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(1) Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer. Surgical resection, tumor ablation, and liver transplantation are curative treatments indicated for early-stage HCC. The management of intermediate and advanced stages of pathology is based on the use of systemic therapies which often show important side effects. Vitamin E-derivative tocotrienols (TTs) play antitumoral properties in different tumors. Here, we analyzed the activity of delta-TT (δ-TT) on HCC human cell lines. (2) We analyzed the ability of δ-TT to trigger apoptosis, to induce oxidative stress, autophagy, and mitophagy in HepG2 cell line. We evaluated the correlation between the activation of autophagy with the ability of δ-TT to induce cell death. (3) The data obtained demonstrate that δ-TT exerts an antiproliferative and proapoptotic effect in HCC cells. Furthermore, δ-TT induces the release of mitochondrial ROS and causes a structural and functional alteration of the mitochondria compatible with a fission process. Finally, δ-TT triggers selective autophagy process removing dysfunctional mitochondria. Inhibition of autophagy reversed the cytotoxic action of δ-TT. (4) Our results demonstrate that δ-TT through the activation of autophagy could represent a potential new approach in the treatment of advanced HCC.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a K d value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

Introduction: Protein kinase type C-ε (PKCε) plays an important role in the sensitization of primary afferent nociceptors, promoting mechanical hyperalgesia. In accordance, we showed that PKCε is present in sensory neurons of the peripheral nervous system (PNS), participating in the control of pain onset and chronification. Recently, it was found that PKCε is also implicated in the control of cell proliferation, promoting mitogenesis and metastatic invasion in some types of cancer. However, its role in the main glial cell of the PNS, the Schwann cells (SCs), was still not investigated., Methods: Rat primary SCs culture were treated with different pharmacologic approaches, including the PKCε agonist dicyclopropyl-linoleic acid (DCP-LA) 500 nM, the human recombinant brain derived neurotrophic factor (BDNF) 1 nM and the TrkB receptor antagonist cyclotraxin B 10 nM. The proliferation (by cell count), the migration (by scratch test and Boyden assay) as well as some markers of SCs differentiation and epithelial-mesenchymal transition (EMT) process (by qRT-PCR and western blot) were analyzed., Results: Overall, we found that PKCε is constitutively expressed in SCs, where it is likely involved in the switch from the proliferative toward the differentiated state. Indeed, we demonstrated that PKCε activation regulates SCs proliferation, increases their migration, and the expression of some markers (e.g., glycoprotein P0 and the transcription factor Krox20) of SCs differentiation. Through an autocrine mechanism, BDNF activates TrkB receptor, and controls SCs proliferation via PKCε. Importantly, PKCε activation likely promoted a partial EMT process in SCs., Discussion: PKCε mediates relevant actions in the neuronal and glial compartment of the PNS. In particular, we posit a novel function for PKCε in the transformation of SCs, assuming a role in the mechanisms controlling SCs' fate and plasticity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mohamed, Colciago, Montagnani Marelli, Moretti and Magnaghi.)

The aim of this study was to investigate the chemical profile and the cytotoxic activity in two castration-resistant prostate cancer (CRPC) cell lines of the leaf essential oil in Myrtus communis subsp. tarentina (L.) Nyman (EO MT), which was cultivated at the Ghirardi Botanical Garden (Toscolano Maderno, Brescia, Italy). The leaves were air-dried and extracted by hydrodistillation with a Clevenger-type apparatus, and the EO profile was characterized by GC/MS. For the cytotoxic activity investigation, we analyzed the cell viability by MTT assay, and the apoptosis induction by Annexin V/propidium iodide assay/Western blot analysis of cleaved caspase 3 and cleaved PARP proteins. Moreover, the cellular migration was analyzed by Boyden's chamber assay and the distribution of actin cytoskeleton filaments by immunofluorescence. We identified 29 total compounds; the main compound classes were oxygenated monoterpenes, monoterpene hydrocarbons, and sesquiterpenes. The main constituents were α-pinene, α-humulene, α-terpineol, durohydroquinon, linalool, geranyl acetate, and β-caryophyllene. We found that EO MT was able to reduce cellular viability, activating an apoptotic process, and to decrease the migratory capacity of CRPC cells. These results suggest that it might be interesting to further investigate the effects of single compounds present in EO MT for their possible use in prostate cancer treatment.

Prostate cancer (PCa) represents the fifth cause of cancer death in men. Currently, chemotherapeutic agents for the treatment of cancers, including PCa, mainly inhibit tumor growth by apoptosis induction. However, defects in apoptotic cellular responses frequently lead to drug resistance, which is the main cause of chemotherapy failure. For this reason, trigger non-apoptotic cell death might represent an alternative approach to prevent drug resistance in cancer. Several agents, including natural compounds, have been shown to induce necroptosis in human cancer cells. In this study we evaluated the involvement of necroptosis in anticancer activity of delta-tocotrienol (δ-TT) in PCa cells (DU145 and PC3). Combination therapy is one tool used to overcome therapeutic resistance and drug toxicity. Evaluating the combined effect of δ-TT and docetaxel (DTX), we found that δ-TT potentiates DTX cytotoxicity in DU145 cells. Moreover, δ-TT induces cell death in DU145 cells that have developed DTX resistance (DU-DXR) activating necroptosis. Taken together, obtained data indicate the ability of δ-TT to induce necroptosis in both DU145, PC3 and DU-DXR cell lines. Furthermore, the ability of δ-TT to induce necroptotic cell death may represent a promising therapeutical approach to overcome DTX chemoresistance in PCa.

Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Some specific driver mutations have been described in multiple oncogenes including BRAF and NRAS that are mutated in 60-70% and 15-20% of melanoma, respectively. The aim of this study was to evaluate the role of Small Heat Shock Protein B8 (HSPB8) on cell growth and migration of both BLM (BRAF wt /NRAS Q61R ) and A375 (BRAF V600E /NRAS wt ) human melanoma cell lines. HSPB8 is a member of the HSPB family of chaperones involved in protein quality control (PQC) system and contributes to chaperone assisted selective autophagy (CASA) as well as in the regulation of mitotic spindle. In cancer, HSPB8 has anti- or pro-tumoral action depending on tumor type. In melanoma cell lines characterized by low HSPB8 levels, we demonstrated that the restoration of HSPB8 expression causes cell growth arrest, reversion of EMT (Epithelial-Mesenchymal Transition)-like phenotype switching and antimigratory effect, independently from the cell mutational status. We demonstrated that HSPB8 regulates the levels of the active prenylated form of NRAS in NRAS-mutant and NRAS-wild-type melanoma cell lines. Consequently, the inhibition of NRAS impairs the activation of Akt/mTOR pathway inducing autophagy activation. Autophagy can play a dual role in regulating cell death and survival. We have therefore demonstrated that HSPB8-induced autophagy is a crucial event that counteracts cell growth in melanoma. Collectively, our results suggest that HSPB8 has an antitumoral action in melanoma cells characterized by BRAF and NRAS mutations., (© 2022. The Author(s).)

The impairment of development/migration of hypothalamic gonadotropin-releasing hormone (GnRH) neurons is the main cause of Kallmann's syndrome (KS), an inherited disorder characterized by hypogonadism, anosmia, and other developmental defects. Olfactorin is an extracellular matrix protein encoded by the UMODL1 (uromodulin-like 1) gene expressed in the mouse olfactory region along the migratory route of GnRH neurons. It shares a combination of WAP and FNIII repeats, expressed in complementary domains, with anosmin-1, the product of the ANOS1 gene, identified as the causative of KS. In the present study, we have investigated the effects of olfactorin in vitro and in vivo models. The results show that olfactorin exerts an anosmin-1-like strong chemoattractant effect on mouse-immortalized GnRH neurons (GN11 cells) through the activation of the FGFR and MAPK pathways. In silico analysis of olfactorin and anosmin-1 reveals a satisfactory similarity at the N-terminal region for the overall arrangement of corresponding WAP and FNIII domains and marked similarities between WAP domains' binding modes of interaction with the resolved FGFR1-FGF2 complex. Finally, in vivo experiments show that the down-modulation of the zebrafish z-umodl1 gene (orthologous of UMODL1 ) in both GnRH3:GFP and omp 2k :gap-CFP rw034 transgenic zebrafish strains leads to a clear disorganization and altered fasciculation of the neurites of GnRH3:GFP neurons crossing at the anterior commissure and a significant increase in olfactory CFP + fibers with altered trajectory. Thus, our study shows olfactorin as an additional factor involved in the development of olfactory and GnRH systems and proposes UMODL1 as a gene worthy of diagnostic investigation in KS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Di Schiavi, Vistoli, Moretti, Corrado, Zuccarini, Gervasoni, Casati, Bottai, Merlo and Maggi.)

