Bcl-2 dependent modulation of Hippo pathway in cancer cells.

INTRODUCTION: Bcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2. METHODS: We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts. RESULTS: We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation. CONCLUSIONS: We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

Bcl-2 family inhibitors sensitize human cancer models to therapy.

BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor, was approved by FDA for the treatment of several hematological malignancies. We have recently discovered IS21, a novel pan BH3 mimetic with preclinical antitumor activity in several tumor types. Here, we evaluated the efficacy of IS21 and other BH3 mimetics, both as single agents and combined with the currently used antineoplastic agents in T-cell acute lymphoblastic leukemia, ovarian cancer, and melanoma. IS21 was found to be active in T-cell acute lymphoblastic leukemia, melanoma, lung, pancreatic, and ovarian cancer cell lines. Bcl-xL and Mcl-1 protein levels predicted IS21 sensitivity in melanoma and ovarian cancer, respectively. Exploring IS21 mechanism of action, we found that IS21 activity depends on the presence of BAX and BAK proteins: complexes between Bcl-2 and Bcl-xL proteins and their main binding partners were reduced after IS21 treatment. In combination experiments, BH3 mimetics sensitized leukemia cells to chemotherapy, ovarian cancer cells and melanoma models to PARP and MAPK inhibitors, respectively. We showed that this enhancing effect was related to the potentiation of the apoptotic pathway, both in hematologic and solid tumors. In conclusion, our data suggest the use of inhibitors of anti-apoptotic proteins as a therapeutic strategy to enhance the efficacy of anticancer treatment.

Discussing end-of-life care issues with terminally ill patients and their relatives: comparisons among physicians, nurses and psychologists.

BACKGROUND AND AIMS: The aim of this study was to analyse the end-of-life topics most frequently discussed by Italian physicians, nurses and psychologists with terminally ill patients and their relatives. Findings were compared with the levels of communication reported by physicians in other countries involved in the EURELD research project, in Europe and elsewhere. METHODS: An ad hoc questionnaire was prepared to measure levels of communication and administered to 716 professionals (181 physicians, 454 nurses and 81 psychologists) employed in geriatric hospital wards, hospices and nursing homes, or registered with professional associations in the Veneto and Trentino Alto-Adige regions of north-east Italy. Statistical analyses (frequency analysis, multivariate logistic regression) were conducted on data from questionnaires returned by standard mail or email. RESULTS: Communication levels vary for the various end-of-life issues which physicians, nurses and psychologists are required to discuss and the individuals with whom they deal. Italian physicians are more communicative with relatives than with patients, whereas psychologists tend to discuss these problems more with patients than with members of their families. Nurses behave in much the same way with both patients and relatives. By comparison with their colleagues elsewhere in Europe, Italian physicians reveal more evident differences in their willingness to discuss end-of-life issues, depending on whether they are communicating with patients or relatives. Having received bio-ethical training helps physicians communicate with their patients. CONCLUSIONS: Communicating is a fundamental part of providing care for terminally ill patients and support for their families. The patient care process involves several kinds of professionals, who are all increasingly called upon to be prepared to discuss the end of a patient's life, and to develop a therapeutic relationship which includes communicating without evading any of the aspects (and problems) relating to this crucial final stage of an individual's life.

Physicians' and nurses' experiences of end-of-life decision-making in geriatric settings.

BACKGROUND AND AIMS: In Italy there is a paucity of empirical data on practices concerning end-of-life decisions (ELDs) in geriatrics. We aimed to investigate the frequency and characteristics of ELDs made by Italian physicians and nurses in the geriatric setting. METHODS: In 2009, an anonymous questionnaire was sent to 54 geriatric units, 21 hospices, and 382 nursing homes in the Veneto and Trentino Alto Adige regions, and to professionals in the area who are members of the Italian Gerontology and Geriatrics Association. RESULTS: This paper reports the results of 552 questionnaires answered by 171 physicians, 368 nurses and 13 professionals who did not state their profession. Death was preceded by decisions to start or continue treatments likely to prolong the patients' life in 51.3% of cases. The proportion of deaths preceded by a decision to end life (DEL) was 20.8%; 18% of DELs concerned non-treatment decisions. There were 9 cases of ending of life without patient's explicit request. No cases of doctor-assisted suicide were reported, while there were 2 cases of euthanasia, one reported by a physician and one by a nurse. CONCLUSION: In geriatrics, DELs often precede the deaths of terminally-ill Italian patients. Nurses report making DELs more often than physicians, especially in incompetent patients. Continuous deep sedation was adopted by 39.5% of the Italian physicians for deaths not occurring suddenly and unexpectedly. Our report on physicians' and nurses' experiences of ELD making in geriatric settings can offer a valuable contribution to the current debate on end-of-life treatment, an issue that goes beyond national borders.

