NEW INSIGHTS ON ESTROGENS AND VITAMIN E DERIVATIVE TOCOTRIENOLS ON HUMAN MALIGNANT MELANOMA: TOWARDS NOVEL THERAPEUTIC INTERVENTIONS.

Malignant melanoma represents the deadliest form of skin cancer: despite surgical resection of cutaneous in situ melanoma warrants a good prognosis, metastatic melanoma is mostly an incurable disease, because of the fast developing of resistance to existing therapies. For this reason, the identification of novel molecular targets and more effective antitumor compounds might be helpful in the management of the pathology. Clinical observations indicate that estrogen receptor \u3b2 (ER\u3b2) is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its anticancer activity. Moreover, this receptor was found to mediate the antitumor effect of the vitamin E derivative \u3b4-tocotrienol on breast cancer cell lines. This project firstly focused on the characterization of the estrogenic system on human melanoma cell lines. ER\u3b2 is expressed in melanoma cell lines but one, and proliferation and transactivation studies indicated that specific ER\u3b2 ligands exert an antiproliferative activity inducing the classical, nuclear mechanism of action of steroid hormones receptors. This antitumor effect is due to a G1/S cell cycle blockade, evidenced by the induction of the cell cycle inhibitor p27 and the reduction of the expression of cyclin D1 and D3, rather than to the activation of apoptotic mechanisms; furthermore, the activation of the receptor can influence the DNA methylation pattern of melanoma cells, suggesting that it could also exert antitumor effects through epigenetic regulation. However, the antiproliferative activity is dependent on the specific Ras/Raf mutational status of the cells and/or the expression of specific ER\u3b2 isoforms. MTT assays indicated that \u3b4-tocotrienol induces a significant reduction in melanoma cell viability, independently of the specific mutational status of the cells, and the expression of ER\u3b2. Gene-reporter assays failed to associate the antitumor activity of \u3b4-tocotrienol and the activation of the estrogen receptor \u3b2 in melanoma cells. The molecular mechanisms underlying \u3b4-tocotrienol activity were then analysed, evidencing a cytotoxic/apoptotic effect, through inhibition of colony-formation and cleavage of caspase-3 and PARP. Such apoptotic effect was found to be related to the activation of the endoplasmic reticulum (ER) stress pathways, as demonstrated by the induction of ER stress markers PERK, IRE1\u3b1, ATF4 and CHOP and the cleavage of caspase-4. Furthermore, the apoptotic effect was partially reverted by the ER stress inhibitor salubrinal. However, the mitochondrial apoptotic way was also activated by \u3b4-tocotrienol, since cytochrome c release and Bax/Bcl-2 ratio increment were observed. In vivo studies were also performed, and the reduction of tumor mass and volume and the delay of tumor progression strongly support the effectiveness of this compound on melanoma. Finally, the population of cancer stem cells was characterized in the A375 melanoma cell line, through the ability to grow on suspended melanospheres, and the expression of the melanoma stem cell marker CD271 and the embryonic stem cell marker Oct4. The cytotoxic activity of \u3b4-tocotrienol was assessed on this population of cells, showing the reduction in the number of spheroids and in the expression of CD271. In conclusion, this doctoral project demonstrated two important points. 1- ER\u3b2 has a tumor suppressive role on melanoma cells, depending of the specific mutation found in the Ras/Raf pathway: these results enforce the usefulness of genetic profiling of melanoma samples in dictating the more effective therapeutic strategy. Future efforts should be done to assess the clinical application of ER\u3b2-based therapies. 2- The vitamin E derivative \u3b4-tocotrienol has a potent antitumor effect, independent of ER\u3b2 activation. The translation of these results to the clinics might be an important tool for the management of metastatic melanoma, at least as adjuvant therapy

Anticancer properties of tocotrienols : a review of cellular mechanisms and molecular targets

Vitamin E is composed of two groups of compounds: \u3b1-, \u3b2-, \u3b3-, and \u3b4-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, \u3b3-TT and \u3b4-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, \u201ctargeted\u201d 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