Prostate cancer (PCa) is the most common malignancy in men and represents the second leading cause of cancer deaths in Western countries. PCa is initially androgen-dependent, however, this tumor inevitably progresses as castration-resistant prostate cancer (CRPC), which represents the most aggressive phase of the pathology. In this work, in two CRPC cell lines (DU145 and PC3), we studied the in vitro inhibitory properties of the tryptophan-derived endogenous metabolite kynurenic acid (KYNA) and of the lactam form of 3-2'-pyrrilonidinyl-kynurenic acid (3-PKA-L), alkaloids usually present in combination in chestnut honey. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell colony formation assay, and Western blot analysis of the major mediator proteins involved in apoptotic processes. In all experiments, KYNA was scarcely or not active while 3-PKA-L showed anticancer activity in the high concentration range (0.01 mM - 1 mM) from 24 to 72 h. The results obtained showed that cell death was induced by extrinsic apoptotic pathway, by cell morphological changes and reduction of cell colonies number. These novel results represent the first promising step to the accurate description of 3-PKA-L cytotoxic effect, not observed with KYNA, paving the way to the search of new anticancer agents, as well as to the better understanding of the physiopathological role of this interesting natural product.

The cellular response to cancer-induced stress is one of the major aspects regulating cancer development and progression. The Heat Shock Protein B8 (HSPB8) is a small chaperone involved in chaperone-assisted selective autophagy (CASA). CASA promotes the selective degradation of proteins to counteract cell stress such as tumor-induced stress. HSPB8 is also involved in (i) the cell division machinery regulating chromosome segregation and cell cycle arrest in the G0/G1 phase and (ii) inflammation regulating dendritic cell maturation and cytokine production. HSPB8 expression and role are tumor-specific, showing a dual and opposite role. Interestingly, HSPB8 may be involved in the acquisition of chemoresistance to drugs. Despite the fact the mechanisms of HSPB8-mediated CASA activation in tumors need further studies, HSPB8 could represent an important factor in cancer induction and progression and it may be a potential target for anticancer treatment in specific types of cancer. In this review, we will discuss the molecular mechanism underlying HSPB8 roles in normal and cancer conditions. The basic mechanisms involved in anti- and pro-tumoral activities of HSPB8 are deeply discussed together with the pathways that modulate HSPB8 expression, in order to outline molecules with a beneficial effect for cancer cell growth, migration, and death.

Pituitary Gonadotropin-Releasing Hormone receptors (GnRH-R) mediate the activity of the hypothalamic decapeptide GnRH, thus playing a key role in the regulation of the reproductive axis. Early-stage prostate cancer (PCa) is dependent on serum androgen levels, and androgen-deprivation therapy (ADT), based on GnRH agonists and antagonists, represents the standard therapeutic approach for PCa patients. Unfortunately, the tumor often progresses towards the more aggressive castration-resistant prostate cancer (CRPC) stage. GnRH receptors are also expressed in CRPC tissues, where their binding to both GnRH agonists and antagonists is associated with significant antiproliferative/proapoptotic, antimetastatic and antiangiogenic effects, mediated by the Gαi/cAMP signaling cascade. GnRH agonists and antagonists are now considered as an effective therapeutic strategy for CRPC patients with many clinical trials demonstrating that the combined use of these drugs with standard therapies (i.e., docetaxel, enzalutamide, abiraterone) significantly improves disease-free survival. In this context, GnRH-based bioconjugates (cytotoxic drugs covalently linked to a GnRH-based decapeptide) have been recently developed. The rationale of this treatment is that the GnRH peptide selectively binds to its receptors, delivering the cytotoxic drug to CRPC cells while sparing nontumor cells. Some of these compounds have already entered clinical trials.

Objective:  To evaluate the agreement between the histopathological diagnoses of preoperative endometrial samples and surgical specimens and correlate the agreement between the diagnoses with the impact on surgical management and the survival of patients with endometrial adenocarcinomas., Methods:  Sixty-two patients treated for endometrial cancer at a university hospital from 2002 to 2011 were retrospectively evaluated. The histopathological findings of preoperative endometrial samples and of surgical specimens were analyzed. The patients were subjected to hysterectomy as well as adjuvant treatment, if necessary, and clinical follow-up, according to the institutional protocol. Lesions were classified as endometrioid tumor (type 1) grades 1, 2, or 3 or non-endometrioid carcinoma (type 2)., Results:  The agreement between the histopathological diagnoses based on preoperative endometrial samples and surgical specimens was fair (Kappa: 0.40; p  < 0.001). However, the agreement was very significant for tumor type and grade, in which a higher concordance occurred at a higher grade. The percentage of patients with lymph nodes affected was 19.2%. Although most patients presenting with disease remission or cure were in the early stages (90.5%), there were no significant differences between those patients who had a misdiagnosis (11/16; 68.8%) and those who had a correct diagnosis (25/33; 75.8%) based on preoperative endometrial sampling ( p  = 0.605)., Conclusion:  Our findings corroborate the literature and confirm the under staging of preoperative endometrial samples based on histopathological assessment, especially for lower grade endometrial tumors. We suggest that the preoperative diagnosis should be complemented with other methods to better plan the surgical management strategy., Competing Interests: The authors have no conflict of interests to declare., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/).)

Competing Interests: Competing interests: None declared.

Purpose: The objective of this review was to address the barriers limiting access to genetic cancer risk assessment and genetic testing for individuals with suspected hereditary breast and ovarian cancer (HBOC) through a review of the diagnosis and management steps of HBOC., Methods: A selected panel of Brazilian experts in fields related to HBOC was provided with a series of relevant questions to address before the multiday conference. During this conference, each narrative was discussed and edited by the entire group, through numerous drafts and rounds of discussion, until a consensus was achieved., Results: The authors propose specific and realistic recommendations for improving access to early diagnosis, risk management, and cancer care of HBOC specific to Brazil. Moreover, in creating these recommendations, the authors strived to address all the barriers and impediments mentioned in this article., Conclusion: There is a great need to expand hereditary cancer testing and counseling in Brazil, and changing current policies is essential to accomplishing this goal. Increased knowledge and awareness, together with regulatory actions to increase access to this technology, have the potential to improve patient care and prevention and treatment efforts for patients with cancer across the country.

