Differentiation States of Phenotypic Transition of Melanoma Cells Are Revealed by 3D Cell Cultures.

Melanoma is characterized by high metastatic potential favored by the epithelial-to-mesenchymal transition (EMT), leading melanoma cells to exhibit a spectrum of typical EMT markers. This study aimed to analyze the expression of EMT markers in A375 and BLM melanoma cell lines cultured in 2D monolayers and 3D spheroids using morphological and molecular methods. The expression of EMT markers was strongly affected by 3D arrangement and revealed a hybrid phenotype for the two cell lines. Indeed, although E-cadherin was almost undetectable in both A375 and BLM cells, cortical actin was detected in A375 2D monolayers and 3D spheroids and was strongly expressed in BLM 3D spheroids. The mesenchymal marker N-cadherin was significantly up-regulated in A375 3D spheroids while undetectable in BLM cells, but vimentin was similarly expressed in both cell lines at the gene and protein levels. This pattern suggests that A375 cells exhibit a more undifferentiated/mesenchymal phenotype, while BLM cells have more melanocytic/differentiated characteristics. Accordingly, the Zeb1 and 2, Slug, Snail and Twist gene expression analyses showed that they were differentially expressed in 2D monolayers compared to 3D spheroids, supporting this view. Furthermore, A375 cells are characterized by a greater invasive potential, strongly influenced by 3D arrangement, compared to the BLM cell line, as evaluated by SDS-zymography and TIMPs gene expression analysis. Finally, TGF-ß1, a master controller of EMT, and lysyl oxidase (LOX), involved in melanoma progression, were strongly up-regulated by 3D arrangement in the metastatic BLM cells alone, likely playing a role in the metastatic phases of melanoma progression. Overall, these findings suggest that A375 and BLM cells possess a hybrid/intermediate phenotype in relation to the expression of EMT markers. The former is characterized by a more mesenchymal/undifferentiated phenotype, while the latter shows a more melanocytic/differentiated phenotype. Our results contribute to the characterization of the role of EMT in melanoma cells and confirm that a 3D cell culture model could provide deeper insight into our understanding of the biology of melanoma.

Adipocytes reprogram prostate cancer stem cell machinery.

It is now well-established that an obese condition correlates with a higher risk of prostate cancer (PCa). A crosstalk between adipose tissue and PCa has been observed but is still poorly characterized. Herein, we demonstrated that 3T3-L1 adipocyte conditioned media (CM) could endow PC3 and DU145 PCa cells with stemness properties, by stimulating their sphere formation ability and promoting CD133 and CD44 expression. Moreover, after exposure to adipocyte CM both PCa cell lines underwent partial epithelial-to-mesenchymal transition (EMT), with E-/N-cadherin switch and Snail upregulation. Specifically, these changes in PC3 and DU145 cell phenotype were accompanied by increased tumor clonogenic activity and survival, as well as by enhanced invasion, anoikis resistance and matrix metalloproteinase (MMP) production. Finally, adipocyte CM-treated PCa cells exhibited reduced responsiveness to both docetaxel and cabazitaxel, demonstrating greater chemoresistance. Overall, these data indicate that adipose tissue can effectively contribute to PCa aggressiveness by reprogramming the cancer stem cell (CSC) machinery. Adipocytes endow prostate cancer cells with stem-like properties and mesenchymal traits, increasing their tumorigenicity, invasion and chemoresistance.

Unraveling the Peculiar Features of Mitochondrial Metabolism and Dynamics in Prostate Cancer.

