Effects of Resveratrol on p66Shc phosphorylation in cultured prostate cells.

There is increasing evidence that diet plays a crucial role in age-related diseases and cancer. Oxidative stress is a conceivable link between diet and diseases, thus food antioxidants, counteracting the damage caused by oxidation, are potential tools for fight age-related diseases and cancer. Resveratrol (RSV), a polyphenolic antioxidant from grapes, has gained enormous attention particularly because of its ability to induce growth arrest and apoptosis in cancer cells, and it has been proposed as both chemopreventive and therapeutic agent for cancer and other diseases. Even though the effects of RSV have been studied in prostate cancer cells and animal models, little is known about its effects on normal cells and tissues. To address this issue, we have investigated the effects of RSV on EPN cells, a human non-transformed prostate cell line, focusing on the relationship between RSV and p66Shc, a redox enzyme whose activities strikingly intersect those of RSV. p66Shc activity is regulated by phosphorylation of serine 36 (Ser36) and has been related to mitochondrial oxidative stress, apoptosis induction, regulation of cell proliferation and migration. Here we show that RSV inhibits adhesion, proliferation and migration of EPN cells, and that these effects are associated to induction of dose- and time-dependent p66Shc-Ser36 phosphorylation and ERK1/2 de-phosphorylation. Moreover, we found that RSV is able to activate also p52Shc, another member of the Shc protein family. These data show that RSV affects non-transformed prostate epithelial cells and suggest that Shc proteins may be key contributors of RSV effects on prostate cells.

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.

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.

MS-2 fibrosarcoma characterization by laser induced autofluorescence.

BACKGROUND AND OBJECTIVE: MS-2 fibrosarcoma implanted in BALB-CDF1 mice was investigated by frequency and time domain measurements of the autofluorescence (AF) radiation emitted upon excitation by a N(2) laser beam (337.1 nm). STUDY DESIGN/MATERIALS AND METHODS: AF spectra were obtained by using a spectrograph, a multichannel plate and an optical multichannel analyzer for the steady state detection. Time-resolved spectra were performed by means of a monochromator, a photomultiplier, and a digital signal analyzer. RESULTS: Spectral measurements show that the autofluorescence intensity of pathologic tissue is lower than that of healthy one in the 400- to 500- spectral region. In the same spectral range, we found the fluorescence decay to be the sum of a fast and a slow component. The lifetime of the fast component of tumoral tissue is significantly lower than that of healthy samples. CONCLUSION: Frequency and time domain measurements used in combination show that MS2-fibrosarcoma is characterized by the probable presence of the free form of NADH.

In vitro photo-activation of newly synthesized chlorin derivatives with red-light-emitting diodes.

We investigated the in vitro photo-activation properties of two chlorin derivatives, i.e. 8-cis-heptylchlorin dicarboxylic acid and 3-trans-heptylchlorin bisamidoglucose derivative, which exhibit lipophilic properties similar to those of the active fractions of Photofrin II, on a normal epithelial cell line (FRTL-5). We used as an irradiation source an array of diodes emitting red light (lambda = 675 nm), which produced a fluence of 7mW cm-2 on the cells. We found that photo-activation with chlorin derivatives in the concentration range 1-100 ng ml-1 greatly enhanced the mortality of the irradiated cells (energy density, 0.25 J cm-2) with respect to the control cells kept in the dark. This response is immediate and appears to be an "all or none' effect. Taking into account that compounds exhibit a strong absorbance peak in the long wavelength region of visible light where tissues are relatively transparent, our results suggest that chlorins can be considered to be good candidates for application in photodynamic therapy.

Kinetic transport analysis of daunorubicin by LoVo and LoVo/DX cells.

We describe a fluorometric technique for the measurement of transport parameters of fluorescent drugs through cellular membranes. Unlike other procedures, this method gives an accurate measure of drug accumulated in the cells and measures the fraction of free and bound drug in the cell. The kinetic parameters of transport through cellular membranes are determined using a simple three-compartment model combined with fluorescence measurements performed on the extracellular medium and on Triton-permeabilized cells during daunorubicin incorporation. With this technique we found that LoVo cells have a greater daunorubicin uptake, a similar input rate constant and a lower output rate constant than the drug-resistant LoVo/DX cells.

Fluorometric determination of the kinetics of anthracyclines uptake by cells.

Fluorometric measurements on extracellular medium are shown to allow kinetic parameters of in vitro anthracycline uptake by cells to be calculated. The method provides influx and efflux rates, as well as the time dependence of both influx and efflux. It is applied to a normal thyroid epithelial cell line (FRTL-5) and a cell line (MPTK-6) derived from the lung metastases of a thyroid carcinoma exposed to daunorubicin at concentrations within the range of 250 to 1000 ng/ml. The results show that the number of cells influences the dependence of the kinetics upon the extracellular drug concentration and that the MPTK-6 cells are endowed with very efficient efflux mechanisms.

Enhanced response to daunomycin of normal, tumor and metastatic cell lines via drug photoactivation.

We have compared the cytotoxicity of daunomycin in vitro to highly differentiated normal epithelial cells (Fisher rat thyroid cells, FRTL-5) and to two neoplastic cell lines, a thyroid carcinoma (TK-6) and its lung metastasis (MPTK-6). Whereas the cell lines are equally sensitive to the drug in the dark, if irradiated during incubation with daunomycin (86 J/cm2 at 488 nm), they become more and differently sensitive. Namely, the drug doses producing 50% mortality decrease by factors of about 22, 28 and 16 for FRTL-5, TK-6 and MPTK-6 cell lines, respectively. This result correlates with differences in drug uptake and resistance observed in the normal and neoplastic cell lines.

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.