Targeting the anti-apoptotic Bcl-2 family proteins: machine learning virtual screening and biological evaluation of new small molecules.

Bcl-2 family anti-apoptotic proteins are overexpressed in several hematological and solid tumors, and contribute to tumor formation, progression, and resistance to therapy. They represent a promising therapeutic avenue to explore for cancer treatment. Venetoclax, a Bcl-2 inhibitor is currently used for hematological malignancies or is undergoing clinical trials for either hematological or solid tumors. Despite these progresses, ongoing efforts are focusing on the identification and development of new molecules targeting Bcl-2 protein and/or other family members. Methods: Machine learning guided virtual screening followed by surface plasmon resonance, molecular docking and pharmacokinetic analyses were performed to identify new inhibitors of anti-apoptotic members of Bcl-2 family and their pharmacokinetic profile. The sensitivity of cancer cells from different origin to the identified compounds was evaluated both in in vitro (cell survival, apoptosis, autophagy) and in vivo (tumor growth in nude mice) preclinical models. Results: IS20 and IS21 were identified as potential new lead compounds able to bind Bcl-2, Bcl-xL and Mcl-1 recombinant proteins. Molecular docking investigation indicated IS20 and IS21 could bind into the Beclin-1 BH3 binding site of wild type Bcl-2, Bcl-xL and Mcl-1 proteins. In particular, although the IS21 docked conformation did not show a unique binding mode, it clearly showed its ability in flexibly adapting to either BH3 binding sites. Moreover, both IS20 and IS21 reduced cell viability, clonogenic ability and tumor sphere formation, and induced apoptosis in leukemic, melanoma and lung cancer cells. Autophagosome formation and maturation assays demonstrated induction of autophagic flux after treatment with IS20 or IS21. Experiments with z-VAD-fmk, a pan-caspase inhibitor, and chloroquine, a late-stage autophagy inhibitor, demonstrated the ability of the two compounds to promote apoptosis by autophagy. IS21 also reduced in vivo tumor growth of both human leukemia and melanoma models. Conclusion: Virtual screening coupled with in vitro and in vivo experimental data led to the identification of two new promising inhibitors of anti-apoptotic proteins with good efficacy in the binding to recombinant Bcl-2, Bcl-xL and Mcl-1 proteins, and against different tumor histotypes.

Bcl-2-like protein-10 increases aggressive features of melanoma cells.

Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated. Methods: Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis. Results: Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR. Conclusions: Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.

SEMAPHORINS and their receptors: focus on the crosstalk between melanoma and hypoxia.

Hypoxia, a condition of oxygen deprivation, is considered a hallmark of tumor microenvironment regulating several pathways and promoting cancer progression and resistance to therapy. Semaphorins, a family of about 20 secreted, transmembrane and GPI-linked glycoproteins, and their cognate receptors (plexins and neuropilins) play a pivotal role in the crosstalk between cancer and stromal cells present in the tumor microenvironment. Many studies reported that some semaphorins are involved in the development of a permissive tumor niche, guiding cell-cell communication and, consequently, the development and progression, as well as the response to therapy, of different cancer histotypes, including melanoma.In this review we will summarize the state of art of semaphorins regulation by hypoxic condition in cancer with different origin. We will also describe evidence about the ability of semaphorins to affect the expression and activity of transcription factors activated by hypoxia, such as hypoxia-inducible factor-1. Finally, we will focus our attention on findings reporting the role of semaphorins in melanocytes transformation, melanoma progression and response to therapy. Further studies are necessary to understand the mechanisms through which semaphorins induce their effect and to shed light on the possibility to use semaphorins or their cognate receptors as prognostic markers and/or therapeutic targets in melanoma or other malignancies.

