Ras-transfected human mammary tumour cells are resistant to photodynamic therapy by mechanisms related to cell adhesion.

AIMS: Photodynamic therapy (PDT) is a treatment modality for several cancers involving the administration of a tumour-localising photosensitiser (PS) and its subsequent activation by light, resulting in tumour damage. Ras oncogenes have been strongly associated with chemo- and radio-resistance. Based on the described roles of adhesion and cell morphology on drug resistance, we studied if the differences in shape, cell-extracellular matrix and cell-cell adhesion induced by Ras transfection, play a role in the resistance to PDT. MATERIALS AND METHODS: We employed the human normal breast HB4a cells transfected with H-RAS and a panel of five PSs. KEY FINDINGS: We found that resistance to PDT of the HB4a-Ras cells employing all the PSs, increased between 1.3 and 2.5-fold as compared to the parental cells. There was no correlation between resistance and intracellular PS levels or PS intracellular localisation. Even when Ras-transfected cells present lower adherence to the ECM proteins, this does not make them more sensitive to PDT or chemotherapy. On the contrary, a marked gain of resistance to PDT was observed in floating cells as compared to adhesive cells, accounting for the higher ability conferred by Ras to survive in conditions of decreased cell-extracellular matrix interactions. HB4a-Ras cells displayed disorganisation of actin fibres, mislocalised E-cadherin and vinculin and lower expression of E-cadherin and ß1-integrin as compared to HB4a cells. SIGNIFICANCE: Knowledge of the mechanisms of resistance to photodamage in Ras-overexpressing cells may lead to the optimization of the combination of PDT with other treatments.

In Vivo Ischemia Detection by Luminescent Nanothermometers.

There is an urgent need to develop new diagnosis tools for real in vivo detection of first stages of ischemia for the early treatment of cardiovascular diseases and accidents. However, traditional approaches show low sensitivity and a limited penetration into tissues, so they are only applicable for the detection of surface lesions. Here, it is shown how the superior thermal sensing capabilities of near infrared-emitting quantum dots (NIR-QDs) can be efficiently used for in vivo detection of subcutaneous ischemic tissues. In particular, NIR-QDs make possible ischemia detection by high penetration transient thermometry studies in a murine ischemic hindlimb model. NIR-QDs nanothermometers are able to identify ischemic tissues by means of their faster thermal dynamics. In addition, they have shown to be capable of monitoring both the revascularization and damage recovery processes of ischemic tissues. This work demonstrates the applicability of fluorescence nanothermometry for ischemia detection and treatment, as well as a tool for early diagnosis of cardiovascular disease.

Unveiling in Vivo Subcutaneous Thermal Dynamics by Infrared Luminescent Nanothermometers.

The recent development of core/shell engineering of rare earth doped luminescent nanoparticles has ushered a new era in fluorescence thermal biosensing, allowing for the performance of minimally invasive experiments, not only in living cells but also in more challenging small animal models. Here, the potential use of active-core/active-shell Nd(3+)- and Yb(3+)-doped nanoparticles as subcutaneous thermal probes has been evaluated. These temperature nanoprobes operate in the infrared transparency window of biological tissues, enabling deep temperature sensing into animal bodies thanks to the temperature dependence of their emission spectra that leads to a ratiometric temperature readout. The ability of active-core/active-shell Nd(3+)- and Yb(3+)-doped nanoparticles for unveiling fundamental tissue properties in in vivo conditions was demonstrated by subcutaneous thermal relaxation monitoring through the injected core/shell nanoparticles. The reported results evidence the potential of infrared luminescence nanothermometry as a diagnosis tool at the small animal level.

Neodymium-doped LaF(3) nanoparticles for fluorescence bioimaging in the second biological window.

The future perspective of fluorescence imaging for real in vivo application are based on novel efficient nanoparticles which is able to emit in the second biological window (1000-1400 nm). In this work, the potential application of Nd(3+) -doped LaF(3) (Nd(3+) :LaF(3) ) nanoparticles is reported for fluorescence bioimaging in both the first and second biological windows based on their three main emission channels of Nd(3+) ions: (4) F(3/2) →(4) I(9/2) , (4) F(3/2) →(4) I(11/2) and (4) F(3/2) →(4) I(13/2) that lead to emissions at around 910, 1050, and 1330 nm, respectively. By systematically comparing the relative emission intensities, penetration depths and subtissue optical dispersion of each transition we propose that optimum subtissue images based on Nd(3+) :LaF(3) nanoparticles are obtained by using the (4) F3/2 →(4) I11/2 (1050 nm) emission band (lying in the second biological window) instead of the traditionally used (4) F(3/2) →(4) I(9/2) (910 nm, in the first biological window). After determining the optimum emission channel, it is used to obtain both in vitro and in vivo images by the controlled incorporation of Nd(3+) :LaF(3) nanoparticles in cancer cells and mice. Nd(3+) :LaF(3)nanoparticles thus emerge as very promising fluorescent nanoprobes for bioimaging in the second biological window.

