Annamaria Kisslinger

Hypericin photosensitization of tumor and metastatic cell lines of human prostate

We have investigated the photoactivating effect of hypericin on two cancer cell lines: PC-3, a prostatic adenocarcinoma non-responsive to androgen therapy and LNCaP, a lymphonodal metastasis of prostate carcinoma responsive to androgen therapy. The two cell lines are incubated for 24 h with hypericin at concentrations ranging from 0.001 to 0.3 microg/ml in cell culture medium. The cells are irradiated at 599 nm (fluence = 11 J/cm2) using a dye laser pumped by an argon laser. Hypericin exerts phototoxic effects on both cell lines, while it does not produce toxic effects in the absence of irradiation. These results suggest that photodynamic therapy (PDT) with hypericin could be an alternative approach to the treatment of prostatic tumors, and could be beneficial in tumors that are non-responsive to androgen therapy.

EPN: A NOVEL EPITHELIAL CELL LINE DERIVED FROM HUMAN PROSTATE TISSUE

SummaryThis work reports the isolation and characterization of a line of human, nontransformed and differentiated prostate epithelial cells (EPN) in continuous culture. Primary cultures of epithelial prostate cells were set up using normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer. After 70 passages, EPN cells did not undergo “Hayflike, crisis” and were free of fibroblast contamination and were thus subcloned and characterized. EPN cells in culture, as prostate epithelial cells in vivo, express high-molecular weight cytokeratin and Pyk2, whereas they do not express desmin. EPN cells are nontransformed because they do not form colonies in semisolid medium and do not form tumors once injected into nude mice. EPN cells express the functional androgen receptors, which can mediate the mitogenic activity of testosterone. Finally, clonal production of the prostate-specific antigen could be detected in EPN cells. The availability of a line of epithelial nontransformed prostate cell in culture will be useful in investigating the complex process regulating normal prostate physiology as well as the development and progression of prostate tumors.

Resveratrol couples apoptosis with autophagy in UVB-irradiated HaCaT cells.

UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm2). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation.

cAMP induced modifications of HOX D gene expression in prostate cells allow the identification of a chromosomal area involved in vivo with neuroendocrine differentiation of human advanced prostate cancers

The acquisition of epithelial‐neuroendocrine differentiation (ND) is a peculiarity of human advanced, androgen‐independent, prostate cancers. The HOX genes are a network of transcription factors controlling embryonal development and playing an important role in crucial adult eukaryotic cell functions. The molecular organization of this 39‐gene network is unique in the genome and probably acts by regulating phenotype cell identity. The expression patterns of the HOX gene network in human prostate cell phenotypes, representing different stages of prostate physiology and prostate cancer progression, make it possible to discriminate between different human prostate cell lines and to identify loci and paralogous groups harboring the HOX genes mostly involved in prostate organogenesis and cancerogenesis. Exposure of prostate epithelial phenotypes to cAMP alters the expression of lumbo‐sacral HOX D genes located on the chromosomal region 2q31‐33 where the cAMP effector genes CREB1, CREB2, and cAMP‐GEFII are present. Interestingly, this same chromosomal area harbors: (i) a global cis‐regulatory DNA control region able to coordinate the expression of HOX D and contiguous phylogenetically unrelated genes; (ii) a prostate specific ncRNA gene associated with high‐risk prostate cancer (PCGEM1); (iii) a series of neurogenic‐related genes involved with epithelial‐neuronal cell conversion. We report the expression of neurexin 1, Neuro D1, dlx1, and dlx2 in untreated and cAMP treated epithelial prostate cells. The in vivo expression of Neuro D1 in human advanced prostate cancers correlate with the state of tumor differentiation as measured by Gleason score. Thus, we suggest that the chromosomal area 2q 31‐33 might be involved in the epithelial‐ND characteristic of human advanced prostate cancers.

Proline-rich tyrosine kinase 2 regulates proliferation and differentiation of prostate cells.

