Photothermal effect by 808-nm laser irradiation of melanin: a proof-of-concept study of photothermal therapy using B16-F10 melanotic melanoma growing in BALB/c mice.

The photothermal effect is undergoing great interest due to advances in new photosensitizing materials and better-suited light sources, but studies are frequently hampered by the need to employ exogenous photothermal agents and expensive irradiation devices. Here we present a simple strategy based on direct NIR irradiation of the melanin pigment with a commercial 808-nm laser pointer. Proof-of-concept studies showed efficient photothermal effects on melanin in vitro and in vivo. After NIR irradiation, BALB/c mice bearing B16-F10 melanotic melanoma tumors revealed severe histopathological damage and massive necrosis in melanin-containing tumor tissue, while surrounding healthy tissues showed no damage. Therefore, the feasibility of this approach may allow implementing direct procedures for photothermal therapy of pigmented tumors.

NIR laser pointer for in vivo photothermal therapy of murine LM3 tumor using intratumoral China ink as a photothermal agent.

The photothermal effect is one of the most promising photonic procedures currently under development to successfully treat several clinical disorders, none the least some kinds of cancer. At present, this field is undergoing a renewed interest due to advances in both photothermal materials and better-suited light sources. However, scientific studies in this area are sometimes hampered by the relative unavailability of state-of-art materials or the complexity of setting up a dedicated optical facility. Here, we present a simple and affordable approach to do research in the photothermal field that relies on a commercial NIR laser pointer and a readily available everyday pigment: China ink. A proof-of-concept study is presented in which mice bearing intradermal LM3 mammary adenocarcinoma tumors were successfully treated in vivo employing China ink and the laser pointer. TUNEL and Ki-67 post-treatment tissue assessment clearly indicates the deleterious action of the photothermal treatment on the tumor. Therefore, the feasibility of this simple approach has been demonstrated, which may inspire other groups to implement simple procedures to further explore the photothermal effect.

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.

In vitro human cell responses to a low-dose photodynamic treatment vs. mild H2O2 exposure.

Photodynamic treatments allow control of the amount of reactive oxygen species (ROS) produced through the photosensitizer concentration and the light dose delivered to the target. In this way low ROS doses can be achieved in situ to study cell responses related to redox regulation. In this study a comparison has been made between different cell responses to a low-dose photodynamic treatment and both low and relatively high concentrations of H2O2 in human immortalized keratinocytes (HaCaT). The obtained results show that the photodynamic treatment induces a stimulating cell response roughly equivalent to that produced by exposing cells to 10(-5)M H2O2. Higher H2O2 concentrations gave rise to concentration-dependent deleterious effects on the cell cultures. Of importance is that the photodynamic treatment did not produce genotoxic damage, as measured by micronuclei frequency, while cultures exposed to 10(-5)M H2O2 displayed a significant increase in the amount of cells with micronuclei. In summary, the low-dose photodynamic treatment promotes cell proliferation but does not incur in the excessive clastogenic lesions observed after H2O2 exposure. It is therefore proposed as a promising alternative to direct H2O2 exposure in the study of cell redox signalling.

Vehiculization determines the endocytic internalization mechanism of Zn(II)-phthalocyanine.

It is generally accepted that compounds of nanomolecular size penetrate into cells by different endocytic processes. The vehiculization strategy of a compound is a factor that could determine its uptake mechanism. Understanding the influence of the vehicle in the precise mechanism of drug penetration into cells makes possible to improve or modify the therapeutic effects. In this study, using human A-549 cells, we have characterized the possible internalization mechanism of the photosensitizer Zn(II)-phthalocyanine (ZnPc), either dissolved in dimethylformamide (ZnPc-DMF) or included in liposomes of dipalmitoyl-phosphatidyl-choline. Specific inhibitors involved in the main endocytic pathways were used. Co-incubation of cells with ZnPc-liposomes and dynasore (dinamin-mediated endocytosis inhibitor) resulted in a significant decrease of photodamage, whereas other inhibitors did not alter the photodynamic effect of ZnPc. On the contrary, cells treated with ZnPc-DMF in the presence of dynasore, genistein (caveolin-mediated endocytosis inhibitor) or cytochalasin D (macropinocytosis and caveolin-mediated endocytosis inhibitor) showed a significant decrease in ZnPc uptake and photodynamic damage. These results suggest that ZnPc-DMF penetrates into cells mainly by caveolin-mediated endocytosis, whereas ZnPc-liposomes are internalized into cells preferentially by clathrin-mediated endocytosis. We conclude that using different drug vehiculization systems, it is possible to modify the internalization mechanism of a therapeutic compound, which could be of great interest in clinical research.