Prostate cancer (PCa) is the second leading cause of cancer deaths among men in Western countries. Mitochondria, the "powerhouse" of cells, undergo distinctive metabolic and structural dynamics in different types of cancer. PCa cells experience peculiar metabolic changes during their progression from normal epithelial cells to early-stage and, progressively, to late-stage cancer cells. Specifically, healthy cells display a truncated tricarboxylic acid (TCA) cycle and inefficient oxidative phosphorylation (OXPHOS) due to the high accumulation of zinc that impairs the activity of m-aconitase, the enzyme of the TCA cycle responsible for the oxidation of citrate. During the early phase of cancer development, intracellular zinc levels decrease leading to the reactivation of m-aconitase, TCA cycle and OXPHOS. PCa cells change their metabolic features again when progressing to the late stage of cancer. In particular, the Warburg effect was consistently shown to be the main metabolic feature of late-stage PCa cells. However, accumulating evidence sustains that both the TCA cycle and the OXPHOS pathway are still present and active in these cells. The androgen receptor axis as well as mutations in mitochondrial genes involved in metabolic rewiring were shown to play a key role in PCa cell metabolic reprogramming. Mitochondrial structural dynamics, such as biogenesis, fusion/fission and mitophagy, were also observed in PCa cells. In this review, we focus on the mitochondrial metabolic and structural dynamics occurring in PCa during tumor development and progression; their role as effective molecular targets for novel therapeutic strategies in PCa patients is also discussed.

Adipocyte-Derived Extracellular Vesicles Promote Prostate Cancer Cell Aggressiveness by Enabling Multiple Phenotypic and Metabolic Changes.

BACKGROUND: In recent decades, obesity has widely emerged as an important risk factor for prostate cancer (PCa). Adipose tissue and PCa cells have been shown to orchestrate a complex interaction network to support tumor growth and evolution; nonetheless, the study of this communication has only been focused on soluble factors, although increasing evidence highlights the key role of extracellular vesicles (EVs) in the modulation of tumor progression. METHODS AND RESULTS: In the present study, we found that EVs derived from 3T3-L1 adipocytes could affect PC3 and DU145 PCa cell traits, inducing increased proliferation, migration and invasion. Furthermore, conditioning of both PCa cell lines with adipocyte-released EVs resulted in lower sensitivity to docetaxel, with reduced phosphatidylserine externalization and decreased caspase 3 and PARP cleavage. In particular, these alterations were paralleled by an Akt/HIF-1α axis-related Warburg effect, characterized by enhanced glucose consumption, lactate release and ATP production. CONCLUSIONS: Collectively, these findings demonstrate that EV-mediated crosstalk exists between adipocytes and PCa, driving tumor aggressiveness.

Melanoma Stem Cells Educate Neutrophils to Support Cancer Progression.

BACKGROUND: It is now well-established that cancer stem cells (CSCs) can support melanoma progression by reshaping the tumor immune microenvironment. However, the molecular mechanisms underlying the crosstalk between melanoma SCs and cancer-associated neutrophils have not been elucidated yet. METHODS: The aim of the present study was to unravel the role of melanoma SCs in neutrophil polarization. HL60 neutrophil-like (dHL60) cells were treated with conditioned medium from A375 melanoma SCs (CSC-CM), and their phenotype was investigated. RESULTS: We demonstrated that CSC-CM could specifically activate immune cells by increasing CD66b and CD11b expression. In particular, we revealed that A375 CSCs could release various soluble factors, namely TGF-ß, IL-6, and IL-8, able to promote the recruitment of neutrophils and their switch toward an N2 phenotype characterized by the activation of ERK, STAT3, and P38 pathways and the overexpression of CXCR2 and NF-kB. Moreover, after exposure to CSC-CM, dHL60 cells exhibited enhanced ROS production and NET release, without undergoing cell death; increased secretion of MMP-9 and pro-inflammatory cytokines was also observed. Finally, CSC-CM-activated neutrophils endowed A375 cells with stemness traits, stimulating both sphere formation and ABCG2 expression. CONCLUSION: Collectively, our results suggest that melanoma SCs can prime neutrophils to support cancer progression.

Exploiting the Metabolic Consequences of PTEN Loss and Akt/Hexokinase 2 Hyperactivation in Prostate Cancer: A New Role for δ-Tocotrienol.

