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.

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.

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.

Epithelial-To-Mesenchymal Transition Markers and CD44 Isoforms Are Differently Expressed in 2D and 3D Cell Cultures of Prostate Cancer Cells.

Three-dimensional (3D) cell cultures allow the mimic of functions of living tissues andprovide key information encoded in tissue architecture. Considered the pivotal role of epithelial-tomesenchymaltransition (EMT) in carcinoma progression, including prostate cancer (PCa), weaimed at investigating the effect of the 3D arrangement on the expression of some key markers ofEMT in cultured human prostate cancer (PCa) cells, to better understand PCa cell behavior. PC3 andDU145 PCa cells were cultured in RPMI cell culture medium either in 2D-monolayers or in 3Dspheroids.The main EMT markers E-cadherin, N-cadherin, α-smooth muscle actin (αSMA),vimentin, Snail, Slug, Twist and Zeb1 were evaluated by confocal microscopy, real-time PCR andWestern blot. Confocal microscopy revealed that E-cadherin was similarly expressed at the cellboundaries on the plasma membrane of PCa cells grown in 2D-monolayers, as well as in 3Dspheroids,but resulted up-regulated in 3D-spheroids, compared to 2D-monolayers, at the mRNAand protein level. Moreover, markers of the mesenchymal phenotype were expressed at very lowlevels in 3D-spheroids, suggesting important differences in the phenotype of PCa cells grown in 3Dspheroidsor in 2D-monolayers. Considered as a whole, our findings contribute to a clarification ofthe role of EMT in PCa and confirm that a 3D cell culture model could provide deeper insight intothe understanding of the biology of PCa.

Tocotrienols and Cancer: From the State of the Art to Promising Novel Patents.

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

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.