This is the accepted version of the journal article: Blanco Vaca, Francisco; Cedó, Lídia; Julve i Gil, Josep. «Phytosterols in cancer : From molecular mechanisms to preventive and therapeutic potentials». Current medicinal chemistry, Vol. 26 N. 37 (2019). Bentham Science Publishers. DOI 10.2174/0929867325666180607093111 This version is available at https://ddd.uab.cat/record/270407 under the terms of the license 1 Phytosterols in cancer: From molecular mechanisms to preventive and therapeutic potentials 2 Running title: Role of phytosterols in cancer prevention and therapy 3 Authors: Francisco Blanco-Vaca* †, Lídia Cedó*, Josep Julve† 4 Author affiliation: Institut d’Investigacions Biomèdiques Sant Pau (IIB-Sant Pau), Barcelona, Spain 5 (FB-V, LC, and JJ). CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Madrid, Spain (FB-V, 6 LC, and JJ). Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 7 Barcelona, Spain (FB-V, and JJ). 8 * These authors contributed equally to this work. † 9 These authors are co-corresponding authors of this work. 10 Address correspondence to: Dr. Francisco Blanco-Vaca or Josep Julve, IIB-Sant Pau, C/Sant Antoni M. 11 Claret 167, 08025 Barcelona, Spain 12 13 -1- 1 Abstract 2 Cancer is the second leading cause of death worldwide. Compelling evidence supports the hypothesis that 3 the manipulation of dietary components, including plant compounds termed phytochemicals, 4 demonstrates certain important health benefits in humans, including those in cancer. In fact, beyond their 5 well-known cardiovascular applications, phytosterols may also possess anticancer properties, as has been 6 demonstrated by several studies. Although the mechanism of action by which phytosterols (and 7 derivatives) may prevent cancer development is still under investigation, data from multiple experimental 8 studies support the hypothesis that they may modulate proliferation and apoptosis of tumor cells. 9 Phytosterols are generally considered safe for human consumption and may also be added to a broad 10 spectrum of food matrices; further, they could be used in primary and secondary prevention. However, 11 few interventional studies have evaluated the relationship between the efficacy of different types and 12 forms of phytosterols in cancer prevention. In this context, the purpose of this review was to revisit and 13 update the current knowledge on the molecular mechanisms involved in the anticancer action of 14 phytosterols and their potential in cancer prevention or treatment. 15 16 Keywords: edible vegetables – sitosterol – antitumor therapies – angiogenesis – proliferation – metastasis 17 – apoptosis – phytotherapy 18 -2- 1 1. Introduction 2 Cancer is the second leading cause of death and morbidity worldwide, accounting for 8.7 million deaths 3 in 2015 (1). As per the projections for 2030, these figures will double by the year 2030 (2). 4 The pathophysiology of cancer is complex; however, it may be defined as uncontrolled cell growth that 5 may spread to other body parts from the original site, resulting in tissue disorganization and destruction of 6 these other body parts (3). This change is attributable to the dysregulated expression of genes involved in 7 cell cycle control (4). Conventional therapies frequently fail, partly because they generally target single 8 processes, often causing adverse health effects. 9 Diet has been identified as an important and modifiable risk factor for cancer (5-7). Therefore, 10 modification in dietary habits that involves the inclusion of functional food components with 11 chemopreventive properties has been identified as a potential strategy for halting or reversing the early 12 phases before the manifestation of a malignancy. Supporting this theory, diets high in fruits and 13 vegetables, legumes, and whole grains are chemopreventive (8, 9). Moreover, there is considerable 14 epidemiologic data suggesting that vegetarians tend to have lower overall rates of cancer than the general 15 population (4), being one of the most convincing data coming from long-term association studies of the 16 Seventh-Day Adventist community (10, 11). Further, adherence to the traditional Mediterranean diet has 17 also been evidenced to be protective against the risk of cancer, mainly owing to its higher content of 18 abundant and variable plant foods, high consumption of cereals, and predominant use of olive oil as the 19 main fat (12-14). Therefore, it is not surprising that the intake of plant-origin foods appears in the current 20 guidelines as one of the main evidence-based recommendations for reducing the risk of cancer (2, 15-18). 21 Overall, these studies further reveal the presence of dietary functional components with cancer-preventive 22 potential in these diets (19). 23 The functional components of food can be defined as non-conventional biomolecules present in 24 foodstuffs that are closely associated to health benefits (20). Research has proved that functional dietary 25 components can be effectively used in the treatment and especially, in the prevention of diseases. Studies 26 have shown that supplementation with specific plant metabolites (e.g., antioxidants, dietary fiber, and 27 different species of phytochemicals) may reduce the individual risk and alleviate the risk of specific types 28 of cancers (2). In particular, in terms of anticancer therapies, dietary phytochemicals have received 29 increasing attention because of their high potency and low toxicity in modulating crucial intracellular 30 signaling pathways than the common chemotherapy agents (21). Phytosterols are one such class of -3- 1 bioactive dietary phytochemicals (4). This class of molecules has been shown to be effective in improving 2 the blood lipid profile, thereby protecting against cardiovascular disease. In addition to their 3 cardioprotective effects, phytosterols have also shown to have a protective effect against different forms 4 of cancer (22). These compounds are naturally present in Western diets in amounts similar to those of 5 dietary cholesterol (23); the amount of these compounds in vegetarian diets is about 50% higher (24). In 6 this context, we herein will critically review the potential role for dietary phytosterols in cancer 7 prevention. 8 2. Dietary phytosterols 9 2.1. Molecular characteristics 10 Phytosterols are the plant equivalents of animal cholesterol. These compounds are structurally similar to 11 cholesterol except for the extra double bonds and methyl and/or ethyl groups in their side-chain (25). 