Tumour cell death induced by the bulk photovoltaic effect of LiNbO3:Fe under visible light irradiation.

This work reports a pioneer application of the bulk photovoltaic effect in the biomedical field. Massive necrotic cell death was induced in human tumour cell cultures grown on a bulk photovoltaic material (iron-doped lithium niobate, LiNbO(3):Fe) after irradiation with visible light. Lethal doses (≈100% cell death) were obtained with low-intensity visible light sources (10-100 mW cm(-2) irradiances) and short exposure times of the order of minutes. The wavelength dependence to induce the lethal effect observed is consistent with that corresponding to the bulk photovoltaic effect generation in LiNbO(3):Fe. Necrosis also occurred when cultured tumour cells were exposed to LiNbO(3):Fe microparticles and visible light.

Do folate-receptor targeted liposomal photosensitizers enhance photodynamic therapy selectivity?

One of the current goals in photodynamic therapy research is to enhance the selective targeting of tumor cells in order to minimize the risk and the extension of unwanted side-effects caused by normal cell damage. Special attention is given to receptor mediated delivery systems, in particular, to those targeted to folate receptor. Incorporation of a model photosensitizer (ZnTPP) into a folate-targeted liposomal formulation has been shown to lead an uptake by HeLa cells (folate receptor positive cells) 2-fold higher than the non-targeted formulation. As a result, the photocytotoxicity induced by folate-targeted liposomes was improved. This selectivity was completely inhibited with an excess of folic acid present in the cell culture media. Moreover, A549 cells (folate receptor deficient cells) have not shown variations in the liposomal incorporation. Nevertheless, the differences observed were slighter than expected. Both folate-targeted and non-targeted liposomes localize in acidic lysosomes, which confirms that the non-specific adsorptive pathway is also involved. These results are consistent with the singlet oxygen kinetics measured in living cells treated with both liposomal formulations.

Photosensitization ability of a water soluble zinc(II)tetramethyltetrapyridinoporphyrazinium salt in aqueous solution and biomimetic reverse micelles medium.

The spectroscopic properties and the photodynamic activity of a highly water soluble zinc(II)tetramethyltetrapyridino[2,3-b:2',3'-g:2",3"-l:2"',3'''-q]porphyrazinium salt (ZnTM2,3PyPz) were investigated in aqueous homogeneous solution and in biomimetic reverse micelles medium bearing photooxidizable biological substrates. Absorption and fluorescence spectroscopic studies indicate that ZnTM2,3PyPz is dissolved as monomer in water and in n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT, 0.1 M)/water (W0 = 30) micellar system. Fluorescence quantum yields (phi F) of 0.29 and 0.27 were calculated for ZnTM2,3PyPz in water and in AOT micelles, respectively. Spectroscopic analysis at different AOT concentrations showed interaction between ZnTM2,3PyPz and AOT reverse micelles with a binding constant (Kb) of 1.7 x 10(3) M(-1). The photosensitization ability of ZnTM2,3PyPz was evaluated using 9,10-dimethylanthracene (DMA). Singlet molecular oxygen, O2(1 delta g), production yielded values of phi(delta) = 0.65 for ZnTM2,3PyPz in AOT micelles. Also, ZnTM2,3PyPz induced efficiently the decomposition of the amino acid L-tryptophan (Trp) and the nucleotide guanosine 5'-monophosphate (GMP) in both media. A value of approximately 3.6 x 10(7) s(-1) M(-1) was found for the second order rate constant of Trp (k(r)(Trp)) decomposition in the AOT system, which is near to that found in pure water. Moreover, ZnTM2,3PyPz formed stable complexes with GMP with a binding constant of K(GMP) = 1.0 x 10(3) M(-1). 1H NMR studies indicated that ZnTM2,3PyPz interacts mainly with the guanine moiety more than the sugar part of GMP. On the other hand, the photodynamic activity of ZnTM2,3PyPz produced decomposition of GMP. Quantification of GMP by HPLC indicates that the nucleotide is photooxidized with a k(obs)(GMP) = 2.6 x 10(-4) s(-1) in water. Photooxidation of GMP considerably increases in deuteriated water indicating that ZnTM2,3PyPz appears to perform its photosensitizing action via the intermediacy of O2(1delta g). Also, efficient sensitized decomposition was observed in micellar media resembling that in pure water. These results provide a better understanding of the effective photodynamic action produced by ZnTM2,3PyPz like a potential phototherapeutic agent for the treatment of neoplastic diseases by photodynamic therapy.

Disorganisation of cytoskeleton in cells resistant to photodynamic treatment with decreased metastatic phenotype.