The Warburg effect is commonly recognized as a hallmark of nearly all tumors. In prostate cancer (PCa), it has been shown to be driven by PTEN loss- and Akt hyperactivation-associated upregulation of hexokinase 2 (HK2). δ-Tocotrienol (δ-TT) is an extensively studied antitumor compound; however, its role in affecting PCa glycolysis is still unclear. Herein, we demonstrated that δ-TT inhibits glucose uptake and lactate production in PTEN-deficient LNCaP and PC3 PCa cells, by specifically decreasing HK2 expression. Notably, this was accompanied by the inhibition of the Akt pathway. Moreover, the nutraceutical could synergize with the well-known hypoglycemic agent metformin in inducing PCa cell death, highlighting the crucial role of the above metabolic phenotype in δ-TT-mediated cytotoxicity. Collectively, these results unravel novel inhibitory effects of δ-TT on glycolytic reprogramming in PCa, thus providing new perspectives into the mechanisms of its antitumor activity and into its use in combination therapy.

Molecular Mechanisms of Cancer Drug Resistance: Emerging Biomarkers and Promising Targets to Overcome Tumor Progression.

Cancer still represents a major global burden, being the second leading cause of death worldwide [...].

Molecular mechanisms and genetic alterations in prostate cancer: From diagnosis to targeted therapy.

Prostate cancer remains one of the most lethal malignancies among men worldwide. Although the primary tumor can be successfully managed by surgery and radiotherapy, advanced metastatic carcinoma requires better therapeutic approaches. In this context, a deeper understanding of the molecular mechanisms that underlie the initiation and progression of this disease is urgently needed, leading to the identification of new diagnostic/prognostic markers and the development of more effective treatments. Herein, the current state of knowledge of prostate cancer genetic alterations is discussed, with a focus on their potential in tumor detection and staging as well as in the screening of novel therapeutics.

The multifaceted roles of mitochondria at the crossroads of cell life and death in cancer.

Mitochondria are the cytoplasmic organelles mostly known as the "electric engine" of the cells; however, they also play pivotal roles in different biological processes, such as cell growth/apoptosis, Ca2+ and redox homeostasis, and cell stemness. In cancer cells, mitochondria undergo peculiar functional and structural dynamics involved in the survival/death fate of the cell. Cancer cells use glycolysis to support macromolecular biosynthesis and energy production ("Warburg effect"); however, mitochondrial OXPHOS has been shown to be still active during carcinogenesis and even exacerbated in drug-resistant and stem cancer cells. This metabolic rewiring is associated with mutations in genes encoding mitochondrial metabolic enzymes ("oncometabolites"), alterations of ROS production and redox biology, and a fine-tuned balance between anti-/proapoptotic proteins. In cancer cells, mitochondria also experience dynamic alterations from the structural point of view undergoing coordinated cycles of biogenesis, fusion/fission and mitophagy, and physically communicating with the endoplasmic reticulum (ER), through the Ca2+ flux, at the MAM (mitochondria-associated membranes) levels. This review addresses the peculiar mitochondrial metabolic and structural dynamics occurring in cancer cells and their role in coordinating the balance between cell survival and death. The role of mitochondrial dynamics as effective biomarkers of tumor progression and promising targets for anticancer strategies is also discussed.

δ-Tocotrienol sensitizes and re-sensitizes ovarian cancer cells to cisplatin via induction of G1 phase cell cycle arrest and ROS/MAPK-mediated apoptosis.

