Poncirus trifoliata (L.) Raf. Seed Extract Induces Cell Cycle Arrest and Apoptosis in the Androgen Receptor Positive LNCaP Prostate Cancer Cells.

Prostate cancer (PCa) is the second most common male cancer. Its incidence derives from the interaction between modifiable and non-modifiable factors. The progression of prostate cancer into a more aggressive phenotype is associated with chronic inflammation and increased ROS production. For their biological properties, some phytochemicals from fruits and vegetable emerge as a promise strategy for cancer progression delay. These bioactive compounds are found in the highest amounts in peels and seeds. Poncirus trifoliata (L.) Raf. (PT) has been widely used in traditional medicine and retains anti-inflammatory, anti-bacterial, and anticancer effects. The seeds of P. trifoliata were exhaustively extracted by maceration with methanol as the solvent. The cell proliferation rate was performed by MTT and flow cytometry, while the apoptosis signals were analyzed by Western blotting and TUNEL assay. P. trifoliata seed extract reduced LNCaP and PC3 cell viability and induced cell cycle arrest at the G0/G1phase and apoptosis. In addition, a reduction in the AKT/mTOR pathway has been observed together with the up-regulation of stress-activated MAPK (p38 and c-Jun N-terminal kinase). Based on the study, the anti-growth effects of PT seed extract on prostate tumor cells give indications on the potential of the phytochemical drug for the treatment of this type of cancer. However, future in-depth studies are necessary to identify which components are mainly responsible for the anti-neoplastic response.

Cdk4 Regulates Glioblastoma Cell Invasion and Stemness and Is Target of a Notch Inhibitor Plus Resveratrol Combined Treatment.

Glioblastoma multiforme (GBM) is one of the most aggressive types of cancer characterized by poor patient outcomes. To date, it is believed that the major cause of its recurrence and chemoresistance is represented by the enrichment of GBM stem cells (GSCs) sustained by the abnormal activation of a number of signaling pathways. In this study, we found that in GBM cells, treatment with low toxicity doses of the γ-secretase inhibitor RO4929097 (GSI), blocking the Notch pathway activity, in combination with resveratrol (RSV) was able to reverse the basal mesenchymal phenotype to an epithelial-like phenotype, affecting invasion and stemness interplay. The mechanism was dependent on cyclin D1 and cyclin-dependent kinase (CDK4), leading to a reduction of paxillin (Pxn) phosphorylation. Consequently, we discovered the reduced interaction of Pxn with vinculin (Vcl), which, during cell migration, transmits the intracellular forces to the extracellular matrix. The exogenous expression of a constitutively active Cdk4 mutant prevented the RSV + GSI inhibitory effects in GBM cell motility/invasion and augmented the expression of stemness-specific markers, as well as the neurosphere sizes/forming abilities in untreated cells. In conclusion, we propose that Cdk4 is an important regulator of GBM stem-like phenotypes and invasive capacity, highlighting how the combined treatment of Notch inhibitors and RSV could be prospectively implemented in the novel therapeutic strategies to target Cdk4 for these aggressive brain tumors.

Valproic acid inhibits cell growth in both MCF-7 and MDA-MB231 cells by triggering different responses in a cell type-specific manner.