Antitumor effect of Melaleuca alternifolia essential oil and its main component terpinen-4-ol in combination with target therapy in melanoma models.

Essential oils (EOs) have been recently emerging for their promising biological activities in preventing tumorigenesis or progression of different tumor histotypes, including melanoma. In this study, we investigated the antitumor activity of a panel of EOs in different tumor models. The ability of Melaleuca alternifolia (tea tree oil) and its main component, terpinen-4-ol, to sensitize the target therapy currently used for melanoma treatment was also assessed. Our results demonstrated that EOs differently affect the viability of human cancer cells and led us to select six EOs effective in melanoma and lung cancer cells, without toxic effects in human fibroblasts. When combined with dabrafenib and/or trametinib, Melaleuca alternifolia synergistically reduced the viability of melanoma cells by activating apoptosis. Through machine learning classification modeling, α-terpineol, tepinolene, and terpinen-4-ol, three components of Melaleuca alternifolia, were identified as the most likely relevant components responsible for the EO's antitumor effect. Among them, terpinen-4-ol was recognized as the Melaleuca alternifolia component responsible for its antitumor and proapoptotic activity. Overall, our study holds promise for further analysis of EOs as new anticancer agents and supports the rationale for their use to improve target therapy response in melanoma.

Inhibition of lysine acetyltransferases impairs tumor angiogenesis acting on both endothelial and tumor cells.

BACKGROUND: Understanding the signalling pathways involved in angiogenesis, and developing anti-angiogenic drugs are one of the major focuses on cancer research. Herein, we assessed the effect of CPTH6, a lysine acetyltransferase inhibitor and anti-tumoral compound, on angiogenesis-related properties of both endothelial and cancer cells. METHODS: The in vitro effect of CPTH6 on protein acetylation and anti-angiogenic properties on endothelial and lung cancer cells was evaluated via wound healing, trans-well invasion and migration, tube formation, immunoblotting and immunofluorescence. Matrigel plug assay, zebrafish embryo and mouse xenograft models were used to evaluate in vivo anti-angiogenic effect of CPTH6. RESULTS: CPTH6 impaired in vitro endothelial angiogenesis-related functions, and decreased the in vivo vascularization both in mice xenografts and zebrafish embryos. Mechanistically, CPTH6 reduced α-tubulin acetylation and induced accumulation of acetylated microtubules in the perinuclear region of endothelial cells. Interestingly, CPTH6 also affected the angiogenesis-related properties of lung cancer cells, and conditioned media derived from CPTH6-treated lung cancer cells impaired endothelial cells morphogenesis. CPTH6 also modulated the VEGF/VEGFR2 pathway, and reshaped cytoskeletal organization of lung cancer cells. Finally, anti-migratory effect of CPTH6, dependent on α-tubulin acetylation, was also demonstrated by genetic approaches in lung cancer cells. CONCLUSION: Overall, this study indicates that α-tubulin acetylation could play a role in the anti-angiogenic effect of CPTH6 and, more in general, it adds information to the role of histone acetyltransferases in tumor angiogenesis, and proposes the inhibition of these enzymes as an antiangiogenic therapy of cancer.

Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages.