OBJECTIVES: Among gynaecologic malignancies, ovarian cancer (OC) represents the leading cause of death for women worldwide. Current OC treatment involves cytoreductive surgery followed by platinum-based chemotherapy, which is associated with severe side effects and development of drug resistance. Therefore, new therapeutic strategies are urgently needed. Herein, we evaluated the anti-tumour effects of Vitamin E-derived δ-tocotrienol (δ-TT) in two human OC cell lines, IGROV-1 and SKOV-3 cells. MATERIALS AND METHODS: MTT and Trypan blue exclusion assays were used to assess δ-TT cytotoxicity, alone or in combination with other molecules. δ-TT effects on cell cycle, apoptosis, ROS generation and MAPK phosphorylation were investigated by flow cytometry, Western blot and immunofluorescence analyses. The synergism between δ-TT and chemotherapy was evaluated by isobologram analysis. RESULTS: We demonstrated that δ-TT could induce cell cycle block at G1-S phase and mitochondrial apoptosis in OC cell lines. In particular, we found that the proapoptotic activity of δ-TT correlated with mitochondrial ROS production and subsequent JNK and p38 activation. Finally, we observed that the compound was able to synergize with cisplatin, not only enhancing its cytotoxicity in IGROV-1 and SKOV-3 cells but also re-sensitizing IGROV-1/Pt1 cell line to its anti-tumour effects. CONCLUSIONS: δ-TT triggers G1 phase cell cycle arrest and ROS/MAPK-mediated apoptosis in OC cells and sensitizes them to platinum treatment, thus representing an interesting option for novel chemopreventive/therapeutic strategies for OC.

In Vitro 3D Cultures to Model the Tumor Microenvironment.

It is now well established that the tumor microenvironment plays a key role in determining cancer growth, metastasis and drug resistance. Thus, it is fundamental to understand how cancer cells interact and communicate with their stroma and how this crosstalk regulates disease initiation and progression. In this setting, 3D cell cultures have gained a lot of interest in the last two decades, due to their ability to better recapitulate the complexity of tumor microenvironment and therefore to bridge the gap between 2D monolayers and animal models. Herein, we present an overview of the 3D systems commonly used for studying tumor-stroma interactions, with a focus on recent advances in cancer modeling and drug discovery and testing.

Dissecting the Hormonal Signaling Landscape in Castration-Resistant Prostate Cancer.

Understanding the molecular mechanisms underlying prostate cancer (PCa) progression towards its most aggressive, castration-resistant (CRPC) stage is urgently needed to improve the therapeutic options for this almost incurable pathology. Interestingly, CRPC is known to be characterized by a peculiar hormonal landscape. It is now well established that the androgen/androgen receptor (AR) axis is still active in CRPC cells. The persistent activity of this axis in PCa progression has been shown to be related to different mechanisms, such as intratumoral androgen synthesis, AR amplification and mutations, AR mRNA alternative splicing, increased expression/activity of AR-related transcription factors and coregulators. The hypothalamic gonadotropin-releasing hormone (GnRH), by binding to its specific receptors (GnRH-Rs) at the pituitary level, plays a pivotal role in the regulation of the reproductive functions. GnRH and GnRH-R are also expressed in different types of tumors, including PCa. Specifically, it has been demonstrated that, in CRPC cells, the activation of GnRH-Rs is associated with a significant antiproliferative/proapoptotic, antimetastatic and antiangiogenic activity. This antitumor activity is mainly mediated by the GnRH-R-associated Gαi/cAMP signaling pathway. In this review, we dissect the molecular mechanisms underlying the role of the androgen/AR and GnRH/GnRH-R axes in CRPC progression and the possible therapeutic implications.

Ca2+ overload- and ROS-associated mitochondrial dysfunction contributes to δ-tocotrienol-mediated paraptosis in melanoma cells.