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

Estrogen receptor β 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 \u3b2 (ER\u3b2) 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 ER\u3b2 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. ER\u3b2 transcriptional activity was evaluated by gene reporter assay. Global DNA methylation was analyzed by restriction enzyme assay and ER\u3b2 isoforms were identified by qRT-PCR. We demonstrated that ER\u3b2 is expressed in a panel of human melanoma cell lines (BLM, WM115, A375, WM1552). In BLM (NRAS-mutant) cells, ER\u3b2 agonists significantly and specifically inhibited cell proliferation. ER\u3b2 activation triggered its cytoplasmic-To-nuclear translocation and transcriptional activity. Moreover, the antiproliferative activity of ER\u3b2 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 ER\u3b2 activation. ER\u3b2 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 ER\u3b2 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 ER\u3b2 agonists. Conclusions Our results demonstrate that ER\u3b2 is expressed in melanoma cell lines and that ER\u3b2 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. Copyright

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 enzyme1 (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

Gonadotropin-Releasing Hormone Agonists Sensitize, and Resensitize, Prostate Cancer Cells to Docetaxel in a p53-Dependent Manner

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 docetaxelresistant CRPC patients expressing a functional p53 protein

PO-078 Role of the ER stress-autophagy axis and mitochondrial metabolism reprogramming in the apoptosis induced by δ-TOCOTRIENOL in prostate cancer

Introduction Castration resistant prostate cancer (CRPC) is an aggressive tumour with still limited therapeutic outcomes. Tocotrienols (TT), vitamin E derivatives, were reported to exert anticancer activity in different tumours. The aim of this study was to assess the effects of δ-TT on human CRPC cells growth and the molecular mechanisms associated with its activity. Material and methods In human normal prostate (RWPE-1) and CRPC (PC3 and DU145) cell lines the effect of δ-TT on cell viability was evaluated by MTT assay; in PC3 and DU145 cells Trypan blue exclusion and colony formation assays were also performed. The expression of apoptosis-, ER stress- and autophagy-related proteins was analysed by Western blot and immunofluorescence assays, and the cytotoxic effect of δ-TT was also assessed using specific inhibitors of these pathways. The effect on mitochondrial metabolism was evaluated analysing the expression of the OXPHOS complexes (Western blot), the mitochondrial activity and mass (flow cytometry), the oxygen consumption (Clark-type oxygen electrode) and the ATP production (colorimetric assay). Results and discussions We demonstrated that δ-TT exerts a cytotoxic effect on PC3 and DU145 but not on RWPE-1 cells. In particular, δ-TT induces caspase 3 and PARP cleavage and cytochrome c release from mitochondria, and its cytotoxic effect is partially blocked by co-treatment with the pan-caspase inhibitor z-VAD-FMK, confirming that δ-TT exerts a pro-apoptotic effect on CRPC cells. We also observed that δ-TT significantly increases the expression of ER stress (BiP, IRE1α, PERK, pEIF2α, ATF4 and CHOP) and autophagy mediators (LC3-II and p62). Using the ER stress inhibitors salubrinal and 4-phenylbutyrate (4-PBA) and the autophagic flux inhibitors 3-methyladenine and chloroquine, we confirmed that the effect of δ-TT is mediated by both these mechanisms. In addition, treatment with salubrinal or 4-PBA impairs δ-TT-induced LC3-II expression, demonstrating that this compound triggers the ER stress-autophagy axis. Finally, we observed that δ-TT severely alters mitochondrial metabolism: the expression of the OXPHOS protein complexes, the mitochondrial activity/mass ratio, the oxygen consumption and the ATP production were significantly reduced after δ-TT treatment. Conclusion These results demonstrate that δ-TT exerts a selective pro-apoptotic effect on human CRPC cells through the activation of the ER stress-autophagy axis and the rewiring of mitochondrial metabolism

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 \uce\ub4-TT might target melanoma CSCs. We demonstrated that melanoma cells escaping the antitumor activity of \uce\ub4-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. \uce\ub4-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 \uce\ub4-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma

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 \u3b4-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. \u3b4-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, \u3b4-TT exerted its antitumor effect through activation of the PERK/p-eIF2\u3b1/ATF4/CHOP, IRE1\u3b1 and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of \u3b4-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that \u3b4-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by \u3b4-TT treatment. In conclusion, \u3b4-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. \u3b4-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma