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.

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.

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.

New porphyrin amino acid conjugates: Synthesis and photodynamic effect in human epithelial cells

The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT) were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates stable in liposomes are able to penetrate efficiently in the cytoplasm of cultured cancer and normal cells. No dark cytotoxicity is observed at the same concentration used for PDT cell treatment and during long incubation time (24h). The cell survival after the PDT treatment with visible light is dependent upon light exposure level and compound concentration. The tested compounds show higher photocytotoxicity in tumoral HeLa cells than in no tumoral HaCaT cells. The results suggest that these amino acid porphyrin conjugates are potential photosensitizers for PDT.

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 β‐catenin), cell‐substrate adhesion proteins (β1‐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. J. Cell. Biochem. 112: 2266–2278, 2011.

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.

A secretion of the mollusc Cryptomphalus aspersa promotes proliferation, migration and survival of keratinocytes and dermal fibroblasts in vitro

Regenerative properties of skin decrease with age, and thus, the search for substances that minimize cutaneous ageing has increased in the last few years. The secretion of the mollusc Cryptomphalus Aspersa (SCA) is a natural product that bears regenerative properties when applied topically. The purpose of this work is to study the in vitro effects of SCA on cell proliferation and migration, as well as on cell–cell (E‐cadherin and β‐catenin) and cell–substrate (vinculin and β1‐integrin) adhesion proteins expression, using a human keratinocyte cell line (HaCaT cells) and primary dermal fibroblasts (HF). We tested the effects of SCA on cell proliferation using a colorimetric assay. In addition, SCA‐induced changes on cell migration were studied by wound‐healing assays. Besides, Western blot and immunofluorescence microscopy were carried out to test the expression of different cell adhesion proteins. We found that SCA promotes proliferation and migration of HaCaT cells in a time‐ and dose‐dependent manner. Moreover, treatment with SCA increases the migratory behaviour and the expression of adhesion molecules in both HaCaT and HF. Finally, SCA also improves cell survival and promotes phosphorylation of FAK and nuclear localization of β‐catenin. These results shed light on the molecular mechanisms underlying the regenerative properties of SCA, based on its promoting effect on skin cell migration, proliferation and survival. Moreover, these results support future clinical uses of SCA in the regeneration of wounded tissues.

Isolation and Initial Characterization of Resistant Cells to Photodynamic Therapy

After photodynamic therapy (PDT), the apparition of resistant tumor cells can occur. Laboratory models are being developed in order to understand the potential mechanisms implicated in such resistance. In this sense, we describe the methods published for the isolation and characterization of tumor cells resistant to PDT. We also propose other unpublished procedures that could be of interest for the study of cells resistant to PDT. Factors such as the parental cell line, the photosensitizer (PS) (or prodrug), the photodynamic treatment conditions, the treatment interval, and the clonal or total population selection have to be taken into consideration. Treatment doses are generally high and repeated over time. The development of resistant cells to PDT could take several months. The characterization of resistant cell populations vs parental cells can be performed by using different cellular and molecular techniques, including: cell morphology analysis, intracellular PS content measurement, PS localization, migration and invasion capacity, expression and distribution of adhesion proteins, death proteins and evaluation of specific genes implicated in cell proliferation and survival. Transplantation mouse models also contribute to determine the biological activity of the PDT-resistant cells in vivo, allowing the evaluation of their tumorigenicity and aggressiveness. Laboratory cell models will help us to understand how resistance to anticancer PDT affects the biological and functional aspects of tumorigenicity in vitro and in vivo, which are necessary to improve the clinical results.

Preclinical photodynamic therapy research in Spain 4: Cytoskeleton and adhesion complexes of cultured tumor cells as targets of photosensitizers

Tumor cell death induced by photodynamic therapy (PDT) with different photosensitizers (PSs) is due to the selective damage of several membranous organelles including mitochondria, lysosomes and Golgi apparatus. Other cell structures such as the cytoskeleton (CSK) (microtubules, actin microfilaments and cytokeratin intermediate filaments) and the cell adhesion components (cadherins and integrins) are also implicated in cell death induced by PSs. CSK and adhesion components are responsible for many cellular functions such as the maintenance of morphology, motility, division and adhesion, all of them of fundamental importance for growth and dissemination of tumors. Therefore, they are considered very important targets for anticancer therapies, including PDT. In addition, similarly to the rest of the anticancer therapies, PDT often leaves a significant number of surviving tumor cells. The reorganization of CSK as well as the adhesion proteins in the PDT resistant cells affect their invasive migratory capabilities. Taking into account all these features, both CSK and adhesion proteins are crucial targets of PDT.

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.