BACKGROUND: A bidirectional crosstalk between tumor cells and the surrounding microenvironment contributes to tumor progression and response to therapy. Our previous studies have demonstrated that bcl-2 affects melanoma progression and regulates the tumor microenvironment. The aim of this study was to evaluate whether bcl-2 expression in melanoma cells could influence tumor-promoting functions of tumor-associated macrophages, a major constituent of the tumor microenvironment that affects anticancer immunity favoring tumor progression. METHODS: THP-1 monocytic cells, monocyte-derived macrophages and melanoma cells expressing different levels of bcl-2 protein were used. ELISA, qRT-PCR and Western blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Chromatin immunoprecipitation assay was performed to evaluate transcription factor recruitment at specific promoters. Boyden chamber was used for migration experiments. Cytofluorimetric and immunohistochemical analyses were carried out to evaluate infiltrating macrophages and T cells in melanoma specimens from patients or mice. RESULTS: Higher production of tumor-promoting and chemotactic factors, and M2-polarized activation was observed when macrophages were exposed to culture media from melanoma cells overexpressing bcl-2, while bcl-2 silencing in melanoma cells inhibited the M2 macrophage polarization. In agreement, the number of melanoma-infiltrating macrophages in vivo was increased, in parallel with a greater expression of bcl-2 in tumor cells. Tumor-derived interleukin-1ß has been identified as the effector cytokine of bcl-2-dependent macrophage reprogramming, according to reduced tumor growth, decreased number of M2-polarized tumor-associated macrophages and increased number of infiltrating CD4+IFNγ+ and CD8+IFNγ+ effector T lymphocytes, which we observed in response to in vivo treatment with the IL-1 receptor antagonist kineret. Finally, in tumor specimens from patients with melanoma, high bcl-2 expression correlated with increased infiltration of M2-polarized CD163+ macrophages, hence supporting the clinical relevance of the crosstalk between tumor cells and microenvironment. CONCLUSIONS: Taken together, our results show that melanoma-specific bcl-2 controls an IL-1ß-driven axis of macrophage diversion that establishes tumor microenvironmental conditions favoring melanoma development. Interfering with this pathway might provide novel therapeutic strategies.

microRNA-378a-5p iS a novel positive regulator of melanoma progression.

Evaluating the expression levels of miR-378a-5p both in a large melanoma patient cohort from The Cancer Genome Atlas database and in melanoma patients from our Institute, we found that miR-378a-5p is upregulated in metastatic melanoma specimens. miR-378a-5p expression was also increased in melanoma cells resistant to target therapy, and decreased in response to drug treatment. We also demonstrated that overexpression of miR-378a-5p enhances in vitro cell invasion and migration, and facilitates the ability of melanoma cells to form de novo vasculogenic structures. While performing downstream targeting studies, we confirmed the ability of miR-378a-5p to modulate the expression of known target genes, such as SUFU, FUS-1, and KLF9. Luciferase-3'UTR experiments also identified STAMBP and HOXD10 as new miR-378a-5p target genes. MMP2 and uPAR, two HOXD10 target genes, were positively regulated by miR-378a-5p. Genetic and pharmacologic approaches inhibiting uPAR expression and activity evidenced that the in vitro tumor-promoting functions of miR-378a-5p, were in part mediated by uPAR. Of note miR-378a-5p was also able to increase VEGF, as well as in vitro and in vivo angiogenesis. Finally, genetic and pharmacologic modulation of Bcl-2 evidenced Bcl-2 ability to regulate miR-378a-5p expression. In conclusion, to the best of our knowledge, this is the first study demonstrating that miR-378a-5p acts as an oncogenic microRNA in melanoma.

DNA Hydroxymethylation Levels Are Altered in Blood Cells From Down Syndrome Persons Enrolled in the MARK-AGE Project.

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the aging process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work, we investigated the levels of 5-hydroxymethylcytosine and of the Ten-eleven translocation dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in aging. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease in 5-hydroxymethylcytosine, TET1, and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS.

Semaphorin 5A drives melanoma progression: role of Bcl-2, miR-204 and c-Myb.

BACKGROUND: Melanoma, the most aggressive form of skin cancer, is characterized by high rates of metastasis, drug resistance and mortality. Here we investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation. METHODS: Western blotting, qRT-PCR, Chromatin immunoprecipitation (ChIP) assay, immunohistochemistry of melanoma patient specimens and xenograft tissues, in vitro Transwell assay for cell migration and invasion evaluation, in vitro capillary-like structure formation analysis. RESULTS: A significant correlation of Sema5A mRNA expression and melanoma progression was observed by analyzing GEO profile dataset. Endogenous Sema5A protein was detected in 95% of human melanoma cell lines tested, in 70% of metastatic specimens from patients affected by melanoma, and 16% of in situ melanoma specimens showed a focal positivity. We demonstrated that Sema5A regulates in vitro cell migration and invasion and the formation of vasculogenic structures. We also found an increase of Sema5A at both mRNA and protein level after forced expression of Bcl-2. By use of transcriptional and proteasome inhibitors, we showed that Bcl-2 increases the stability of Sema5A mRNA and protein. Moreover, by ChIP we demonstrated that Sema5A expression is under the control of the transcription factor c-Myb and that c-Myb recruitment on Sema5A promoter is increased after Bcl-2 overexpression. Finally, a concomitant decrease in the expression of Sema5A, Bcl-2 and c-Myb proteins was observed in melanoma cells after miR-204 overexpression. CONCLUSION: Overall our data provide evidences supporting the role of Sema5A in melanoma progression and the involvement of Bcl-2, miR-204 and c-Myb in regulating its expression.