BACKGROUND: Breast cancer is the second leading cause of death among women after lung cancer. Despite the improvement in prevention and in therapy, breast cancer still remains a threat, both for pre- and postmenopausal women, due to the development of drug resistance. To counteract that, novel agents regulating gene expression have been studied in both hematologic and solid tumors. The Histone Deacetylase (HDAC) inhibitor Valproic Acid (VA), used for epilepsy and other neuropsychiatric diseases, has been demonstrated a strong antitumoral and cytostatic activity. In this study, we tested the effects of Valproic Acid on the signaling pathways involved in breast cancer cells viability, apoptosis and in Reactive Oxygen Species (ROS) production using ER-α positive MCF-7 and triple negative MDA-MB-231 cells. METHODS: Cell proliferation assay was performed by MTT Cell cycle, ROS levels and apoptosis were analyzed by flow cytometry, protein levels were detected by Western Blotting. RESULTS: Cell treatment with Valproic Acid reduced cell proliferation and induced G0/G1 cell cycle arrest in MCF-7 and G2/M block in MDA-MB-231 cells. In addition, in both cells the drug enhanced the generation of ROS by the mitochondria. In MCF-7 treated cells, it has been observed a reduction in mitochondrial membrane potential, a down regulation of the anti-apoptotic marker Bcl-2 and an increase of Bax and Bad, leading to release of cytochrome C and PARP cleavage. Less consistent effects are recorded in MDA-MB-231 cells, in which the greater production of ROS, compared to MCF-7cells, involves an inflammatory response (activation of p-STAT3, increased levels of COX2). CONCLUSIONS: Our results have demonstrated that in MCF-7 cells the Valproic Acid is a suitable drug to arrest cell growth, to address apoptosis and mitochondrial perturbations, all factors that are important in determining cell fate and health. In a triple negative MDA-MB 231 cells, valproate directs the cells towards the inflammatory response with a sustained expression of antioxidant enzymes. Overall, the not always unequivocal data between the two cellular phenotypes indicate that further studies are needed to better define the use of the drug, also in combination with other chemotherapy, in the treatment of breast tumors.

Resveratrol, Epigallocatechin Gallate and Curcumin for Cancer Therapy: Challenges from Their Pro-Apoptotic Properties.

Plant-derived bioactive compounds are gaining wide attention for their multiple health-promoting activities and in particular for their anti-cancer properties. Several studies have highlighted how they can prevent cancer initiation and progression, improve the effectiveness of chemotherapy, and, in some cases, limit some of the side effects of chemotherapy agents. In this paper, we provide an update of the literature on the anti-cancer effects of three extensively studied plant-derived compounds, namely resveratrol, epigallocatechin gallate, and curcumin, with a special focus on the anti-cancer molecular mechanisms inducing apoptosis in the major types of cancers globally.

The Involvement of Natural Polyphenols in Molecular Mechanisms Inducing Apoptosis in Tumor Cells: A Promising Adjuvant in Cancer Therapy.

Various literature data show how a diet rich in vegetables could reduce the incidence of several cancers due to the contribution of the natural polyphenols contained in them. Polyphenols are attributed multiple pharmacological actions such as anti-inflammatory, anti-oxidant, antibiotic, antiseptic, anti-allergic, cardioprotective and even anti-tumor properties. The multiple mechanisms involved in their anti-tumor action include signaling pathways modulation associated with cell proliferation, differentiation, migration, angiogenesis, metastasis and cell death. Since the dysregulation of death processes is involved in cancer etiopathology, the natural compounds able to kill cancer cells could be used as new anticancer agents. Apoptosis, a programmed form of cell death, is the most potent defense against cancer and the main mechanism used by both chemotherapy agents and polyphenols. The aim of this review is to provide an update of literature data on the apoptotic molecular mechanisms induced by some representative polyphenol family members in cancer cells. This aspect is particularly important because it may be useful in the design of new therapeutic strategies against cancer involving the polyphenols as adjuvants.

Notch Signaling in Breast Tumor Microenvironment as Mediator of Drug Resistance.

Notch signaling dysregulation encourages breast cancer progression through different mechanisms such as stem cell maintenance, cell proliferation and migration/invasion. Furthermore, Notch is a crucial driver regulating juxtracrine and paracrine communications between tumor and stroma. The complex interplay between the abnormal Notch pathway orchestrating the activation of other signals and cellular heterogeneity contribute towards remodeling of the tumor microenvironment. These changes, together with tumor evolution and treatment pressure, drive breast cancer drug resistance. Preclinical studies have shown that targeting the Notch pathway can prevent or reverse resistance, reducing or eliminating breast cancer stem cells. In the present review, we will summarize the current scientific evidence that highlights the involvement of Notch activation within the breast tumor microenvironment, angiogenesis, extracellular matrix remodeling, and tumor/stroma/immune system interplay and its involvement in mechanisms of therapy resistance.

Estrogen Receptors-Mediated Apoptosis in Hormone-Dependent Cancers.