Malignant melanoma is a tumor characterized by a very high level of heterogeneity, responsible for its malignant behavior and ability to escape from standard therapies. In this review we highlight the molecular and biological features of the subpopulation of cancer stem cells (CSCs), well known to be characterized by self-renewal properties, deeply involved in triggering the processes of tumor generation, metastasis, progression and drug resistance. From the molecular point of view, melanoma CSCs are identified and characterized by the expression of stemness markers, such as surface markers, ATP-binding cassette (ABC) transporters, embryonic stem cells and intracellular markers. These cells are endowed with different functional features. In particular, they play pivotal roles in the processes of tumor dissemination, epithelial-to-mesenchymal transition (EMT) and angiogenesis, mediated by specific intracellular signaling pathways; moreover, they are characterized by a unique metabolic reprogramming. As reported for other types of tumors, the CSCs subpopulation in melanoma is also characterized by a low immunogenic profile as well as by the ability to escape the immune system, through the expression of a negative modulation of T cell functions and the secretion of immunosuppressive factors. These biological features allow melanoma CSCs to escape standard treatments, thus being deeply involved in tumor relapse. Targeting the CSCs subpopulation is now considered an attractive treatment strategy; in particular, combination treatments, based on both CSCs-targeting and standard drugs, will likely increase the therapeutic options for melanoma patients. The characterization of CSCs in liquid biopsies from single patients will pave the way towards precision medicine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Melanoma is the most fatal form of skin cancer. Current therapeutic approaches include surgical resection, chemotherapy, targeted therapy and immunotherapy. However, these treatment strategies are associated with development of drug resistance and severe side effects. In recent years, natural compounds have also been extensively studied for their anti-melanoma effects, including tumor growth inhibition, apoptosis induction, angiogenesis and metastasis suppression and cancer stem cell elimination. Moreover, a considerable number of studies reported the synergistic activity of phytochemicals and standard anti-melanoma agents, as well as the enhanced effectiveness of their synthetic derivatives and novel formulations. However, clinical data confirming these promising effects in patients are still scanty. This review emphasizes the anti-tumor mechanisms and potential application of the most studied natural products for melanoma prevention and treatment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: Prostate cancer, after the phase of androgen dependence, may progress to the castration-resistant prostate cancer (CRPC) stage, with resistance to standard therapies. Vitamin E-derived tocotrienols (TTs) possess a significant antitumour activity. Here, we evaluated the anti-cancer properties of δ-TT in CRPC cells (PC3 and DU145) and the related mechanisms of action., Materials and Methods: MTT, Trypan blue and colony formation assays were used to assess cell viability/cell death/cytotoxicity. Western blot, immunofluorescence and MTT analyses were utilized to investigate apoptosis, ER stress and autophagy. Morphological changes were investigated by light and transmission electron microscopy., Results: We demonstrated that δ-TT exerts a cytotoxic/proapoptotic activity in CRPC cells. We found that in PC3 cells: (a) δ-TT triggers both the endoplasmic reticulum (ER) stress and autophagy pathways; (b) autophagy induction is related to the ER stress, and this ER stress/autophagy axis is involved in the antitumour activity of δ-TT; in autophagy-defective DU145 cells, only the ER stress pathway is involved in the proapoptotic effects of δ-TT; (c) in both CRPC cell lines, δ-TT also induces an intense vacuolation prevented by the ER stress inhibitor salubrinal and the protein synthesis inhibitor cycloheximide, together with increased levels of phosphorylated JNK and p38, supporting the induction of paraptosis by δ-TT., Conclusions: These data demonstrate that apoptosis, involving ER stress and autophagy (in autophagy positive PC3 cells), and paraptosis are involved in the anti-cancer activity of δ-TT in CRPC cells., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Cancer represents a serious global health problem, and its incidence and mortality are rapidly growing worldwide. One of the main causes of the failure of an anticancer treatment is the development of drug resistance by cancer cells. Therefore, it is necessary to develop new drugs characterized by better pharmacological and toxicological profiles. Natural compounds can represent an optimal collection of bioactive molecules. Many natural compounds have been proven to possess anticancer effects in different types of tumors, but often the molecular mechanisms associated with their cytotoxicity are not completely understood. The endoplasmic reticulum (ER) is an organelle involved in multiple cellular processes. Alteration of ER homeostasis and its appropriate functioning originates a cascade of signaling events known as ER stress response or unfolded protein response (UPR). The UPR pathways involve three different sensors (protein kinase RNA(PKR)-like ER kinase (PERK), inositol requiring enzyme1α (IRE1) and activating transcription factor 6 (ATF6)) residing on the ER membranes. Although the main purpose of UPR is to restore this organelle's homeostasis, a persistent UPR can trigger cell death pathways such as apoptosis. There is a growing body of evidence showing that ER stress may play a role in the cytotoxicity of many natural compounds. In this review we present an overview of different plant-derived natural compounds, such as curcumin, resveratrol, green tea polyphenols, tocotrienols, and garcinia derivates, that exert their anticancer activity via ER stress modulation in different human cancers.

Vitamin E is composed of two groups of compounds: α-, β-, γ-, and δ-tocopherols (TPs), and the corresponding unsaturated tocotrienols (TTs). TTs are found in natural sources such as red palm oil, annatto seeds, and rice bran. In the last decades, TTs (specifically, γ-TT and δ-TT) have gained interest due to their health benefits in chronic diseases, based on their antioxidant, neuroprotective, cholesterol-lowering, anti-inflammatory activities. Several in vitro and in vivo studies pointed out that TTs also exert a significant antitumor activity in a wide range of cancer cells. Specifically, TTs were shown to exert antiproliferative/proapoptotic effects and to reduce the metastatic or angiogenic properties of different cancer cells; moreover, these compounds were reported to specifically target the subpopulation of cancer stem cells, known to be deeply involved in the development of resistance to standard therapies. Interestingly, recent studies pointed out that TTs exert a synergistic antitumor effect on cancer cells when given in combination with either standard antitumor agents (i.e., chemotherapeutics, statins, "targeted" therapies) or natural compounds with anticancer activity (i.e., sesamin, epigallocatechin gallate (EGCG), resveratrol, ferulic acid). Based on these observations, different TT synthetic derivatives and formulations were recently developed and demonstrated to improve TT water solubility and to reduce TT metabolism in cancer cells, thus increasing their biological activity. These promising results, together with the safety of TT administration in healthy subjects, suggest that these compounds might represent a new chemopreventive or anticancer treatment (i.e., in combination with standard therapies) strategy. Clinical trials aimed at confirming this antitumor activity of TTs are needed., (© 2018 Wiley Periodicals, Inc.)

Background: Tocotrienols (TTs) are vitamin E derivatives naturally occurring in several plants and vegetable oils. Like Tocopherols (TPs), they comprise four isoforms, α, β, γ and δ, but unlike TPs, they present an unsaturated isoprenoid chain. Recent studies indicate that TTs provide important health benefits, including neuroprotective, anti-inflammatory, anti-oxidant, cholesterol lowering and immunomodulatory effects. Moreover, they have been found to possess unique anti-cancer properties., Objective: The purpose of this review is to present an overview of the state of the art of TTs role in cancer prevention and treatment, as well as to describe recent patents proposing new methods for TTs isolation, chemical modification and use in cancer prevention and/or therapy., Methods: Recent literature and patents focusing on TTs anti-cancer applications have been identified and reviewed, with special regard to their scientific impact and novelty., Results: TTs have demonstrated significant anti-cancer activity in multiple tumor types, both in vitro and in vivo. Furthermore, they have shown synergistic effects when given in combination with standard anti-cancer agents or other anti-tumor natural compounds. Finally, new purification processes and transgenic sources have been designed in order to improve TTs production, and novel TTs formulations and synthetic derivatives have been developed to enhance their solubility and bioavailability., Conclusion: The promising anti-cancer effects shown by TTs in several preclinical studies may open new opportunities for therapeutic interventions in different tumors. Thus, clinical trials aimed at confirming TTs chemopreventive and tumor-suppressing activity, particularly in combination with standard therapies, are urgently needed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Prostate cancer (PC) is one of the leading causes of death in males. Available treatments often lead to the appearance of chemoresistant foci and metastases, with mechanisms still partially unknown. Within tumour mass, autophagy may promote cell survival by enhancing cancer cells tolerability to different cell stresses, like hypoxia, starvation or those triggered by chemotherapic agents. Because of its connection with the apoptotic pathways, autophagy has been differentially implicated, either as prodeath or prosurvival factor, in the appearance of more aggressive tumours. Here, in three PC cells (LNCaP, PC3, and DU145), we tested how different autophagy inducers modulate docetaxel-induced apoptosis. We selected the mTOR-independent disaccharide trehalose and the mTOR-dependent macrolide lactone rapamycin autophagy inducers. In castration-resistant PC (CRPC) PC3 cells, trehalose specifically prevented intrinsic apoptosis in docetaxel-treated cells. Trehalose reduced the release of cytochrome c triggered by docetaxel and the formation of aberrant mitochondria, possibly by enhancing the turnover of damaged mitochondria via autophagy (mitophagy). In fact, trehalose increased LC3 and p62 expression, LC3-II and p62 (p62 bodies) accumulation and the induction of LC3 puncta. In docetaxel-treated cells, trehalose, but not rapamycin, determined a perinuclear mitochondrial aggregation (mito-aggresomes), and mitochondria specifically colocalized with LC3 and p62-positive autophagosomes. In PC3 cells, rapamycin retained its ability to activate autophagy without evidences of mitophagy even in presence of docetaxel. Interestingly, these results were replicated in LNCaP cells, whereas trehalose and rapamycin did not modify the response to docetaxel in the ATG5-deficient (autophagy resistant) DU145 cells. Therefore, autophagy is involved to alter the response to chemotherapy in combination therapies and the response may be influenced by the different autophagic pathways utilized and by the type of cancer cells.