Advanced dementia: opinions of physicians and nurses about antibiotic therapy, artificial hydration and nutrition in patients with different life expectancies.

AIM: The aim of the present study was to investigate the proportion of physicians and nurses who agree with the administration of antibiotic therapy (AT), artificial hydration (AH), and artificial nutrition (AN) in patients with advanced dementia and different life expectancies. Furthermore, we aimed at analyzing the correlates of the opinion according to which medical treatments should no longer be given to advanced dementia patients once their life expectancy falls. METHODS: End-of-life decisions and opinions were measured with a questionnaire that was sent to geriatric units, hospices and nursing homes in three different regions of Italy. Multivariate logistic regressions were carried out to ascertain the correlates of the agreement with the administration of AH, AT or AN. RESULTS: When the patient's life expectancy was 1-6 months, 83% of respondents agreed with AH, 79% with AT and 71% with AN. When the life expectancy was less than 1 month, a large proportion of respondents still agreed with AH and AT (73% and 61%), whereas less than one in two respondents (48%) agreed with AN. CONCLUSIONS: The findings of the present study showed that AN creates more ethical dilemmas in the clinical management of end of life than other treatments, such as AH or AT. Opinions on whether or not these practices are appropriate at the end of life were related with feelings, thoughts and ethical issues that played a different part for physicians and nurses. Geriatr Gerontol Int 2017; 17: 487-493.

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study.

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.

5mC-hydroxylase activity is influenced by the PARylation of TET1 enzyme.

5-hydroxymethylcytosine is a new epigenetic modification deriving from the oxidation of 5-methylcytosine by the TET hydroxylase enzymes. DNA hydroxymethylation drives DNA demethylation events and is involved in the control of gene expression. Deregulation of TET enzymes causes developmental defects and is associated with pathological conditions such as cancer. Little information thus far is available on the regulation of TET activity by post-translational modifications. Here we show that TET1 protein is able to interact with PARP-1/ARTD1 enzyme and is target of both noncovalent and covalent PARylation. In particular, we have demonstrated that the noncovalent binding of ADP-ribose polymers with TET1 catalytic domain decreases TET1 hydroxylase activity while the covalent PARylation stimulates TET1 enzyme. In addition, TET1 activates PARP-1/ARTD1 independently of DNA breaks. Collectively, our results highlight a complex interplay between PARylation and TET1 which may be helpful in coordinating the multiple biological roles played by 5-hydroxymethylcytosine and TET proteins.

PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression.

To overcome cancer cells resistance to pharmacological therapy, the development of new therapeutic approaches becomes urgent. For this purpose, the use of poly(ADP-ribose) polymerase (PARP) inhibitors in combination with other cytotoxic agents could represent an efficacious strategy. Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification that plays a well characterized role in the cellular decisions of life and death. Recent findings indicate that PARP-1 may control the expression of Snail, the master gene of epithelial-mesenchymal transition (EMT). Snail is highly represented in different resistant tumors, functioning as a factor regulating anti-apoptotic programmes. MDA-MB-231 is a Snail-expressing metastatic breast cancer cell line, which exhibits chemoresistance properties when treated with damaging agents. In this study, we show that the PARP inhibitor ABT-888 was capable to modulate the MDA-MB-231 cell response to doxorubicin, leading to an increase in the rate of apoptosis. Our further results indicate that PARP-1 controlled Snail expression at transcriptional level in cells exposed to doxorubicin. Given the increasing interest in the employment of PARP inhibitors as chemotherapeutic adjuvants, our in vitro results suggest that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells in vivo, is targeting Snail expression thus promoting apoptosis.