It is known that estrogen stimulates growth and inhibits apoptosis through estrogen receptor(ER)-mediated mechanisms in many cancer cell types. Interestingly, there is strong evidence that estrogens can also induce apoptosis, activating different ER isoforms in cancer cells. It has been observed that E2/ERα complex activates multiple pathways involved in both cell cycle progression and apoptotic cascade prevention, while E2/ERß complex in many cases directs the cells to apoptosis. However, the exact mechanism of estrogen-induced tumor regression is not completely known. Nevertheless, ERs expression levels of specific splice variants and their cellular localization differentially affect outcome of estrogen-dependent tumors. The goal of this review is to provide a general overview of current knowledge on ERs-mediated apoptosis that occurs in main hormone dependent-cancers. Understanding the molecular mechanisms underlying the induction of ER-mediated cell death will be useful for the development of specific ligands capable of triggering apoptosis to counteract estrogen-dependent tumor growth.

Aberrant Notch signaling in gliomas: a potential landscape of actionable converging targets for combination approach in therapies resistance.

The current therapeutic protocols and prognosis of gliomas still depend on clinicopathologic and radiographic characteristics. For high-grade gliomas, the standard of care is resection followed by radiotherapy plus temozolomide chemotherapy. However, treatment resistance develops due to different mechanisms, among which is the dynamic interplay between the tumor and its microenvironment. Different signaling pathways cause the proliferation of so-called glioma stem cells, a minor cancer cell population with stem cell-like characteristics and aggressive phenotype. In the last decades, numerous studies have indicated that Notch is a crucial pathway that maintains the characteristics of resistant glioma stem cells. Data obtained from preclinical models indicate that downregulation of the Notch pathway could induce multifaceted drug sensitivity, acting on the expression of drug-transporter proteins, inducing epithelial-mesenchymal transition, and shaping the tumor microenvironment. This review provides a brief overview of the published data supporting the roles of Notch in drug resistance and demonstrates how potential novel strategies targeting Notch could become an efficacious action to improve the therapy of high-grade glioma to overcome drug resistance.

Novel Insights into the Antagonistic Effects of Losartan against Angiotensin II/AGTR1 Signaling in Glioblastoma Cells.

New avenues for glioblastoma therapy are required due to the limited mortality benefit of the current treatments. The renin-angiotensin system (RAS) exhibits local actions and works as a paracrine system in different tissues and tumors, including glioma. The glioblastoma cell lines U-87 MG and T98G overexpresses Angiotensin II (Ang II)/Angiotensin II type I receptor (AGTR1) signaling, which enhances in vitro and in vivo local estrogen production through a direct up-regulation of the aromatase gene promoters p I.f and p I.4. In addition, Ang II/AGTR1 signaling transactivates estrogen receptor-α in a ligand-independent manner through mitogen-activated protein kinase (MAPK) activation. The higher aromatase mRNA expression in patients with glioblastoma was associated with the worst survival prognostic, according to The Cancer Genome Atlas (TCGA). An intrinsic immunosuppressive glioblastoma tumor milieu has been previously documented. We demonstrate how Ang II treatment in glioblastoma cells increases programmed death-ligand 1 (PD-L1) expression reversed by combined exposure to Losartan (LOS) in vitro and in vivo. Our findings highlight how LOS, in addition, antagonizes the previously documented neoangiogenetic, profibrotic, and immunosuppressive effects of Ang II and drastically inhibits its stimulatory effects on local estrogen production, sustaining glioblastoma cell growth. Thus, Losartan may represent an adjuvant pharmacological tool to be repurposed prospectively for glioblastoma treatment.

Nutraceuticals in the Mediterranean Diet: Potential Avenues for Breast Cancer Treatment.

