cAMP and Pyk2 interact to regulate prostate cell proliferation and function.

In cultured prostate cancer cells cAMP blocks proliferation and induces neuroendocrine differentiation. Pyk2 expression inversely correlates with malignancy of prostate cancer. The aim of this study was to investigate the interaction between cAMP and Pyk2 in the prostate. EPN cells, a line derived from human normal prostate expressing Pyk2, and EPN-PKM3 cells, an EPN clone bearing a Pyk2 kinase-negative mutant, were adopted as model system. cAMP inhibited cell growth in both prostate cell lines, and activated Pyk2, but not ERK1/2, in EPN cells. cAMP treatment, abolished the activation of AKT1, an important component of the pro-survival pathway, in the EPN cells but not in EPN-PKM3 cells. Finally, upon cAMP treatment, EPN and EPN-PKM3 cells exhibited different expression patterns of HOX genes, an important network controlling cell identity. These data demonstrated for the first time that Pyk2 and cAMP interact in regulating prostate cell functions and in "keeping" prostate identity

A failure mode and effect analysis (FMEA)-based approach for risk assessment of scientific processes in non-regulated research laboratories

AbstractNowadays, Quality Management tools such as failure mode and effect analysis (FMEA) are widely used throughout the aeronautical, automotive, software, food services, health care and many other industries to sustain and improve quality and safety. The increasing complexity of scientific research makes it more difficult to maintain all activities under control, in order to guarantee validity and reproducibility of results. Even in non-regulated research, scientists need to be supported with management tools that maximize study performance and outcomes, while facilitating the research process. Frequently, steps that involve human intervention are the weak links in the process. Risk analysis therefore gives considerable benefit to analytical validation, assessing and avoiding failures due to human error, potential imprecision in applying protocols, uncertainty in equipment function and imperfect control of materials. This paper describes in detail how FMEA methodology can be applied as a performance improvement tool in the field of non-regulated research, specifically on a basic Life Sciences research process. We chose as "pilot process" the selection of oligonucleotide aptamers for therapeutic purposes, as an example of a complex and multi-step process, suitable for technology transfer. We applied FMEA methodology, seeking every opportunity for error and its impact on process output, and then, a set of improvement actions was generated covering most aspects of laboratory practice, such as equipment management and staff training. We also propose a useful tool supporting the risk assessment of research processes and its outputs and that we named "FMEA strip worksheet." These tools can help scientists working in non-regulated research to approach Quality Management and to perform risk evaluation of key scientific procedures and processes with the final aim to increase and better control efficiency and efficacy of their research

Combined effects of radiotherapy and photodynamic therapy on an in vitro human prostate model.

Human prostate cancer cells were evaluated for growth after photodynamic therapy, radiotherapy, and combined treatment. Indocyanine green was tested as a photosensitizer and radiosensitizer. Two human cell lines were used: PC-3 derived from prostate carcinoma, and EPN derived from normal prostate tissue. The light source used for the photoactivation experiments was a diode laser peaked at 805 nm. The light dose incident on cells was 108 J/cm2. Ionizing radiation was produced by a linear accelerator, and the dose was 2, 4 and 6 Gy. Cytotoxicity was evaluated by measuring the colony forming ability of cells. Our results show that indocyanine green induces cell death by photoactivation, but it does not act as a radiosensitizer if used with ionizing radiation. The combined treatment of photodynamic therapy and radiotherapy produces an additive effect which does not depend on the sequence of the two treatments. Combined treatments could be more useful since they allow the reduction of the ionizing radiation dose to obtain the same effect as one obtainable by radiotherapy alone

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/cm(2)). 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

The Impact of Social and Cultural Engagement and Dieting on Well-Being and Resilience in a Group of Residents in the Metropolitan Area of Naples

Social isolation and exclusion are associated with poor health status and premature death. A number of related isolation factors, inadequate transportation system and restrictions in individuals’ life space, have been associated with malnutrition in older adults. Since eating is a social event, isolation can have a negative effect on nutrition. Cultural involvement and participation in interactive activities are essential tools to fight social isolation, and they can counteract the detrimental effects of social isolation on health. To provide data supporting the hypothesis that encouraging participation might represent an innovative preventive and health promoting strategy for healthy living and aging, we developed an ad hoc questionnaire to investigate the relationship between cultural participation, well-being, and resilience in a sample of residents in the metropolitan area of Naples. The questionnaire includes a question on adherence to diet or to a special nutritional regimen; in addition, the participants are asked to mention their height and weight. We investigated the relationship between BMI, adherence to diet, and perceived well-being (PWB) and resilience in a sample of 571 subjects over 60 years of age. Here, we present evidence that engagement into social and cultural activities is associated with higher well-being and resilience, in particular in females over 60 years of age