Papel de KLF6, como factor de predicción precoz en cáncer colorrectal humano / Role of KLF6 as early predictor in human colorectal cancer

El cáncer colorrectal (CCR) constituye el segundo tipo de cáncer más frecuente en la población europea. Actualmente no existe biomarcadores moleculares que se pueden utilizar para la detección temprana del cáncer de CCR. KLF6 es un supresor tumoral relacionado con varios tipos de cánceres. Nuestra hipótesis plantea que KLF6 puede ser un excelente marcador en el diagnóstico precoz de CCR. Para estudiar la implicancia de KLF6 en el CCR, se seleccionaron 15 biopsias de cada estadio (T1,T2 y T3) de los archivos del Servicio de Anatomía Patológica del Hospital Central de la Defensa Gómez-Ulla, las cuales presentaban áreas de tejido afectado (tumor) y áreas sin afectación (no tumorales). Para ello se realizó un estudio histológico, inmunohistoquímico y RT-PCR, basada en la expresión de 3 genes, Ki67 y p53 como marcadores positivos y KLF6 como marcador en estudio. Los resultados mostraron que la expresión de KLF6 está directamente relacionada con el aumento de la malignidad celular en los adenocarcinomas, corroboradas por las RT-PCR, observándose la aparición progresiva de formas de procesado alternativo, no correspondiente a KLF6. Esta proteína, se expresó tanto a nivel citoplasmático como nuclear en los primeros estadios T1 y T2, para desaparecer a nivel nuclear en el estadio más avanzado (T3). Concluimos que KLF6 es un buen marcador tumoral de CCR, debido a que muestra patrones crecientes de expresión a nivel citoplasmático y decrecientes a nivel nuclear...

Colorectal cancer (CRC) is the second most common type of cancer in the European population. Currently there molecular biomarkers that can be used for early detection of cancer of CRC. Is a tumor suppressor KLF6 associated with several types of cancers. Our hypothesis is that KLF6 can be an excellent marker for the early diagnosis of CRC. To study the implication of KLF6 in CRC, we selected 15 biopsies of each stage (T1, T2 and T3) from the archives of the Pathology Department of Defense Central Hospital Gómez-Ulla, which had areas of affected tissue (tumor) and unaffected areas (non-tumor). This study was performed histological, immunohistochemical and RT-PCR, based on the expression of three genes, markers Ki67 and p53 as positive and as a marker in KLF6 study. The results showed that the expression of KLF6 is directly related to increased malignancy cell adenocarcinomas, corroborated by RT-PCR, showing the gradual emergence of alternative forms processing, corresponding to no KLF6. This protein was expressed both cytoplasmic and nuclear in the early stages T1 and T2, disappearing at the nuclear level in the most advanced stage (T3). KLF6 conclude that a good CRC tumor marker because it shows patterns of expression level increased cytoplasmic and nuclear level decreasing...

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.

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.

Photodynamic therapy of cancer. Basic principles and applications.

Photodynamic therapy (PDT) is a minimally invasive therapeutic modality approved for clinical treatment of several types of cancer and non-oncological disorders. In PDT, a compound with photosensitising properties (photosensitiser, PS) is selectively accumulated in malignant tissues. The subsequent activation of the PS by visible light, preferentially in the red region of the visible spectrum (lambda>or=600 nm), where tissues are more permeable to light, generates reactive oxygen species, mainly singlet oxygen ((1)O(2)), responsible for cytotoxicity of neoplastic cells and tumour regression. There are three main mechanisms described by which (1)O(2) contributes to the destruction of tumours by PDT: direct cellular damage, vascular shutdown and activation of immune response against tumour cells. The advantages of PDT over other conventional cancer treatments are its low systemic toxicity and its ability to selectively destroy tumours accessible to light. Therefore, PDT is being used for the treatment of endoscopically accessible tumours such as lung, bladder, gastrointestinal and gynaecological neoplasms, and also in dermatology for the treatment of non-melanoma skin cancers (basal cell carcinoma) and precancerous diseases (actinic keratosis). Photofrin, ALA and its ester derivatives are the main compounds used in clinical trials, though newer and more efficient PSs are being evaluated nowadays.

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.