The traditional Mediterranean Diet constitutes a food model that refers to the dietary patterns of the population living in countries bordering the Mediterranean Sea in the early 1960s. A huge volume of literature data suggests that the Mediterranean-style diet provides several dietary compounds that have been reported to exert beneficial biological effects against a wide spectrum of chronic illnesses, such as cardiovascular and neurodegenerative diseases and cancer including breast carcinoma. Among bioactive nutrients identified as protective factors for breast cancer, natural polyphenols, retinoids, and polyunsaturated fatty acids (PUFAs) have been reported to possess antioxidant, anti-inflammatory, immunomodulatory and antitumoral properties. The multiple anticancer mechanisms involved include the modulation of molecular events and signaling pathways associated with cell survival, proliferation, differentiation, migration, angiogenesis, antioxidant enzymes and immune responses. This review summarizes the anticancer action of some polyphenols, like resveratrol and epigallocatechin 3-gallate, retinoids and omega-3 PUFAs by highlighting the important hallmarks of cancer in terms of (i) cell cycle growth arrest, (ii) apoptosis, (iii) inflammation and (iv) angiogenesis. The data collected from in vitro and in vivo studies strongly indicate that these natural compounds could be the prospective candidates for the future anticancer therapeutics in breast cancer disease.

A Notch inhibitor plus Resveratrol induced blockade of autophagy drives glioblastoma cell death by promoting a switch to apoptosis.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors and the hardest type of cancer to treat. Therapies targeting developmental pathways, such as Notch, eliminate neoplastic glioma cells, but their efficacy can be limited by various mechanisms. Combination regimens may represent a good opportunity for effective therapies with durable effects. We used low doses of the γ-secretase inhibitor RO4929097 (GSI), to block the Notch pathway activity, in combination with Resveratrol (RSV) and we evidenced the mechanisms of autophagy/apoptosis transition in GBM cells. Resveratrol and GSI combination results in the synergistic induction of cell death together with the block of the autophagic flux evidenced by a sustained increase of LC3-II and p62 protein content, due to the dramatic reduction of CDK4, an important regulator of lysosomal function. The ectopic overexpression of the constitutive active CDK4 mutant, greatly counteracted the RSV+GSI induced block of the autophagy. Triggering autophagy in RSV+GSI-treated cells, which have impaired lysosomal function, caused the collapse of the system and a following apoptosis. For instance, by combining the CDK4 mutant as well as the early stage autophagy inhibitor, 3-methyladenina, abolished the RSV+GSI induced caspases activation. The initiator caspases (caspases-8 and -9), effector caspase (caspase-3) and its downstream substrate PARP were induced after RSV+GSI exposure as well as the percentage of the TUNEL positive cells. Moreover, the pro-apoptotic signaling MAPK p38 was activated while the pro-survival MAPK p42/p44 signaling was inhibited. In short, we establish the role of CDK4 in the regulation of autophagy/apoptosis transition induced by RSV and GSI in GBM cells. This new synergistic therapeutic combination, increasing the accumulation of autophagosomes, may have therapeutic value for GBM patients.

Cyclin D1 in Cancer: A Molecular Connection for Cell Cycle Control, Adhesion and Invasion in Tumor and Stroma.

Cyclin D1, an important regulator of cell cycle, carries out a central role in the pathogenesis of cancer determining uncontrolled cellular proliferation. In normal cells, Cyclin D1 expression levels are strictly regulated, conversely, in cancer, its activity is intensified in various manners. Different studies demonstrate that CCDN1 gene is amplified in several tumor types considering it as a negative prognostic marker of this pathology. Cyclin D1 is known for its role in the nucleus, but recent clinical studies associate the amount located in the cytoplasmic membrane with tumor invasion and metastasis. Cyclin D1 has also other functions: it governs the expression of specific miRNAs and it plays a crucial role in the tumor-stroma interactions potentiating most of the cancer hallmarks. In the present review, we will summarize the current scientific evidences that highlight the involvement of Cyclin D1 in the pathogenesis of different types of cancer, best of all in breast cancer. We will also focus on recent insights regarding the Cyclin D1 as molecular bridge between cell cycle control, adhesion, invasion, and tumor/stroma/immune-system interplay in cancer.

FSH-R Human Early Male Genital Tract, Testicular Tumors and Sperm: Its Involvement in Testicular Disorders.