Convergent effects of resveratrol and PYK2 on prostate cells

Resveratrol, a dietary polyphenol, is under consideration as chemopreventive and chemotherapeutic agent for several diseases, including cancer. However, its mechanisms of action and its effects on non-tumor cells, fundamental to understand its real efficacy as chemopreventive agent, remain largely unknown. Proline-rich tyrosine kinase 2 (PYK2), a non-receptor tyrosine kinase acting as signaling mediator of different stimuli, behaves as tumor-suppressor in prostate. Since, PYK2 and RSV share several fields of interaction, including oxidative stress, we have investigated their functional relationship in human non-transformed prostate EPN cells and in their tumor-prone counterpart EPN-PKM, expressing a PYK2 dead-kinase mutant. We show that RSV has a strong biological activity in both cell lines, decreasing ROS production, inducing morphological changes and reversible growth arrest, and activating autophagy but not apoptosis. Interestingly, the PYK2 mutant increases basal ROS and autophagy levels, and modulates the intensity of RSV effects. In particular, the anti-oxidant effect of RSV is more potent in EPN than in EPN-PKM, whereas its anti-proliferative and pro-autophagic effects are more significant in EPN-PKM. Consistently, PYK2 depletion by RNAi replicates the effects of the PKM mutant. Taken together, our results reveal that PYK2 and RSV act on common cellular pathways and suggest that RSV effects on prostate cells may depend on mutational-state or expression levels of PYK2 that emerges as a possible mediator of RSV mechanisms of action. Moreover, the observation that resveratrol effects are reversible and not associated to apoptosis in tumor-prone EPN-PKM cells suggests caution for its use in humans

Resveratrol, UVB or both and HaCaT cells proliferation.

<p>Resveratrol (25 and 100 µM) was added to HaCaT cells for 2 hours (panel A) or 24 hours (panel B) and withdrawn prior to irradiation with UVB (10, 20, 40, and 100 mJ/cm²). Cell count was performed after 48 hours in culture in standard medium. Graphs report mean values ± standard deviation of three independent measurement for each experimental point. RSV: resveratrol; * p<0.05.</p

Multiple pathway analysis in HaCaT cells exposed to resveratrol, UVB or both: earlier time point (15 minutes).

<p>HaCaT cells were pretreated with 25 and 100 µM resveratrol for 2 and 24 hours prior irradiation with UVB (10, 40, and 100 mJ/cm²). Cell lysates were collected 15′ after UVB irradiation. Phosphorylated or not phosphorylated protein levels were analyzed by western blot. The figure shows representative blots analyzing the levels of the phosphorylated forms of ERK1/2, p38, p53, AKT, and S6, and the levels of BAX, and Bcl2.GAPDH was used as loading control.</p

Relative contribution of apoptosis and autophagy to resveratrol enhanced UVB-induced HaCaT cells death.

<p>(A) HaCaT cells were pretreated for 24 hours with 25 µM resveratrol followed by 2 hours in the presence or absence of pan caspase inhibitor Z-VAD (50 µM) prior to irradiation with UVB (30 mJ/cm²). Cell count was performed after additional 24 hours of culture in standard medium. Graphs report mean values ± standard deviation of three independent measurements for each experimental point. RSV: resveratrol; * p<0.05. (B) “Total” represents the difference of cell number between UVB-irradiated HaCaT cells and resveratrol pre-treated and UVB-irradiated HaCaT cells; “Apoptotic” represent the difference of cell number between resveratrol/Z-VAD pre-treated/UVB-irradiated HaCaT cells and resveratrol pre-treated and UVB-irradiated HaCaT cells; Both “Total” and “Apoptotic” have been reported as percentage of “Total”(100% and 45% respectively). “Non-apoptotic” (55%) represent the difference of the percentages between “Total” and “Apoptotic”.</p