Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(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 Kd 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.

Necroptosis Induced by Delta-Tocotrienol Overcomes Docetaxel Chemoresistance in Prostate Cancer Cells.

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.

Apoptosis-mediated anticancer activity in prostate cancer cells of a chestnut honey (Castanea sativa L.) quinoline-pyrrolidine gamma-lactam alkaloid.

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.

Anticancer properties of tocotrienols: A review of cellular mechanisms and molecular targets.

Vitamin E is composed of two groups of compounds: α-, ß-, 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.

Protein kinase C epsilon activation regulates proliferation, migration, and epithelial to mesenchymal-like transition in rat Schwann cells.

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.

The Leaf Essential Oil of Myrtus communis subsp. tarentina (L.) Nyman: From Phytochemical Characterization to Cytotoxic and Antimigratory Activity in Human Prostate Cancer Cells.

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 ß-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.

HSPB8 counteracts tumor activity of BRAF- and NRAS-mutant melanoma cells by modulation of RAS-prenylation and autophagy.

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 (BRAFwt/NRASQ61R) and A375 (BRAFV600E/NRASwt) 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.

Anosmin-1-Like Effect of UMODL1/Olfactorin on the Chemomigration of Mouse GnRH Neurons and Zebrafish Olfactory Axons Development.

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.

δ-Tocotrienol induces apoptosis, involving endoplasmic reticulum stress and autophagy, and paraptosis in prostate cancer cells.

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.

Semi-preparative HPLC purification of δ-tocotrienol (δ-T3) from Elaeis guineensis Jacq. and Bixa orellana L. and evaluation of its in vitro anticancer activity in human A375 melanoma cells.

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 1H 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.

Dual role of autophagy on docetaxel-sensitivity in prostate cancer cells.

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.

Targeting melanoma stem cells with the Vitamin E derivative δ-tocotrienol.

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.

GnRH and GnRH receptors in the pathophysiology of the human female reproductive system.

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.

Estrogen Receptor ß in Melanoma: From Molecular Insights to Potential Clinical Utility.

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 ERß) that affect cancer growth in an opposite way: ERα is associated with a proliferative action and ERß with an anticancer effect. ERß is the predominant ER in melanoma, and its expression decreases in melanoma progression, supporting its role as a tumor suppressor. Thus, ERß is now considered as an effective molecular target for melanoma treatment. 17ß-estradiol was reported to inhibit melanoma cells proliferation; however, clinical trials did not provide the expected survival benefits. In vitro studies demonstrate that ERß ligands inhibit the proliferation of melanoma cells harboring the NRAS (but not the BRAF) mutation, suggesting that ERß activation might impair melanoma development through the inhibition of the PI3K/Akt pathway. These data suggest that ERß 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 ERß 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 ERß activating function, these compounds might impair melanoma development through additional mechanisms. A better identification of the role of ERß in melanoma development will help increase the therapeutic options for this aggressive pathology.