The follicle-stimulating hormone receptor (FSH-R) expression was always considered human gonad-specific. The receptor has also been newly detected in extragonadal tissues. In this finding, we evaluated FSH-R expression in the human male early genital tract, in testicular tumors, and in sperm from healthy and varicocele patients. In sperm, we also studied the mechanism of FSH-R action. Immunohystochemistry and Western blot analysis showed FSH-R presence in the first pathways of the human genital tract, in embryonal carcinoma, and in sperm, but it was absent in seminoma and in lower varicocele. In sperm, FSH/FSH-R activity is mediated by G proteins activating the PKA pathway, as we observed by using the H89. It emerged that increasing FSH treatments induced motility, survival, capacitation, and acrosome reaction in both sperm samples. The different FSH-R expression in tumor testicular tissues may be discriminate by tumor histological type. In spermatozoa, FSH-R indicates a direct action of FSH in these cells, which could be beneficial during semen preparation for in vitro fertilization procedures. For instance, FSH positive effects could be relevant in idiopathic infertility and in the clinic surgery of varicocele. In conclusion, FSH-R expression may be considered a molecular marker of testicular disorders.

The Tumor Suppressor PTEN as Molecular Switch Node Regulating Cell Metabolism and Autophagy: Implications in Immune System and Tumor Microenvironment.

Recent studies conducted over the past 10 years evidence the intriguing role of the tumor suppressor gene Phosphatase and Tensin Homolog deleted on Chromosome 10 PTEN in the regulation of cellular energy expenditure, together with its capability to modulate proliferation and survival, thus expanding our knowledge of its physiological functions. Transgenic PTEN mice models are resistant to oncogenic transformation, present decreased adiposity and reduced cellular glucose and glutamine uptake, together with increased mitochondrial oxidative phosphorylation. These acquisitions led to a novel understanding regarding the role of PTEN to counteract cancer cell metabolic reprogramming. Particularly, PTEN drives an "anti-Warburg state" in which less glucose is taken up, but it is more efficiently directed to the mitochondrial Krebs cycle. The maintenance of cellular homeostasis together with reduction of metabolic stress are controlled by specific pathways among which autophagy, a catabolic process strictly governed by mTOR and PTEN. Besides, a role of PTEN in metabolic reprogramming and tumor/stroma interactions in cancer models, has recently been established. The genetic inactivation of PTEN in stromal fibroblasts of mouse mammary glands, accelerates breast cancer initiation and progression. This review will discuss our novel understanding in the molecular connection between cell metabolism and autophagy by PTEN, highlighting novel implications regarding tumor/stroma/immune system interplay. The newly discovered action of PTEN opens innovative avenues for investigations relevant to counteract cancer development and progression.

AIB1 sequestration by androgen receptor inhibits estrogen-dependent cyclin D1 expression in breast cancer cells.

BACKGROUND: Androgens, through their own receptor, play a protective role on breast tumor development and progression and counterbalance estrogen-dependent growth stimuli which are intimately linked to breast carcinogenesis. METHODS: Cell counting by trypan blu exclusion was used to study androgen effect on estrogen-dependent breast tumor growth. Quantitative Real Time RT-PCR, western blotting, transient transfection, protein immunoprecipitation and chromatin immunoprecipitation assays were carried out to investigate how androgen treatment and/or androgen receptor overexpression influences the functional interaction between the steroid receptor coactivator AIB1 and the estrogen- or androgen receptor which, in turn affects the estrogen-induced cyclin D1 gene expression in MCF-7 breast cancer cells. Data were analyzed by ANOVA. RESULTS: Here we demonstrated, in estrogen receptor α (ERα)-positive breast cancer cells, an androgen-dependent mechanism through which ligand-activated androgen receptor (AR) decreases estradiol-induced cyclin D1 protein, mRNA and gene promoter activity. These effects involve the competition between AR and ERα for the interaction with the steroid receptor coactivator AIB1, a limiting factor in the functional coupling of the ERα with the cyclin D1 promoter. Indeed, AIB1 overexpression is able to reverse the down-regulatory effects exerted by AR on ERα-mediated induction of cyclin D1 promoter activity. Co-immunoprecipitation studies indicated that the preferential interaction of AIB1 with ERα or AR depends on the intracellular expression levels of the two steroid receptors. In addition, ChIP analysis evidenced that androgen administration decreased E2-induced recruitment of AIB1 on the AP-1 site containing region of the cyclin D1 gene promoter. CONCLUSIONS: Taken together all these data support the hypothesis that AIB1 sequestration by AR may be an effective mechanism to explain the reduction of estrogen-induced cyclin D1 gene activity. In estrogen-dependent breast cancer cell proliferation, these findings reinforce the possibility that targeting AR signalling may potentiate the effectiveness of anti-estrogen adjuvant therapies.