Objective:  To analyze the perioperative results and safety of performing gynecological surgeries using robot-assisted laparoscopy during implementation of the technique in a community hospital over a 6-year period., Methods:  This was a retrospective observational study in which the medical records of 274 patients who underwent robotic surgery from September 2008 to December 2014 were analyzed. We evaluated age, body mass index (BMI), diagnosis, procedures performed, American Society of Anesthesiologists (ASA) classification, the presence of a proctor (experienced surgeon with at least 20 robotic cases), operative time, transfusion rate, perioperative complications, conversion rate, length of stay, referral to the intensive care unit (ICU), and mortality. We compared transfusion rate, perioperative complications and conversion rate between procedures performed by experienced and beginner robotic surgeons assisted by an experienced proctor., Results:  During the observed period, 3 experienced robotic surgeons performed 187 surgeries, while 87 surgeries were performed by 20 less experienced teams, always with the assistance of a proctor. The median patient age was 38 years, and the median BMI was 23.3 kg/m 2 . The most frequent diagnosis was endometriosis (57%) and the great majority of the patients were classified as ASA I or ASA II (99.6%). The median operative time was 225 minutes, and the median length of stay was 2 days. We observed a 5.8% transfusion rate, 0.8% rate of perioperative complications, 1.1% conversion rate to laparoscopy or laparotomy, no patients referred to ICU, and no deaths. There were no differences in transfusion, complications and conversion rates between experienced robotic surgeons and beginner robotic surgeons assisted by an experienced proctor., Conclusion:  In our casuistic, robot-assisted laparoscopy demonstrated to be a safe technique for gynecological surgeries, and the presence of an experienced proctor was considered a highlight in the safety model adopted for the introduction of the robotic gynecological surgery in a high-volume hospital and, mainly, for its extension among several surgical teams, assuring patient safety., Competing Interests: The authors have no conflicts of interest to disclose., (Thieme Revinter Publicações Ltda Rio de Janeiro, Brazil.)

In this work, we report a rapid and convenient HPLC-UV-DAD method for the isolation of δ-T3 on semi-preparative scale from two different vitamin E rich processed, commercially available products obtained from the fruits of Elaeis guineensis Jacq. (oil palm) and from the seeds of Bixa orellana L. (achiote tree). Chromatography was run using reverse phase (RP) C-18 columns and HPLC-grade acetonitrile as mobile phase. The purity of the isolated δ-T3, assessed by GC-MS and 1 H NMR was above 98%. The δ-T3 cytotoxic activity found in vitro against the proliferation of human A375 melanoma cells compared to that of the other δ-T3 free tocols mixture suggest its primary role in the experimental anticancer activity observed for palm oil derived products. Taken altogether, the results of this study highlight the importance of the application of suitable purification systems for the preparations of tocotrienols prior to their experimental or clinical testing.

The prognosis of metastatic melanoma is very poor, due to the development of drug resistance. Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse. We first characterized CSCs in melanoma cell lines. We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells. We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture. Based on these data, we investigated whether δ-TT might target melanoma CSCs. We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres. In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker. These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

Cutaneous melanoma is an aggressive tumor; its incidence has been reported to increase fast in the past decades. Melanoma is a heterogeneous tumor, with most patients harboring mutations in the BRAF or NRAS oncogenes, leading to the overactivation of the MAPK/ERK and PI3K/Akt pathways. The current therapeutic approaches are based on therapies targeting mutated BRAF and the downstream pathway, and on monoclonal antibodies against the immune checkpoint blockade. However, treatment resistance and side effects are common events of these therapeutic strategies. Increasing evidence supports that melanoma is a hormone-related cancer. Melanoma incidence is higher in males than in females, and females have a significant survival advantage over men. Estrogens exert their effects through estrogen receptors (ERα and ERβ) that affect cancer growth in an opposite way: ERα is associated with a proliferative action and ERβ with an anticancer effect. ERβ is the predominant ER in melanoma, and its expression decreases in melanoma progression, supporting its role as a tumor suppressor. Thus, ERβ is now considered as an effective molecular target for melanoma treatment. 17β-estradiol was reported to inhibit melanoma cells proliferation; however, clinical trials did not provide the expected survival benefits. In vitro studies demonstrate that ERβ ligands inhibit the proliferation of melanoma cells harboring the NRAS (but not the BRAF) mutation, suggesting that ERβ activation might impair melanoma development through the inhibition of the PI3K/Akt pathway. These data suggest that ERβ agonists might be considered as an effective treatment strategy, in combination with MAPK inhibitors, for NRAS mutant melanomas. In an era of personalized medicine, pretreatment evaluation of the expression of ER isoforms together with the concurrent oncogenic mutations should be considered before selecting the most appropriate therapeutic intervention. Natural compounds that specifically bind to ERβ have been identified. These phytoestrogens decrease the proliferation of melanoma cells. Importantly, these effects are unrelated to the oncogenic mutations of melanomas, suggesting that, in addition to their ERβ activating function, these compounds might impair melanoma development through additional mechanisms. A better identification of the role of ERβ in melanoma development will help increase the therapeutic options for this aggressive pathology.

Cervical cancer incidence and mortality rates are significantly higher in low- and middle-income countries compared with the United States and other developed countries. This disparity is caused by decreased access to screening, often coupled with low numbers of trained providers offering cancer prevention and treatment services. However, similar disparities are also found in underserved areas of the United States, such as the Texas-Mexico border, where cervical cancer mortality rates are 30% higher than in the rest of Texas. To address these issues, we have adopted the Project ECHO (Extension for Community Healthcare Outcomes) program, a low-cost telementoring model previously proven to be successful in increasing local capacity, improving patient management skills, and ultimately improving patient outcomes in rural and underserved areas. We use the Project ECHO model to educate local providers in the management of cervical dysplasia in a low-resource region of Texas and have adapted it to inform strategies for the management of advanced cervical and breast cancer in Latin America and sub-Saharan Africa. This innovative approach, using ECHO, is part of a larger strategy to enhance clinical skills and develop collaborative projects between academic centers and partners in low-resource regions., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/site/ifc. Melissa S. LopezNo relationship to discloseEllen S. BakerStock or Other Ownership: MerckAndrea M. MilbourneNo relationship to discloseRose M. GowenNo relationship to discloseAna M. RodriguezNo relationship to discloseCesaltina LorenzoniNo relationship to discloseCatherine MwabaResearch Funding: Mylan Zambia (Inst) Travel, Accommodations, Expenses: Fresenius Kabi South AfricaSusan Citonje MsadabweTravel, Accommodations, Expenses: AstraZenecaJosé Humberto TavaresNo relationship to discloseGeorgia Fontes-CintraNo relationship to discloseGustavo Zucca-MatthesNo relationship to discloseDonato Callegaro-FilhoNo relationship to discloseDanielle Ramos-MartinNo relationship to discloseIcaro Thiago de CarvalhoNo relationship to discloseRobson CoelhoNo relationship to discloseRenato Moretti-MarquesNo relationship to discloseThiago ChulamTravel, Accommodations, Expenses: A.C. Camargo Cancer CenterMila Pontremoli-SalcedoNo relationship to discloseFernanda NozarNo relationship to discloseVeronica FiolTravel, Accommodations, Expenses: UrufarmaMauricio MazaNo relationship to discloseSanjeev AroraNo relationship to discloseErnest T. HawkEmployment: MD Anderson Cancer Center Honoraria: National Cancer Institute, Huntsman Cancer Institute, Mayo Clinic Cancer Center, Kansas University Medical Center, Ohio State University Comprehensive Cancer Center, Roswell Park Cancer Institute, Simmons Comprehensive Cancer Center, University of Nebraska Medical Center, University of South Alabama Mitchell Cancer Institute, Johns Hopkins University, Oncology Nursing Society Conference, Weill Cornell Medical College, ASCO, American Association for Cancer Research Consulting or Advisory Role: Cancer Prevention Pharmaceuticals, PLx Pharma, POZEN Research Funding: National Institutes of Health/National Cancer Institute (Inst), the Cancer Prevention Research Institute of Texas (Inst), Cancer Prevention Pharmaceuticals (Inst) Patents, Royalties, Other Intellectual Property: John Wiley & Sons Travel, Accommodations, Expenses: National Cancer Institute, American Association for Cancer Research, Huntsman Cancer Institute, Kansas University Medical Center, Mayo Clinic Cancer Center, Ohio State University Comprehensive Cancer Center, Roswell Park Cancer Institute, Simmons Comprehensive Cancer Center, University of Nebraska Medical Center, Johns Hopkins University, Oncology Nursing Society Conference, Weill Cornell Medical College, ASCO, American Association for Cancer Research, American Cancer Society, Association of American Cancer Institutes—Cancer Center Administrators Forum, Alliance Prevention Committee, First Annual Meta-ECHO MeetingKathleen M. SchmelerResearch Funding: Becton Dickinson Patents, Royalties, Other Intellectual Property: UpToDate

Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma.