Melanoma is an aggressive tumor with still poor therapy outcomes. δ-tocotrienol (δ-TT) is a vitamin E derivative displaying potent anti-cancer properties. Previously, we demonstrated that δ-TT triggers apoptosis in human melanoma cells. Here, we investigated whether it might also activate paraptosis, a non-canonical programmed cell death. In accordance with the main paraptotic features, δ-TT was shown to promote cytoplasmic vacuolization, associated with endoplasmic reticulum/mitochondrial dilation and protein synthesis, as well as MAPK activation in A375 and BLM cell lines. Moreover, treated cells exhibited a significant reduced expression of OXPHOS complex I and a marked decrease in oxygen consumption and mitochondrial membrane potential, culminating in decreased ATP synthesis and AMPK phosphorylation. This mitochondrial dysfunction resulted in ROS overproduction, found to be responsible for paraptosis induction. Additionally, δ-TT caused Ca2+ homeostasis disruption, with endoplasmic reticulum-derived ions accumulating in mitochondria and activating the paraptotic signaling. Interestingly, by using both IP3R and VDAC inhibitors, a close cause-effect relationship between mitochondrial Ca2+ overload and ROS generation was evidenced. Collectively, these results provide novel insights into δ-TT anti-melanoma activity, highlighting its ability to induce mitochondrial dysfunction-mediated paraptosis. δ-tocotrienol induces paraptotic cell death in human melanoma cells, causing endoplasmic reticulum dilation and mitochondrial swelling. These alterations induce an impairment of mitochondrial function, ROS production and calcium overload.

Cancer Stem Cells-Key Players in Tumor Relapse.

Tumor relapse and treatment failure are unfortunately common events for cancer patients, thus often rendering cancer an uncurable disease. Cancer stem cells (CSCs) are a subset of cancer cells endowed with tumor-initiating and self-renewal capacity, as well as with high adaptive abilities. Altogether, these features contribute to CSC survival after one or multiple therapeutic approaches, thus leading to treatment failure and tumor progression/relapse. Thus, elucidating the molecular mechanisms associated with stemness-driven resistance is crucial for the development of more effective drugs and durable responses. This review will highlight the mechanisms exploited by CSCs to overcome different therapeutic strategies, from chemo- and radiotherapies to targeted therapies and immunotherapies, shedding light on their plasticity as an insidious trait responsible for their adaptation/escape. Finally, novel CSC-specific approaches will be described, providing evidence of their preclinical and clinical applications.

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.

Three-Dimensional Cell Cultures as an In Vitro Tool for Prostate Cancer Modeling and Drug Discovery.

In the last decade, three-dimensional (3D) cell culture technology has gained a lot of interest due to its ability to better recapitulate the in vivo organization and microenvironment of in vitro cultured cancer cells. In particular, 3D tumor models have demonstrated several different characteristics compared with traditional two-dimensional (2D) cultures and have provided an interesting link between the latter and animal experiments. Indeed, 3D cell cultures represent a useful platform for the identification of the biological features of cancer cells as well as for the screening of novel antitumor agents. The present review is aimed at summarizing the most common 3D cell culture methods and applications, with a focus on prostate cancer modeling and drug discovery.

Mitochondrial functional and structural impairment is involved in the antitumor activity of δ-tocotrienol in prostate cancer cells.

The therapeutic options for castration-resistant prostate cancer (CRPC) are still limited. Natural bioactive compounds were shown to possess pro-death properties in different tumors. We previously reported that δ-tocotrienol (δ-TT) induces apoptosis, paraptosis and autophagy in CRPC cells. Here, we investigated whether δ-TT might exert its activity by impairing mitochondrial functions. We demonstrated that, in PC3 and DU145 cells, δ-TT impairs mitochondrial respiration and structural dynamics. In both cell lines, δ-TT triggers mitochondrial Ca2+ and ROS overload. In PC3 cells, both Ca2+ and ROS mediate the δ-TT-related anticancer activities (decrease of cell viability, apoptosis, paraptosis, autophagy and mitophagy). As expected, in autophagy-defective DU145 cells, Ca2+ overload was involved in δ-TT-induced pro-death effects but not in autophagy and mitophagy. In this cell line, we also demonstrated that ROS overload is not involved in the anticancer activities of δ-TT, supporting a low susceptibility of these cells to ROS-related oxidative stress. Taken together, these data demonstrate that, in CRPC cells, δ-TT triggers cell death by inducing mitochondrial functional and structural impairments, providing novel mechanistic insights in its antitumor activity.