Progesterone Receptor B signaling Reduces Breast Cancer Cell Aggressiveness: Role of Cyclin-D1/Cdk4 Mediating Paxillin Phosphorylation.

Progesterone-Receptor (PR) positivity is related with an enhanced response to breast cancer therapy, conversely cyclin D1 (CD1) is a retained marker of poor outcome. Herein, we demonstrate that hydroxyprogesterone (OHPg) through progesterone receptor B (PR-B) reduces breast cancer cell aggressiveness, by targeting the cytoplasmic CD1. Specifically, OHPg diminishes CD1 expression by a transcriptional regulation due to the recruitment of PR-B at a canonical half-PRE site of the CD1 promoter, together with HDAC1, determining a chromatin conformation less prone for gene transcription. CD1, together with its kinase partner Cdk4, regulates cell migration and metastasis, through the association with key components of focal adhesion, such as Paxillin (Pxn). Kaplan-Meier analysis shows that low Pxn expression was associated with increased distant metastasis-free survival in luminal A PR+ breast carcinomas. Interestingly, OHPg treatment reduced Pxn content in T47-D and MCF-7 cells; besides, the interaction between endogenous cytoplasmic CD1/Cdk4 with Pxn was reduced. This was consistent with the reduction of p-Ser83Pxn levels, crucially causing the delay in cell migration and a concomitant inhibition of Rac1 activity and p-PAK. Collectively, these findings support the role of PR-B in breast epithelial cell integrity and reinforce the importance in targeting PR-B as a potential strategy to restrict breast tumor cell invasion and metastasis.

Progress to Improve Oral Bioavailability and Beneficial Effects of Resveratrol.

Resveratrol (3,5,4'-trihydroxystilbene; RSV) is a natural nonflavonoid polyphenol present in many species of plants, particularly in grapes, blueberries, and peanuts. Several in vitro and in vivo studies have shown that in addition to antioxidant, anti-inflammatory, cardioprotective and neuroprotective actions, it exhibits antitumor properties. In mammalian models, RSV is extensively metabolized and rapidly eliminated and therefore it shows a poor bioavailability, in spite it of its lipophilic nature. During the past decade, in order to improve RSV low aqueous solubility, absorption, membrane transport, and its poor bioavailability, various methodological approaches and different synthetic derivatives have been developed. In this review, we will describe the strategies used to improve pharmacokinetic characteristics and then beneficial effects of RSV. These methodological approaches include RSV nanoencapsulation in lipid nanocarriers or liposomes, nanoemulsions, micelles, insertion into polymeric particles, solid dispersions, and nanocrystals. Moreover, the biological results obtained on several synthetic derivatives containing different substituents, such as methoxylic, hydroxylic groups, or halogens on the RSV aromatic rings, will be described. Results reported in the literature are encouraging but require additional in vivo studies, to support clinical applications.

Steroid Receptor Signallings as Targets for Resveratrol Actions in Breast and Prostate Cancer.