For symptomatic deep infiltrating endometriosis, surgery is often required to achieve symptom relief and restore fertility. A minimally invasive approach using laparoscopy is considered the gold standard. However, specific limitations of the laparoscopic approach deep in the pelvis keep challenging even surgeons with a solid experience with minimally invasive techniques. Robotic surgery has the potential to compensate for technical drawbacks inherent in conventional laparoscopic surgery, such as limited degree of freedom, two-dimensional vision, and the fulcrum effect. In the present report, we aim at demonstrating the central role of robotic surgery for deep infiltrating endometriosis, with special emphasis in the ability to practice organ (rectal) preservation. A 45-year-old white female with a 4-month history of chronic pelvic pain, dyschezia, and dysmenorrhea, refractory to hormonal therapy was referred to our unit. MRI findings were diagnostic of deep infiltrating endometriosis (retrocervical and rectovaginal) extending to the anterior rectal serosal layer (partial-thickness rectal invasion). Using a fully robotic approach, appropriate dissection of the rectovaginal septum and of the extraperitoneal rectum followed by complete excision of the endometriotic rectal nodule with organ (rectal) preservation was undertaken. It is our belief that using a robotic approach, the potential to boost rectal preservation might be established. Moreover, it is possible that in many cases, a robotic operation may allow the surgeon to perform the intervention with greater accuracy and comfort. As a result, more patients with deep infiltrating endometriosis may benefit from rectal sparing procedures.

Background: Human reproduction depends on an intact hypothalamic-pituitary-gonadal (HPG) axis. Hypothalamic gonadotrophin-releasing hormone (GnRH) has been recognized, since its identification in 1971, as the central regulator of the production and release of the pituitary gonadotrophins that, in turn, regulate the gonadal functions and the production of sex steroids. The characteristic peculiar development, distribution and episodic activity of GnRH-producing neurons have solicited an interdisciplinary interest on the etiopathogenesis of several reproductive diseases. The more recent identification of a GnRH/GnRH receptor (GnRHR) system in both the human endometrium and ovary has widened the spectrum of action of the peptide and of its analogues beyond its hypothalamic function., Methods: An analysis of research and review articles published in international journals until June 2015 has been carried out to comprehensively summarize both the well established and the most recent knowledge on the physiopathology of the GnRH system in the central and peripheral control of female reproductive functions and diseases., Results: This review focuses on the role of GnRH neurons in the control of the reproductive axis. New knowledge is accumulating on the genetic programme that drives GnRH neuron development to ameliorate the diagnosis and treatment of GnRH deficiency and consequent delayed or absent puberty. Moreover, a better understanding of the mechanisms controlling the episodic release of GnRH during the onset of puberty and the ovulatory cycle has enabled the pharmacological use of GnRH itself or its synthetic analogues (agonists and antagonists) to either stimulate or to block the gonadotrophin secretion and modulate the functions of the reproductive axis in several reproductive diseases and in assisted reproduction technology. Several inputs from other neuronal populations, as well as metabolic, somatic and age-related signals, may greatly affect the functions of the GnRH pulse generator during the female lifespan; their modulation may offer new possible strategies for diagnostic and therapeutic interventions. A GnRH/GnRHR system is also expressed in female reproductive tissues (e.g. endometrium and ovary), both in normal and pathological conditions. The expression of this system in the human endometrium and ovary supports its physiological regulatory role in the processes of trophoblast invasion of the maternal endometrium and embryo implantation as well as of follicular development and corpus luteum functions. The GnRH/GnRHR system that is expressed in diseased tissues of the female reproductive tract (both benign and malignant) is at present considered an effective molecular target for the development of novel therapeutic approaches for these pathologies. GnRH agonists are also considered as a promising therapeutic approach to counteract ovarian failure in young female patients undergoing chemotherapy., Conclusions: Increasing knowledge about the regulation of GnRH pulsatile release, as well as the therapeutic use of its analogues, offers interesting new perspectives in the diagnosis, treatment and outcome of female reproductive disorders, including tumoral and iatrogenic diseases., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aim of the Video / Introduction: Pelvic congestion syndrome is a well-known cause of cyclic pelvic pain (Ganeshan et al., Cardiovasc Intervent Radiol 30(6):1105-11, 2007). What is much less well known is that dilated or malformed branches of the internal or external iliac vessels can entrap the nerves of the sacral plexus against the pelvic sidewalls, producing symptoms that are not commonly seen in gynecological practice, such as sciatica, or refractory urinary and anorectal dysfunction (Possover et al., Fertil Steril 95(2):756-8. 2011). The objective of this video is to explain and describe the symptoms suggestive of vascular entrapment of the sacral plexus, as well as the technique for the laparoscopic decompression of these nerves., Method: Two anecdotal cases of intrapelvic vascular entrapment are used to review the anatomy of the lumbosacral plexus and demonstrate the laparoscopic surgical technique for decompression at two different sites, one on the sciatic nerve and one on the sacral nerve roots., Result: After surgery, the patient with the sciatic entrapment showed full recovery of the sciatica and partial recovery of the myofascial pain. The patient with sacral nerve root entrapment showed full recovery with resolution of symptoms., Conclusion: The symptoms suggestive of intrapelvic nerve entrapment are: perineal pain or pain irradiating to the lower limbs in the absence of a spinal disorder, and lower urinary tract symptoms in the absence of prolapse of a bladder lesion. In the presence of such symptoms, the radiologist should provide specific MRI sequences of the intrapelvic portion of the sacral plexus and a team and equipment to expose and decompress the sacral nerves should be prepared.

Introduction and Hypothesis: The laparoscopic implantation of neuromodulation electrodes--the LION procedure--was first described as a rescue procedure in patients with local complications of a Brindley procedure. The objective of this video article is to demonstrate the technique for the laparoscopic implantation of electrodes for bilateral neuromodulation of femoral, sciatic and pudendal nerves and describe our initial experience with two multiple sclerosis (MS) patients., Method: This is a retrospective analysis of two patients with MS and neurogenic detrusor overactivity. A quadripolar electrode was implanted with two poles into the Alcock's canal and the two other laying over the lumbosacral trunk. The other two electrodes were implanted posteriorly to the femoral nerves., Results: At the neuromodulation trial, both patients presented a full recovery of urinary symptoms. One of them found it easier to stand up for transfers and daily activities and the other managed to advance from the wheelchair to the walker and both patients received the permanent implant. At 1-year follow up, urinary results were maintained and 1 patient presented a disease relapse, demanding multiple reprogramming sessions., Conclusion: Our initial observations are encouraging and indicate that the LION procedure seems to produce in MS patients similar results to those observed in patients with spinal cord injury. Patients, however, should be advised that MS is a progressive disease and that the positive effects of neuromodulation can potentially fade with time and that multiple reprogramming sessions might be necessary.