The appearance of cells resistant to photodynamic therapy (PDT) is crucial for the outcome of this antitumoral treatment. We had previously isolated two sublines resistant to PDT derived from the mammary adenocarcinoma LM3 [A. Casas, C. Perotti, B. Ortel, G. Di Venosa, M. Saccoliti, A. Batlle, T. Hasan, Induction of murine tumour cell lines resistant to ALA-mediated Photodynamic Therapy, Int. J. Oncol. 29 (2006) 397-405.]. These clones have severely impaired its metastatic potential in vivo together with decreased general anchorage-dependent adhesion and invasion. In the present work we analyzed the differential expression and distribution of cytoskeleton and adhesion proteins in these cell lines. In both resistant clones, loss of actin stress fibers and disorganization of the actin cortical rim was observed. E-cadherin and beta-catenin and vinculin distribution was also disorganized. However, Western blot assays did not show differential expression of actin, E-cadherin, vinculin or beta-catenin. It was demonstrated that PDT strongly affects cell-cell and cell-substrate adhesion through the reorganization of some cytoskeletal and adhesion proteins. Changes in the metastasis phenotypes previously found are likely to be ascribed to these differences.

Caspase-2: a possible trigger of apoptosis induced in A-549 tumor cells by ZnPc photodynamic treatment.

Photodynamic therapy applied to cell cultures represents a widely accepted experimental method to investigate molecular mechanisms that lead to apoptotic cell death. In this context the subcellular localization of photosensitizers seems to be a significant factor in order to determine the apoptotic pathway that could be activated. We have characterized the experimental conditions that induce apoptotic cell death in A-549 cells incubated with ZnPc and irrradiated with red light. Previously we have found that in this cell line the drug is localized in the Golgi apparatus after 3-h incubation. Indirect immunofluorescence analysis of the events that lead to apoptosis made possible the detection of caspase-2 activation in the Golgi region immediately after photodynamic treatments. A few minutes later, the morphology of this organelle starts to disrupt and just 6 h after treatment the nuclei appear affected showing the fragmented appearance typical of apoptotic cell death. From this results we assume that following the photodynamic treatment of A-549 cells with ZnPc, the activation of caspase-2 in the Golgi apparatus could begin to initiate immediately the apoptotic process.

Fluorescent cationic probes for nuclei of living cells: why are they selective? A quantitative structure-activity relations analysis.

Selectivity of nuclear probes is controlled by competitive accumulation of the probe by cellular organelles as well as the high affinity for nucleic acids. Physicochemical features of probes which favor nucleic acid binding include cationic character and a planar aromatic system above a minimum size. Features of probes which permit entry into cells are low protein and lipid binding. Features which reduce accumulation in non-nuclear sites include high base strength and hydrophilicity of the cation. The overall quantitative structure-activity (QSAR) model specifying nuclear accumulation may be expressed as follows: CBN<40; 8>log P (neutral species)>0; AI<8; Z>0; -510; LCF>17; LCF/CBN>0.70 (where CBN is the conjugated bond number, log P (x )the logarithm of the water-octanol partition coefficient of species x, AI the amphilicity index, Z the electric charge, pK ( a ) the negative logarithm of the equilibrium constant for the free base-protonated base reaction, and LCF the largest conjugated fragment). Preliminary applications of the QSAR model--to the selection of anticancer drugs, minimization of dye and drug toxicity and the designed synthesis of fluorescent probes-are outlined.

Long-term regression of the murine mammary adenocarcinoma, LM3, by repeated photodynamic treatments using meso-tetra (4-N-methylpyridinium) porphine.

Photodynamic therapy (PDT) is based on the cytotoxic effect induced by a photosensitizer in the presence of light and molecular oxygen, with production of reactive oxygen species which cause cell death and tumor destruction. Here we describe the response of the murine mammary adenocarcinoma, LM3, to repeated PDT treatments using the synthetic porphyrin derivative, meso-tetra (4-N-methylpyridinium) porphine (TMPyP). Intradermal LM3 tumors in BALB/c mice were left untreated, only treated with light, only injected with 0.9% NaCl solution, or with TMPyP alone (10 microg in 0.1 ml of 0.9% NaCl). For PDT, the intratumoral TMPyP injection was followed 1 h later by blue-red light irradiation for 50 min (80 mW/cm2 total dose: 240 J/cm2). In all cases, control and PDT treatments were performed on the depilated and glycerol-covered skin over the tumor of anesthetized mice and repeated four times (every two days). In a pilot experiment, no significant differences were found in the growth rate of untreated tumors (n=4) and tumors only treated with light (n=4), 0.9% NaCl (n=3) or TMPyP (n=3). PDT-treated tumors (n=3) showed transitory regression and growth delay. In a second approach, the average diameter (mean, mm +/- SEM) of control (drug alone, n=15) vs PDT tumors (n=17) was 2.13+/-0.11 vs 2.02+/-0.10 at day 0, and 4.00+/-0.17 vs 0.20+/-0.07 at day 9, p<0.0001. At day 37 the average diameter of tumors from control vs the PDT group was 10.98+/-0.59 vs 6.31+/-0.82, p<0.0001. PDT caused partial regression of tumors in one from a total of 17 mice, long-term regression in 15, and cure in one animal. Significant differences in the survival and tumor size at death were found between control and PDT-treated mice. Histopathological analysis of LM3 tumors one day after a unique PDT treatment showed extensive hemorrhage and necrotic areas. These results indicate the considerable potential of intratumoral injection of photosensitizers and repeated PDT protocols.