Natural Compounds in Prostate Cancer Prevention and Treatment: Mechanisms of Action and Molecular Targets.

Prostate cancer (PCa) represents a major cause of cancer mortality among men in developed countries. Patients with recurrent disease initially respond to androgen-deprivation therapy, but the tumor eventually progresses into castration-resistant PCa; in this condition, tumor cells acquire the ability to escape cell death and develop resistance to current therapies. Thus, new therapeutic approaches for PCa management are urgently needed. In this setting, natural products have been extensively studied for their anti-PCa activities, such as tumor growth suppression, cell death induction, and inhibition of metastasis and angiogenesis. Additionally, numerous studies have shown that phytochemicals can specifically target the androgen receptor (AR) signaling, as well as the PCa stem cells (PCSCs). Interestingly, many clinical trials have been conducted to test the efficacy of nutraceuticals in human subjects, and they have partially confirmed the promising results obtained in vitro and in preclinical models. This article summarizes the anti-cancer mechanisms and therapeutic potentials of different natural compounds in the context of PCa prevention and treatment.

The emerging role of paraptosis in tumor cell biology: Perspectives for cancer prevention and therapy with natural compounds.

Standard anti-cancer therapies promote tumor growth suppression mainly via induction of apoptosis. However, in most cases cancer cells acquire the ability to escape apoptotic cell death, thus becoming resistant to current treatments. In this setting, the interest in alternative cell death modes has recently increased. Paraptosis is a new form of programmed cell death displaying endoplasmic reticulum (ER) and/or mitochondria dilation, generally due to proteostasis disruption or redox and ion homeostasis alteration. Recent studies have highlighted that several natural compounds can trigger paraptosis in different tumor cell lines. Here, we review the molecular mechanisms underlying paraptotic cell death, as well as the natural products inducing this kind of cell death program. A better understanding of paraptosis should facilitate the development of new therapeutic strategies for cancer prevention and treatment.

Beneficial effects of δ-tocotrienol against oxidative stress in osteoblastic cells: studies on the mechanisms of action.

PURPOSE: Natural antioxidants are considered as promising compounds in the prevention/treatment of osteoporosis. We studied the ability of purified δ-tocotrienol (δ-TT) isolated from a commercial palm oil (Elaeis guineensis) fraction to protect osteoblast MC3T3-E1 and osteocyte MLO-Y4 cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage and the mechanisms involved in its protective action in MC3T3-E1. METHODS: MC3T3-E1 and MLO-Y4 cells were treated with δ-TT (1.25-20 µg/ml for 2 h) followed by t-BHP at 250 µM or 125 µM for 3 h, respectively. MTT test was used to measure cell viability. Apoptotic cells were stained with Hoechst-33258 dye. Intracellular ROS levels were measured by dichlorofluorescein CM-DCFA. The OPT fluorimetric assay was used to detect the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) contents. RESULTS: δ-TT significantly prevented the effects of t-BHP on cell viability and apoptosis reaching a maximum protective activity at 10 and 5 µg/ml in MC3T3-E1 and MLO-Y4 cells, respectively. This protective effect was due to a reduction of intracellular ROS levels and an increase in the defense systems shown by the increase in the GSH/GSSG. GSH loss induced by an inhibitor of GSH synthesis significantly reduced the δ-TT-positive effect on ROS levels. δ-TT prevention of oxidative damage was completely removed by combined treatment with the specific inhibitors of PI3K/AKT (LY294002) and Nrf2 (ML385). CONCLUSIONS: The δ-TT protective effect against oxidative damage in MC3T3-E1 cells is due to a reduction of intracellular ROS levels and an increase of the GSH/GSSG ratio, and involves an interaction between the PI3K/Akt-Nrf2 signaling pathways.