Objective: To demonstrate the laparoscopic neuroanatomy of the autonomic nerves of the pelvis using the laparoscopic neuronavigation technique, as well as the technique for a nerve-sparing radical endometriosis surgery., Design: Step-by-step explanation of the technique using videos and pictures (educational video) to demonstrate the anatomy of the intrapelvic bundles of the autonomic nerve system innervating the bladder, rectum, and pelvic floor., Setting: Tertiary referral center., Patient(s): One 37-year-old woman with an infiltrative endometriotic nodule on the anterior third of the left uterosacral ligament and one 34-year-old woman with rectovaginal endometriosis., Intervention(s): Exposure and preservation by direct visualization of the hypogastric nerve and the inferior hypogastric plexus., Main Outcome Measure(s): Visual control and identification of the autonomic nerve branches of the posterior pelvis., Result(s): Exposure and preservation of the hypogastric nerve and the superficial part of the left hypogastric nerve were achieved on the first patient. Nerve roots S2, S3, and S4 were identified on the second patient, allowing for the exposure and preservation of the pelvic splanchnic nerves and the deep portion inferior hypogastric plexus., Discussion(s): Radical surgery for endometriosis can induce urinary dysfunction in 2.4%-17.5% of patients owing to lesion of the autonomic nerves. The surgeon's knowledge of the anatomy of these nerves is the main factor for preserving postoperative urinary function. The following nerves are the intrapelvic part of the autonomic nervous system: the hypogastric nerves, which derive from the superior hypogastric plexus and carry the sympathetic signals to the internal urethral and anal sphincters as well as to the pelvic visceral proprioception; and the pelvic splanchnic nerves, which arise from S2 to S4 and carry nociceptive and parasympathetic signals to the bladder, rectum, and the sigmoid and left colons. The hypogastric and pelvic splanchnic nerves merge into the pararectal fossae to form the inferior hypogastric plexus. Most of the nerve-sparing techniques involve the dissection and exposure of the pelvic splanchnic nerves and the inferior hypogastric plexus. However, knowledge of the topographic anatomy and awareness of the landmarks for avoiding intraoperative nerve injuries seem to be the most important factors in avoiding postoperative bladder and bowel dysfunction, although this latter nerve-sparing technique seems to be associated with reduced radicality and symptom persistence., Conclusion(s): This video demonstrates a technique to expose the sympathetic and parasympathetic nerves of the pelvis to preserve them in radical pelvic surgery, by means of direct visualization, in a similar fashion to the technique used to preserve the ureters. An alternative to this technique is to use landmarks for limiting dissection and avoiding intraoperative nerve injury. Despite being safe and more easily reproducible, this latter technique is associated with a higher rate of symptom persistence., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Cutaneous melanoma is an aggressive malignancy; its incidence is increasing worldwide and its prognosis remains poor. Clinical observations indicate that estrogen receptor β (ERβ) is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its tumor suppressive function. These experiments were performed to investigate the effects of ERβ activation on melanoma cell growth., Methods and Results: Protein expression was analyzed by Western blot and immunofluorescence assays. Cell proliferation was assessed by counting the cells by hemocytometer. ERβ transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ERβ isoforms were identified by qRT-PCR. We demonstrated that ERβ is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552). In BLM (NRAS-mutant) cells, ERβ agonists significantly and specifically inhibited cell proliferation. ERβ activation triggered its cytoplasmic-to-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ERβ agonists was associated with an altered expression of G1-S transition-related proteins. In these cells, global DNA was found to be hypomethylated when compared to normal melanocytes; this DNA hypomethylation status was reverted by ERβ activation. ERβ agonists also decreased the proliferation of WM115 (BRAF V600D-mutant) cells, while they failed to reduce the growth of A375 and WM1552 (BRAF V600E-mutant) cells. Finally, we could observe that ERβ isoforms are expressed at different levels in the various cell lines. Specific oncogenic mutations or differential expression of receptor isoforms might be responsible for the different responses of cell lines to ERβ agonists., Conclusions: Our results demonstrate that ERβ is expressed in melanoma cell lines and that ERβ agonists differentially regulate the proliferation of these cells. These data confirm the notion that melanoma is a heterogeneous tumor and that genetic profiling is mandatory for the development of effective personalized therapeutic approaches for melanoma patients.

It is well established that gonadotropin-releasing hormone receptors (GnRH-R) are expressed in different types of cancers, including castration-resistant prostate cancer (CRPC) and mediate the antiproliferative effect of GnRH analogs. Thus, these compounds are employed as targeting moieties to selectively deliver chemotherapeutic agents to cancer cells. GnRH-III, the decapeptide isolated from the sea lamprey brain, has lower potency than GnRH in stimulating gonadotropin secretion, but it exerts antiproliferative effects on many tumors expressing the GnRH-R. GnRH-III-based peptides are considered promising targeting moieties for the preparation of anticancer drug delivery systems. These studies were aimed at i) evaluating the antitumor activity of two cytotoxic oxime bond-linked daunorubicin (Dau)-GnRH-III derivative bioconjugates (Dau-GnRH-III, in which daunorubicin was coupled to the 8Lys in the native form of GnRH-III, and Dau-[4Lys(Ac)]-GnRH-III, in which daunorubicin was attached to the 8Lys of a GnRH-III derivative where 4Ser was replaced by an acetylated lysine) on CRPC cells; and ii) to elucidate the involvement of the classical GnRH-R (type I GnRH-R) in this antitumor activity. Our results demonstrated that both Dau-GnRH-III and Dau-[4Lys(Ac)]-GnRH-III were rapidly internalized into DU145 prostate cancer cells and exerted a significant cytostatic effect. Both bioconjugates increased the levels of the active form of caspase-3, indicating the involvement of apoptosis in their antitumor activity. The antiproliferative effect of both Dau-GnRH-III and Dau-[4Lys(Ac)]-GnRH-III was counteracted by the simultaneous treatment of the cells with Antide, an antagonist of the GnRH-R. Moreover, after silencing the type I GnRH-R the antitumor activity of both bioconjugates was completely abolished. These data demonstrate that in CRPC cells, daunorubicin-GnRH-III derivative bioconjugates: i) inhibit tumor cell proliferation, by triggering the apoptosis process; ii) exert their antitumor effect through the activation of the type I GnRH-R expressed on these cells. Cytotoxic-GnRH-III derivative may represent promising targeted chemotherapeutics for the treatment of CRPC patients.

We applied an acute stress model to zebra fish in order to measure the changes in the metabolome due to biological stress. This was done by submitting the fish to fifteen minutes of acute confinement (netting) stress, and then five minutes for the open field and light/dark field tests. A polar extract of the zebra fish was then subjected to (1)H nuclear magnetic spectroscopy. Multivariate data analysis of the spectra showed a clear separation associated to a wide range of metabolites between zebra fish that were submitted to open field and light/dark field tests. Alanine, taurine, adenosine, creatine, lactate, and histidine were high in zebra fish to which the light/dark field test was applied, regardless of stress, while acetate and isoleucine/lipids appeared to be higher in zebra fish exposed to the open field test. These results show that any change in the environment, even for a small period of time, has a noticeable physiological impact. This research provides an insight of how different mechanisms are activated under different environments to maintain the homeostasis of the body. It should also contribute to establish zebra fish as a model for metabolomics studies., (Georg Thieme Verlag KG Stuttgart · New York.)

Gonadotropin-releasing hormone (GnRH) receptors are expressed in prostate cancer, specifically in the most aggressive stage of the tumor (castration-resistant prostate cancer, CRPC) for which the standard treatment, docetaxel-based chemotherapy, can only improve the median survival time by few months. We previously showed that GnRH agonists exert an antitumor activity in CRPC cells; however, a link between GnRH receptors and the apoptotic machinery remains to be defined. Aim of this study was to evaluate whether, in CRPC cells, GnRH agonists might affect the expression/activity of apoptosis-related proteins and might sensitize, or resensitize, cancer cells to chemotherapeutics. We demonstrated that, in p53-positive DU145 cells, GnRH agonists: a) increase the expression of the proapoptotic protein Bax; this effect is mediated by the phosphorylation (activation) of p53, triggered by the p38 MAPK; b) potentiate the antiproliferative/proapoptotic activity of docetaxel; c) resensitize docetaxel-resistant cells to the antitumor activity of the cytotoxic drug. These data indicate that GnRH agonists sensitize and, more importantly, resensitize DU145 CRPC cells to chemotherapy in a p53-dependent manner. To confirm the crucial role of p53 in the activity of GnRH agonists, experiments were performed in p53-null PC3 cells. We found that GnRH agonists fail to increase Bax expression and do not potentiate the cytotoxic activity of docetaxel. These results may provide a rationale for novel combination treatment strategies, especially for docetaxel-resistant CRPC patients expressing a functional p53 protein.

It is now well established that hormonal pathways are involved in the development of prostate cancer towards the castration resistant (CRPC) stage and can be effective molecular targets for novel treatment strategies. Most CRPC are sensitive to androgens and this can be due to the intratumoral production of androgens, androgen receptor (AR) amplification/ mutations and epigenetic modifications of AR expression/signaling. Based on these observations, potent agents targeting the AR axis were developed: 1) inhibitors of CYP17 (a key enzyme in the production of androgens), such as abiraterone and orteronel; 2) AR antagonists that bind to AR and impair AR activation, such as enzalutamide and ARN-509. Moreover, gonadotropin-releasing hormone receptors (GnRH-R), associated with a strong antitumor activity, are expressed in CRPC cells, indicating that they might represent an important target for GnRH analog-based therapeutic strategies. In addition to GnRH agonists and antagonists (i.e., degarelix), cytotoxic GnRH-based bioconjugates, delivering chemotherapeutic drugs to cancer cells expressing the GnRH-R, were developed and reported to exert antitumor effects on CRPC cells; some of them (i.e., AN-152) have already entered clinical trials. This review discusses the most relevant patents and recent observations on the anti-cancer efficacy of novel drugs targeting the AR and the GnRH-R pathways in CRPC.

(-)-Carvone is a monoterpene ketone found in spearmint (Mentha spicata var. crispa) essential oil that is widely used as an odor and flavor additive. An intestinal antispasmodic effect was recently reported for (-)-carvone, and it has been shown to be more potent than its (+)-antipode. The mechanism of (-)-carvone action in the intestines has not been investigated. To gain a better understanding of the (-)-carvone antispasmodic effect, we investigated its pharmacological effects in the guinea pig ileum. Terminal portions of the ileum were mounted for isotonic contraction recordings. The effect of (-)-carvone was compared with that of the classical calcium channel blocker (CCB) verapamil. In isolated ileal smooth muscle, (-)-carvone did not produce direct contractile or relaxation responses and did not modify electrically elicited contractions or low K(+)-evoked contractions. The submaximal contractions induced by histamine (p<0.001), BaCl2 (p<0.05), and carbachol (p<0.01) were significantly reduced by (-)-carvone. The contractile response elicited by high concentrations of carbachol was reduced but not abolished by (-)-carvone. No additive action was detected with co-incubation of (-)-carvone and verapamil on carbachol-induced contraction. (-)-Carvone reduced the contraction induced by high K(+) and was almost 100 times more potent than verapamil. Thus, (-)-carvone showed a typical and potent CCB-like action. Many effects described for both (-)-carvone and spearmint oil can be explained as a CCB-like mode of action., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The crucial role of pituitary GnRH receptors (GnRH-R) in the control of reproductive functions is well established. These receptors are the target of GnRH agonists (through receptor desensitization) and antagonists (through receptor blockade) for the treatment of steroid-dependent pathologies, including hormone-dependent tumors. It has also become increasingly clear that GnRH-R are expressed in cancer tissues, either related (i.e. prostate, breast, endometrial, and ovarian cancers) or unrelated (i.e. melanoma, glioblastoma, lung, and pancreatic cancers) to the reproductive system. In hormone-related tumors, GnRH-R appear to be expressed even when the tumor has escaped steroid dependence (such as castration-resistant prostate cancer). These receptors are coupled to a G(αi)-mediated intracellular signaling pathway. Activation of tumor GnRH-R by means of GnRH agonists elicits a strong antiproliferative, antimetastatic, and antiangiogenic (more recently demonstrated) activity. Interestingly, GnRH antagonists have also been shown to elicit a direct antitumor effect; thus, these compounds behave as antagonists of GnRH-R at the pituitary level and as agonists of the same receptors expressed in tumors. According to the ligand-induced selective-signaling theory, GnRH-R might assume various conformations, endowed with different activities for GnRH analogs and with different intracellular signaling pathways, according to the cell context. Based on these consistent experimental observations, tumor GnRH-R are now considered a very interesting candidate for novel molecular, GnRH analog-based, targeted strategies for the treatment of tumors expressing these receptors. These agents include GnRH agonists and antagonists, GnRH analog-based cytotoxic (i.e. doxorubicin) or nutraceutic (i.e. curcumin) hybrids, and GnRH-R-targeted nanoparticles delivering anticancer compounds.

The antispasmodic effect of 4'-methylepigallocatechin (MEC), which was isolated from Maytenus rigida Mart (Celestraceae), was investigated in vitro in guinea pig intestinal segments. In the isolated ileum, MEC (1 nM-100 μM) did not modify the ileal spontaneous tonus or the electrically elicited contractions. MEC (8 μM) significantly (p<0.01) reduced the submaximal contractions induced by histamine (2 μM), carbachol (100 μM) and BaCl₂ (0.03 M). An additive relaxing action (p<0.001) was observed by co-incubation of verapamil (10 nM) and MEC (8 μM). Although MEC (1 nM-100 μM) did not modify the contractions elicited by 60 mM KCl, it significantly reduced the CaCl₂ contractile response without changing the EC₅₀ (effective concentration of CaCl₂ causing 50% of maximum response). In brief, these results show that MEC has a potent ileal spasmolytic effect and blocks spasms induced by specific and nonspecific stimuli. Importantly, the spasmolytic effects were attained at low concentrations and might be related to the symptomatic relief of abdominal pain that is obtained from the use of the M. rigida stem bark., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: Heparanase 2 (HPSE2) is expressed in various tissues, including the brain, intestine, prostate, breast, and endometrium. The aim of this study was to investigate the role of HPSE2 in cervical carcinogenesis, which has not been clarified to date., Materials and Methods: The immunoexpression of HPSE2 in normal and neoplastic cervical squamous epithelia was determined using a semiquantitative (SQ) method and an index of expression (IE) method, using Image Lab Software. A total of 230 cervical tissue samples were analyzed and segregated into the following diagnostic groups: normal (27.4%), cervical intraepithelial neoplasia 1 (CIN 1, 15.2%), CIN 2 (16.5%), CIN 3 (15.2%), and invasive neoplasia (25.7%). The mean HPSE2 expression in the normal group was significantly lower than that of the other groups individually or combined (p < .001, for all combinations). The immunoexpression via the SQ method was significantly greater in the CIN 3 group compared with that in the CIN 1 group (p = .02). The mean immunoexpression of the high-grade squamous intraepithelial lesion groups was significantly greater than those of the normal and low-grade squamous intraepithelial lesion groups (p < .001) and lower compared with that of the invasive neoplasia group (p < .001). There were no statistically significant differences in the immunoexpression of HPSE2 among the different clinical states within the invasive neoplasia group., Conclusions: The SQ method produced a greater sensitivity and specificity than did the index of expression method. There was a progressive increase in the mean HPSE2 immunoexpression according to the severity of the cervical lesion from the low-grade squamous intraepithelial lesion group to the invasive neoplasm group, whereas the normal group displayed the lowest level of expression. This is a novel study concerning HPSE2 in the cervix and cervical cancer carcinogenesis.

Context: Erythrina velutina (EV) Willd (Fabaceae-Faboideae) is a medicinal tree that is commonly used in Brazil for the treatment of several central nervous system disorders., Objective: The anticholinesterase activity of EV is described in this work., Methods: Concentration-response curves (0-1.6 mg/mL) for EV leaf aqueous extract (AE) and alkaloid-rich extracts (AKEs) were performed in vitro. Cholinesterase inhibition was examined in mouse brains, as the cholinesterase source, and in pure acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE). Mice were treated with AE or AKE (100, 200, and 400 mg/kg, p.o.) and their brains were used for the measurement of cholinesterase activity (CA) ex vivo., Results: CA was inhibited by AE (IC(50) = 0.57 [0.43-0.75] mg/mL) and AKE (IC(50) = 0.52 [0.39-0.70] mg/mL) in brain homogenates in a concentration-dependent manner. The ex vivo experiments indicated that AE (400 mg/kg, p < 0.05, 32.2 ± 3.9% of inhibition) and AKE (all doses: p < 0.05-p < 0.001, 29.6 ± 3.2% as the maximum inhibition) significantly inhibited CA in the central nervous system after oral administration. AE and AKE inhibited AChE and BuChE activities in a concentration-dependent manner (AE: IC(50AChE) = 0.56 [0.38-0.81] mg/mL, IC(50BuChE) = 2.95 [1.51-5.76] mg/mL, AKE: IC(50AChE) = 0.87 [0.60-12.5] mg/mL, IC(50BuChE) = 2.67 [0.87-8.11] mg/mL)., Discussion and Conclusions: These data indicated that AE and AKE crossed the blood-brain barrier to inhibit CA in the brain. AE and AKE also exhibited a dual inhibitory action on acetyl- and BuChE.

The proapoptotic activity of nuclear clusterin (nCLU) in cancer cells is now well established. We previously showed that nCLU decreases the motility of prostate cancer cells by triggering a dramatic dismantling of the actin cytoskeleton. Here, we sought to unravel the molecular mechanisms of the antimetastatic activity of nCLU. We found that nCLU: i) decreases LIMK1 expression, thus increasing the levels of the active (unphosphorylated) form of cofilin, the well known actin depolymerizing factor; ii) binds to vimentin, sequestering the protein from its adhesion sites at the cell periphery, thus interfering with its role in cell motility and adhesion; iii) affects the intracellular distribution of E-cadherin (the major component of epithelial adherens junctions) which appears to be diffusely distributed in the cells. Through these mechanisms nCLU reduces the migratory/invasive behavior of PC3 cells; this effect is further demonstrated by a decreased secretion of active MMP-2 from the cells. Thus, in addition to its proapoptotic function, nCLU also exerts a strong anti-migratory/anti-invasive activity in prostate cancer cells, by interfering with the cytoskeletal components and by decreasing MMP-2 activity.