Loss of E-cadherin mediated cell-cell adhesion as an early trigger of apoptosis induced by photodynamic treatment.

Photodynamic treatment with different photosensitizers (PSs) can result in the specific induction of apoptosis in many cell types. It is commonly accepted that this apoptotic response depends on the mitochondrial accumulation of the PS. Accumulation in other cellular organelles, such as lysosomes or the Golgi complex, and subsequent photodamage resulting in an apoptotic process has been also described. However, the role played by cell adhesion in apoptosis induced in epithelial cells after photodynamic treatment is not well characterized. Here, we have used a murine keratinocyte line, showing a strong dependence on E-cadherin for cell-cell adhesion and survival, to analyze the relevance of this adhesion complex in the context of zinc(II)-phthalocyanine (ZnPc) photodynamic treatment. We report that under apoptotic conditions, ZnPc phototreatment induces a rapid disorganization of the E-cadherin mediated cell-cell adhesion, which largely preceded both the detachment of cells from the substrate, via beta-1 integrins and the induction of apoptotic mitochondrial markers. Therefore, the alteration in E-cadherin, alpha- and beta-catenins adhesion proteins preceded the release of cytochrome c (cyt c) from mitochondria to the cytosol and the activation of caspase 3. In addition, blocking E-cadherin function with a specific antibody (Decma-1) induced apoptosis in this cell system. These results strongly suggest that the E-cadherin adhesion complex could be the primary target of ZnPc phototreatment, and that loss of E-cadherin mediated cell adhesion after early photodamage triggers an apoptotic response.

Human DNA methyltransferase 1 is required for maintenance of the histone H3 modification pattern.

DNA methyltransferase 1 (DNMT1) plays an essential role in murine development and is thought to be the enzyme primarily responsible for maintenance of the global methylation status of genomic DNA. However, loss of DNMT1 in human cancer cells affects only the methylation status of a limited number of pericentromeric sequences. Here we show that human cancer cells lacking DNMT1 display at least two important differences with respect to wild type cells: a profound disorganization of nuclear architecture, and an altered pattern of histone H3 modification that results in an increase in the acetylation and a decrease in the dimethylation and trimethylation of lysine 9. Additionally, this phenotype is associated with a loss of interaction of histone deacetylases (HDACs) and HP1 (heterochromatin protein 1) with histone H3 and pericentromeric repetitive sequences (satellite 2). Our data indicate that DNMT1 activity, via maintenance of the appropriate histone H3 modifications, contributes to the preservation of the correct organization of large heterochromatic regions.

Photodynamic toxicity and its prevention by antioxidative agents in Bufo arenarum embryos.

In this work we describe an experimental model to evaluate the photodynamic toxicity on amphibian embryos, as well as the protective effect of antioxidants against the lethal oxidative stress induced by photosensitization. Bufo arenarum embryos were treated with 10 mg/l methylene blue (MB) in AMPHITOX solution for 72 h and then irradiated with a red laser or white light for variable times. Both light sources affected the survival of MB-treated animals and lethal effects occurred within the initial 12 h post-irradiation. For white light irradiation, the most effective phototoxic condition in our study, the LD10, 50 and 90 at 6 h post-irradiation corresponded to 13.57, 19.87 and 29.10 J/cm2, respectively. To explore the action of antioxidants against the photogenerated oxidative stress, MB-treated embryos were incubated with 1mM glutathione (GSH) or ascorbic acid (AA) during 48 h before irradiation. For GSH and 21.6 J/cm2 irradiation, the survival increased from 20 to 90%, whereas 100% survival was achieved with AA even after 43.2 J/cm2 irradiation. These results indicate that both the lethal photodynamic effect and its prevention by antioxidants can be evaluated by means of a simple toxicity test employing amphibian embryos.