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.

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.

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.

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.

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.

The Role of HSPB8, a Component of the Chaperone-Assisted Selective Autophagy Machinery, in Cancer.

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.

Gonadotropin-Releasing Hormone Receptors in Prostate Cancer: Molecular Aspects and Biological Functions.

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.

Cellular and molecular biology of cancer stem cells in melanoma: Possible therapeutic implications.

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.

Unraveling the molecular mechanisms and the potential chemopreventive/therapeutic properties of natural compounds in melanoma.

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.

Role of Endoplasmic Reticulum Stress in the Anticancer Activity of Natural Compounds.

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.

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.

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.

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.

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.

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.

Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells.

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.

Estrogen Receptor ß Agonists Differentially Affect the Growth of Human Melanoma Cell Lines.

BACKGROUND: Cutaneous melanoma is an aggressive malignancy; its incidence is increasing worldwide and its prognosis remains poor. Clinical observations indicate that estrogen receptor ß (ERß) 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 ERß 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. ERß transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ERß isoforms were identified by qRT-PCR. We demonstrated that ERß is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552). In BLM (NRAS-mutant) cells, ERß agonists significantly and specifically inhibited cell proliferation. ERß activation triggered its cytoplasmic-to-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ERß 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 ERß activation. ERß 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 ERß 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 ERß agonists. CONCLUSIONS: Our results demonstrate that ERß is expressed in melanoma cell lines and that ERß 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.

Oxime bond-linked daunorubicin-GnRH-III bioconjugates exert antitumor activity in castration-resistant prostate cancer cells via the type I GnRH receptor.

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.

In vitro chronic administration of ERbeta selective ligands and prostate cancer cell growth: hypotheses on the selective role of 3beta-adiol in AR-positive RV1 cells.

Prostate cancer (PC) progression from androgen-dependent (AD) to castration-resistant (CR) disease is a process caused by modifications of different signal transduction pathways within tumor microenvironment. Reducing cell proliferation, estrogen receptor beta (ERbeta) is emerging as a potential target in PC chemoprevention. Among the known selective ERbeta ligands, 3beta-Adiol, the endogenous ligand in the prostate, has been proved to counteract PC progression. This study compares the effects of chronic exposure (1-12 weeks) to different ERbeta selective ligands (DPN, 8beta-VE2, 3beta-Adiol) on proliferation of human androgen-responsive CWR22Rv1 cells, representing an intermediate phenotype between the AD- and CR-PC. 3beta-Adiol (10 nM) is the sole ligand decreasing cell proliferation and increasing p21 levels. In vitro transcriptional activity assays were performed to elucidate different behavior between 3beta-Adiol and the other ligands; in these experiments the endogenous and the main ERbeta subtype activation were considered. It is concluded that ERbeta activation has positive effects also in androgen-responsive PC. The underlying mechanisms are still to be clarified and may include the interplay among different ERbeta subtypes and the specific PC microenvironment. ERbeta agonists might be useful in counteracting PC progression, although the final outcome may depend upon the molecular pattern specific to each PC lesion.