Artificial nutrition and hydration in terminally ill patients with advanced dementia: opinions and correlates among Italian physicians and nurses.

BACKGROUND: Although their benefits are controversial, artificial nutrition and hydration are often administered as a form of basic care to terminally ill patients. An important reason for this may be that these treatments have strong emotional and psychological meanings. AIMS: In the present article we investigated the opinions of Italian physicians and nurses on the administration of artificial nutrition and hydration to terminally ill patients with advanced dementia. We also investigated the antecedents of these opinions, considering feelings and thoughts related to death, ethical issues and training in palliative care. METHOD: A questionnaire was administered to Italian physicians (n=288) and nurses (n=763). We analyzed the percentages of agreement with the administration of artificial nutrition and hydration and, using multivariate logistic regressions, the possible antecedents of these opinions. RESULTS: Agreement with the provision of artificial hydration was higher (73%) than for artificial nutrition (48%), suggesting that artificial hydration may be seen as a form of basic care. Agreement with their administration was generally lower among professionals in northern Italy working in geriatrics wards who had received training in palliative care. We also found that death-related feelings and thoughts and ethical issues played a different part for physicians and nurses. CONCLUSIONS: Our findings suggest that opinions concerning artificial nutrition and hydration not only derive from scientific background, but also relate to cultural, ethical, and psychological issues. Our results also reveal important differences between physicians' and nurses' opinions, providing useful information for interpreting and overcoming obstacles to the effective cooperation between these professionals.

Poly(ADP-ribosyl)ation is involved in the epigenetic control of TET1 gene transcription.

TET enzymes are the epigenetic factors involved in the formation of the sixth DNA base 5-hydroxymethylcytosine, whose deregulation has been associated with tumorigenesis. In particular, TET1 acts as tumor suppressor preventing cell proliferation and tumor metastasis and it has frequently been found down-regulated in cancer. Thus, considering the importance of a tight control of TET1 expression, the epigenetic mechanisms involved in the transcriptional regulation of TET1 gene are here investigated. The involvement of poly(ADP-ribosyl)ation in the control of DNA and histone methylation on TET1 gene was examined. PARP activity is able to positively regulate TET1 expression maintaining a permissive chromatin state characterized by DNA hypomethylation of TET1 CpG island as well as high levels of H3K4 trimethylation. These epigenetic modifications were affected by PAR depletion causing TET1 down-regulation and in turn reduced recruitment of TET1 protein on HOXA9 target gene. In conclusion, this work shows that PARP activity is a transcriptional regulator of TET1 gene through the control of epigenetic events and it suggests that deregulation of these mechanisms could account for TET1 repression in cancer.

Does the Multidimensional Prognostic Index (MPI), based on a Comprehensive Geriatric Assessment (CGA), predict mortality in cancer patients? Results of a prospective observational trial.

OBJECTIVE: Despite the lack of definitive data on the impact of Comprehensive Geriatric Assessment (CGA) in the geriatric oncology setting, the broad use of any form of CGA is strongly recommended before any treatment decision in elderly cancer patients (ECP); currently there is no consensus about the best format for this geriatric assessment. The aim of this study was to firstly test the Multidimensional Prognostic Index (MPI) in ECP with locally advanced or metastatic disease. MATERIALS AND METHODS: Patients aged ≥70years with inoperable or metastatic solid cancer consecutively admitted to our Program of Geriatric Oncology were assessed by a multidisciplinary team and received a basal CGA to calculate the MPI score. RESULTS: A hundred and sixty patients entered the study. In the Cox's regression model, MPI, CIRS-SI, BSA, GDS, MMSE, chemotherapy and a diagnosis of primary lung cancer were associated with mortality at 6 and 12months. The ROC curves confirmed the prognostic value of MPI, with the best discriminatory power for mortality at both 6 and 12months. CONCLUSION: The present study is the first to indicate that the MPI retains its prognostic value even in elderly cancer patients with advanced stage of disease. The CIRS-SI and the GDS may potentiate the prognostic value of MPI.