We showed previously that GnRH receptors are expressed in melanoma cells; their activation reduces cell growth and metastatic behavior. Here, we investigated whether GnRH agonists might affect the expression of genes involved in melanoma progression. By genome-wide transcriptomic and real-time PCR analysis, we first observed that GnRH agonists decrease the expression of the pro-angiogenic factor vascular endothelial growth factor (VEGF) (all isoforms) in BLM melanoma cells. Then, we demonstrated that GnRH agonists specifically decrease the expression of the VEGF165 isoform as well as its secretion from BLM cells. These data suggested that activation of GnRH receptors might reduce the pro-angiogenic behavior of melanoma cells. To verify this hypothesis, we treated BLM cells with a GnRH agonist; the conditioned medium from these cells was tested to assess its capability to stimulate human umbilical vein endothelial cell (HUVEC) motility. The migration of HUVECs towards the conditioned medium of GnRH agonist-treated BLM cells was significantly lower than the migration of HUVECs toward the conditioned medium of untreated cells. Thus, GnRH agonists reduce the pro-angiogenic behavior of melanoma cells through a decreased production of bioactive VEGF. We then found that GnRH receptors are also expressed on HUVECs and that GnRH agonists reduce their ability to proliferate and to form capillary-like tubes when stimulated by VEGF. These findings suggest that GnRH agonists exert an anti-angiogenic activity indirectly by decreasing VEGF secretion from tumor cells and directly by counteracting the pro-angiogenic activity of the growth factor. These data might lead to the development of novel targeted approaches for melanoma.

Malignant glioblastoma is one of the highest proliferative and invasive tumors within the central nervous system (CNS); the therapeutical options for this disease are still very poor. Receptors for gonadotropin-releasing hormone (GnRH) have been reported to be present in glioblastoma tissues. This study aimed to determine the role of these receptors in the control of glioma growth. In two human glioblastoma cell lines, U87MG and U373, we demonstrated the expression of GnRH receptors, both at mRNA and protein levels. We also found that GnRH receptor is expressed in glioblastoma tissues from tumor patients as shown by Western blotting. In U87MG and U373 cell lines, we found the expression of mRNA for GnRH, indicating the presence of an autocrine GnRH-based system in these cell lines. Treatment of the two cell lines with a GnRH agonist resulted in a significant decrease of cell proliferation. Moreover, the GnRH agonist significantly counteracted the forskolin-induced increase of intracellular cAMP levels in these cells. These findings suggest that the GnRH receptor might represent a molecular target for an endocrine therapeutical approach against gliomas.

Background: GnRH-II has been shown to exert a strong antiproliferative action on tumors of the female reproductive system. The data so far reported on the effects of GnRH-II on prostate cancer growth are controversial. Moreover, it is still unclear through which receptor [type I or type II GnRH-receptor (GnRH-R)] GnRH-II might modulate cancer cell proliferation., Objective: The objective of this work was to investigate whether GnRH-II might affect the proliferation of prostate cancer cells and to identify the GnRH-R through which the peptide might exert its activity., Design: We investigated the effects of GnRH-II on prostate cancer cell proliferation. We then transfected PC3 cells with a small interfering RNA targeted to type I GnRH-R. After receptor silencing we evaluated the effects of GnRH-II on cell proliferation and on forskolin-induced intracellular cAMP accumulation. Similar experiments were performed by silencing type II GnRH-R., Results: GnRH-II exerted an antiproliferative activity on prostate cancer cells. Transfection of PC3 cells with a type I GnRH-R small interfering RNA resulted in a significant decrease of the expression of this receptor. After type I GnRH-R silencing: 1) the antiproliferative effect of GnRH-II was completely abrogated; and 2) GnRH-II lost its capacity to counteract the forskolin-induced cAMP accumulation. On the contrary, type II GnRH-R silencing did not counteract the antiproliferative effect of GnRH-II., Conclusions: GnRH-II exerts a specific and significant antiproliferative action on prostate cancer cells. This antitumor effect is mediated by the activation of type I (but not of type II) GnRH-R and by its coupled cAMP intracellular signaling pathway.

Cutaneous melanoma represents the leading cause of skin cancer deaths. The prognosis of highly aggressive, metastatic melanoma is still very poor, due to the resistance of the disseminated tumor to existing therapies. The clarification of the molecular mechanisms regulating melanoma growth and progression might help identify novel molecular targets for the development of new therapeutic interventions. We previously showed that gonadotropin-releasing hormone (GnRH) receptors are expressed in melanoma cells; activation of these receptors by means of GnRH agonists significantly reduces cell proliferation. In the current study, we first showed that GnRH agonists significantly reduced the metastatic behavior of melanoma cells in terms of both cell motility (haptotactic assay using laminin as the chemoattractant) and invasiveness (cell invasion assay evaluating the capacity of the cells to invade a reconstituted extracellular matrix barrier). On the basis of this observation, we then investigated the molecular mechanisms underlying the antimetastatic activity of GnRH agonists. We found that, in melanoma cells, a) the activity of the alpha3 integrin subunit is crucial for the migratory behavior of the cells; b) GnRH agonists significantly reduced alpha3 integrin expression (Western blotting and immunofluorescence studies); c) GnRH agonists significantly reduced MMP-2 expression (comparative RT-PCR) and activity (zymographic analysis performed on cell culture media). These data indicate that GnRH agonists, in addition to the previously reported antiproliferative effect, elicit a strong inhibitory activity on the migratory/invasive behavior of melanoma cells expressing GnRH receptors. These compounds reduce the metastatic potential of melanoma cells by interfering with the expression/activity of cell adhesion molecules (alpha3 integrin) and matrix metalloproteinase (MMP-2).

Besides a fully processed, secreted form of clusterin (sCLU), an alternative proapoptotic form of the protein targeting the nucleus (nCLU) was recently described. The possible differential roles played by the two clusterin forms in growth and motility of nonmalignant and malignant prostate cells are investigated here. sCLU or nCLU was transiently transfected in both androgen-independent prostate cancer cells (PC3 and DU 145) and immortalized prostate epithelial cells (PNT1A, a nontumoral control). Then, cell growth, motility, and cytoskeleton organization were studied. We found that (a) in PNT1A cells, both sCLU and nCLU significantly decreased cell proliferation and motility; (b) in PC3 and DU 145 cancer cells, only nCLU inhibited cell growth and migration, with sCLU being ineffective; and (c) the antimotility effect of nCLU was accompanied by a dramatic dismantling of the actin cytoskeleton. Moreover, transfection with "full-length" CLU cDNA produced both sCLU and nCLU in nonmalignant PNT1A cells, whereas only sCLU was found in cancer cells. Thus, CLU gene expression might play a crucial role in prostate tumorigenesis by exerting differential biological effects on normal versus tumor cells through differential processing of CLU isoforms in the two cell systems. We also found that nCLU binds to alpha-actinin, a key protein for the regulation of actin cytoskeleton, and that nCLU and alpha-actinin colocalize in the cytoplasm. Thus, the antimotility activity of nCLU and its ability to cause dismantling of the actin cytoskeleton seem to be mediated by its binding to alpha-actinin.

Androgen-independent prostate carcinoma is characterized by a high proliferation rate and by a strong metastatic behavior. We have previously shown that GnRH agonists exert a direct and specific inhibitory action on the proliferation of androgen-independent prostate cancer cells (DU 145). These compounds mainly act by interfering with the mitogenic activity of growth factors, such as the insulin-like growth factor-I (IGF-I). The present experiments were performed to clarify whether GnRH agonists might also affect the migratory and the invasive behavior of androgen-independent prostate cancer cells and to define their mechanism of action. First we showed that the GnRH agonist Leuprolide reduces the migration of DU 145 cells towards a chemoattractant and their ability to invade a reconstituted basement membrane. Experiments were then performed to clarify whether the GnRH agonist might act by interfering with the pro-metastatic activity of IGF-I. We found that, in androgen-independent prostate cancer cells, Leuprolide: a) interferes with the IGF-I system (receptor protein expression and tyrosine-phosphorylation); b) abrogates the IGF-I-induced phosphorylation of Akt (a kinase previously shown by us to mediate the pro-metastatic activity of IGF-I in prostate cancer cells); c) counteracts the migration and the invasive activity of the cells stimulated by IGF-I; d) abolishes the effects of IGF-I on cell morphology, on actin cytoskeleton organization and on alphavbeta3 integrin expression/cellular localization. These data indicate that GnRH agonists, in addition to their well known antiproliferative effect, can also exert a significant inhibitory activity on the migratory and invasive behavior of androgen-independent prostate cancer cells, expressing the GnRH receptor. GnRH agonists act by interfering with the pro-metastatic activity of the growth factor IGF-I.