Extensive research over the past 25 years in hormone-dependent cancers, such as breast cancer and prostate cancer, has identified the molecular mechanisms driven by steroid receptors, elucidating the interplay between genomic and non-genomic steroid receptors mechanism of action. Altogether, these mechanisms create the specific gene expression programs that contribute to endocrine therapy resistance and cancer progression. These findings, on the bidirectional molecular crosstalk between steroid and growth factor receptors pathways in endocrine resistance, suggest the use of multi-target inhibitors together with endocrine therapies, for treating resistant disease. In this review we will discuss the novel understanding on the chemopreventive and anti-cancer activities of Resveratrol (3,5,4'-trihydroxy-stilbene) (RSV), a phytoalexin found in grapes acting on a plethora of targets. We will highlight Resveratrol effect on steroid receptors signalling and its potential use in the treatment of hormone-dependent cancer. Understanding the molecular mechanisms by which the bioactive compound influences cancer cell behaviour, by interfering with steroid receptors functional activity, will help to advance the design of combination strategies to increase the rate of complete and durable clinical response in patients.

Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis.

The Grapevine Pinot Gris disease (GPG-d) is a novel disease characterized by symptoms such as leaf mottling and deformation, which has been recently reported in grapevines, and mostly in Pinot gris. Plants show obvious symptoms at the beginning of the growing season, while during summer symptom recovery frequently occurs, manifesting as symptomless leaves. A new Trichovirus, named Grapevine Pinot gris virus (GPGV), which belongs to the family Betaflexiviridae was found in association with infected plants. The detection of the virus in asymptomatic grapevines raised doubts about disease aetiology. Therefore, the primary target of this work was to set up a reliable system for the study of the disease in controlled conditions, avoiding interfering factor(s) that could affect symptom development. To this end, two clones of the virus, pRI::GPGV-vir and pRI::GPGV-lat, were generated from total RNA collected from one symptomatic and one asymptomatic Pinot gris grapevine, respectively. The clones, which encompassed the entire genome of the virus, were used in Agrobacterium-mediated inoculation of Vitis vinifera and Nicotiana benthamiana plants. All inoculated plants developed symptoms regardless of their inoculum source, demonstrating a correlation between the presence of GPGV and symptomatic manifestations. Four months post inoculum, the grapevines inoculated with the pRI::GPGV-lat clone developed asymptomatic leaves that were still positive to GPGV detection. Three to four weeks later (i.e. ca. 5 months post inoculum), the same phenomenon was observed in the grapevines inoculated with pRI::GPGV-vir. This observation perfectly matches symptom progression in infected field-grown grapevines, suggesting a possible role for plant antiviral mechanisms, such as RNA silencing, in the recovery process.

Valproic Acid Addresses Neuroendocrine Differentiation of LNCaP Cells and Maintains Cell Survival.

PURPOSE: Neuroendocrine differentiation of prostate cancer, induced by androgen deprivation therapy, is mainly related to advanced disease and poor clinical outcome. Genetic and epigenetic alterations are the key elements of the prostate carcinogenesis. A group of compounds able to induce changes in this sense is inhibitors of histone deacetylase, to which it belongs valproic acid (VPA). In the present paper, we evaluated the role of this molecule on the neuroendocrine differentiation of LNCaP cells together with the effect on proliferation and survival signals. METHODS: Cell growth was analyzed by MTT and flow cytometry, while expression of proteins through Western blot analysis. RESULTS: Our results have documented that VPA in LNCaP cells reduces cell proliferation, decreases the S phase and Cyclin A, and up-regulates the cyclin-dependent kinase inhibitors p21waf and p27. The acquisition of androgen-independent condition is consistent with an induction of ß-III Tubulin and gamma Enolase, both markers of neuroendocrine phenotype. However, all these features cease with the removal of valproate from the culture medium, demonstrating the transitory nature of the epigenetic event. The VPA treatment does not compromise the survival phosphorylated signals of Akt, ERK1/2 and mTOR/p70S6K that remain up-regulated. Consistently, there is an increase of phospho-FOXO3a, to which corresponds the decreased expression of the corresponding oncosuppressor protein. CONCLUSION: Overall, our findings indicate that VPA in LNCaP prostate tumor cells, although it reduces cell proliferation, is able to drive neuroendocrine phenotype and to maintain the survival of these cells. Keeping in mind that neuroendocrine differentiation of prostate cancer appears to be associated with a poor prognosis, it is necessary to develop new treatments that do not induce neurodifferentiation but able to counteract cell survival.