Proline-rich tyrosine kinase 2 (Pyk2) expression in prostate epithelium inversely correlated with degree of malignancy of prostate cancers, thus the role of Pyk2 in the regulation of prostate cells proliferation and differentiation was investigate in PC3 cells. Pyk2 can be activated by canonic stimuli such as tumor necrosis factoralpha and lysophosphatidic acid (LPA) in PC3 cells, in addition, LPA stimulated Pyk2 phosphorylation also induced extracellular signal-regulated kinase 1 and 2 activation in these cells. Proliferation of PC3 cell clones (PC3-PKM) expressing a dominant negative kinase-defective Pyk2 mutant is consistently decreased in respect to that of wild type PC3 cells. In addition, PC3-PKM clones underwent total block cell proliferation upon treatment with dibutyryl cAMP. Finally, in the presence of sustained levels of intracellular cAMP, PC3-PKM cells, but not wild type PC3 cells, acquired a neuron-like morphology. Taken together our results suggest that Pyk2 plays a role in the regulation of prostate cell proliferation and, more interestingly, its expression may represents a sensitive marker of prostate state of differentiation.

Multiple processor version of a Monte Carlo code for photon transport in turbid media

Abstract Although Monte Carlo (MC) simulations represent an accurate and flexible tool to study the photon transport in strongly scattering media with complex geometrical topologies, they are very often infeasible because of their very high computation times. Parallel computing, in principle very suitable for MC approach because it consists in the repeated application of the same calculations to unrelated and superposing events, offers a possible approach to overcome this problem. It was developed an MC multiple processor code for optical and IR photon transport which was run on the parallel processor computer CRAY-T3E (128 DEC Alpha EV5 nodes, 600 Mflops) at CINECA (Bologna, Italy). The comparison between single processor and multiple processor runs for the same tissue models shows that the parallelization reduces the computation time by a factor of about N , where N is the number of used processors. This means a computation time reduction by a factor ranging from about 10 2 (as in our case where 128 processors are available) up to about 10 3 (with the most powerful parallel computers with 1024 processors). This reduction could make feasible MC simulations till now impracticable. The scaling of the execution time of the parallel code, as a function of the values of the main input parameters, is also evaluated.

Overexpression of chromatin assembly factor-1 (CAF-1) p60 is predictive of adverse behaviour of prostatic cancer

Aims:  Prostatic cancer may remain organ‐confined indefinitely; in a number of patients, however it gives rise to clinical symptoms and death. The biological behaviour of this tumour mostly remains difficult to predict. A promising tool for diagnosis and prognosis of some human tumours is the chromatin assembly factor‐1 (CAF‐1), involved in the control of higher order chromatin organization. The aim was to explore the role of CAF‐1/p60 protein as a new prognostic marker for prostatic cancer.

Loss of proline-rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells†

Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline‐rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non‐transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E‐cadherin and IRS1 and an increase of the expression of α5‐integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell–cell and cell–substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype. J. Cell. Physiol. 209: 74–80, 2006.

Resveratrol regulates p66Shc activation in HaCaT cells.

Please cite this paper as: Resveratrol regulates p66Shc activation in HaCaT cells. Experimental Dermatology 2010; 19: 895–903.

Cell photosensitization by 5-iminodaunomycin activated with red light

5-Iminodaunomycin, an anthracycline antitumor drug exhibiting an absorption peak at 595 nm, is shown to photosensitize in vitro cell kill. The photoactivation is performed irradiating the culture dishes during the incubation with the drug for 2 h with 34 mW/cm2 intensity, that is with light doses of up to 245 J/cm2. Long-term effects of administering 50 ng/ml and light for 2 h are studied in terms of growth curves. We show that photoactivation enhances the dark toxicity by a factor of about 10. Immediate cell death is produced by irradiating the cells in the presence of higher drug concentrations (e.g., 1000 ng/ml) which, however, are not toxic in the short term if administered in the dark. The viable cell percentage decreases at increasing light doses, being about 0.6% at the maximum dosage. Administering lower light doses, such as 30 J/cm2, which corresponds to an exposure duration of 15 min, has a short-term effect on the cell survival that strongly depends on the timing of the exposures within the incubation period.