12 More than 200 phytosterol species exist naturally in plants, and many phytosterols are found in edible 13 foodstuffs (25). In foodstuffs, the most abundant dietary phytosterols are -sitosterol, campesterol, and 14 stigmasterol, with the prevalence of -sitosterol being particularly noteworthy (4). Phytostanols, saturated 15 phytosterols, are less abundant than phytosterols (25). Stanol species can be produced by 5-alpha 16 hydrogenation of the corresponding phytosterols (e.g., sitostanol and campestanol) (26). 17 2.2. Sources 18 In human beings, the diet is the only source of phytosterols. In foods, total phytosterol content is defined 19 by the sum of free sterols as well as the conjugated (i.e., esterified and glycosylated) forms (25, 27). The 20 common dietary sources of phytosterols are vegetables, grains, seeds, and vegetable oils. The content of 21 phytosterols is high in lipid-rich plant foods, such as nuts, legumes, and seeds (4). Similarly, many fruits 22 and grains also contain significant levels of phytosterols although usually at lower concentrations. 23 Additionally, industrial procedures used in food production further affect the phytosterol content of plant 24 foodstuffs. For instance, refining processes, as those involved in the production of refined olive oil (27), 25 can result in significant reduction in their relative phytosterol content. Environmental conditions may also 26 influence the phytosterol content and the composition of conjugated forms (4, 27). Phytosterols are found 27 in many foods and are currently a part of the normal human diets. All plant foods, including algae (28), 28 contain phytosterols (29, 30). In the context of the food industry, phytosterols are currently added to a 29 broad spectrum of foods other than fat spread; they are also added to low-fat milk, bakery products, 30 orange juices, cereal and chocolate bars, as well as low-fat beverages (30, 31). -4- 1 2.3. Bioavailability 2 An important issue related to the potential health benefits of phytosterols is their bioavailability. The main 3 bulk of phytosterols is solubilized in micelles that enable their absorption and transport through the same 4 transporter used for cholesterol transport (23), the Niemann Pick C1-Like 1 transporter. Similar to free 5 cholesterol, non-esterified phytosterols may also accumulate within enterocytes or be rapidly re-secreted 6 into the intestinal lumen via ABCG5/G8 transporters (23). Moreover, phytosterols, which are not fully 7 metabolized by the human body, are rapidly excreted, eventually resulting in a minimal net absorption of 8 the dietary phytosterols. Despite this, measurable circulating levels of phytosterols can be detected, 9 especially in individuals who consume phytosterol-enriched diets (27, 30). 10 Several factors may potentially influence phytosterol absorption (30, 32). In human beings, the absorption 11 efficiency for phytosterols is considerably lower (from less than 5 to approximately 10% depending on 12 the study) compared to that of cholesterol (33-60%) (27, 33-39). Dietary phytostanols are absorbed more 13 poorly absorbed than phytosterols (34, 40). Phytosterol absorption may be also influenced by the 14 quantitative differences in the common food sources (30, 32, 41). The bioavailability of phytosterols is 15 enhanced in oils (unrefined, vegetable, and olive oils) (42). The phytosterol content in foodstuffs may 16 also be reduced by the production processes, including refining of crude edible oils to remove the known 17 contaminants extracted from raw foodstuff or hydrogenation to obtain stanol-enriched food matrices. 18 Further, boiling and storage variably affects the total phytosterol content of vegetables (41). As in the 19 case of cholesterol, thermo-oxidative treatments during food processing or oxidation that occur during 20 storage in bulk oil, oil-in-water emulsions, and infant foods may lead to the formation of phytosterol 21 oxidation products (i.e., keto-, hydroxyl-, and epoxy-derivatives) (30, 41, 43). Furthermore, an increased 22 dietary exposure to these oxidized constituents via the consumption of foods enriched with 23 phytosterols/phytostanols may potentially influence the absorption and hamper the biological properties 24 attributed to phytosterols (44). 25 Liposomes are carriers that improve the molecular stability and permeability and control their release into 26 the target tissues. Liposomalization has been reported as another strategy for oral administration (45). 27 This approach improves the bioavailability of several substances that are insoluble or barely soluble in 28 water. To our knowledge, only one study has reported on the effect of oral administration of liposomal - 29 sitosterol in preventing tumor metastasis in vivo (46), suggesting that liposomal-based vehicles could be 30 potentially good candidates for formulating phytosterols in cancer prevention or treatment. -5- 1 2.4. Safety 2 Studies on phytosterols have been conducted on several population groups, including subjects from 3 Europe, Americas, and Asia (47). No adverse effects have been reported in any of these clinical studies or 4 by the regular consumption of food products containing these compounds. Compared to phytosterols, 5 phytostanols are more stable and are therefore not altered during food manufacturing or processing. These 6 compounds are microbiologically inert because they are not affected by fermentation and are not oxidized 7 when heated (48). In a similar manner, they are not metabolized in the body and are excreted intact (49). 8 Unabsorbed phytosterols can further undergo bacterial transformation by the intestinal microbiota to 9 produce metabolites, such as coprosterol and its immediate precursor, coprostanone (50). Although these 10 metabolites are currently used as biomarkers for the presence of human fecal in the environment, some 11 reports have proposed their potential as biomarkers for certain types of cancer (i.e., colon cancer) (51-53). 12 Consumption of phytosterols hampers the intestinal cholesterol absorption and might also interfere with 13 the appropriate intake of other dietary fats, including fat-soluble vitamins (-tocopherol) and carotenoids 14 (-carotene, -carotene, and lycopene) (54). Given their role in antioxidant prevention, reduced plasma 15 levels of these molecules might have negative health implications. Different meta-analyses of randomized 16 controlled trials have revealed that phytosterols intake significantly reduced plasma levels of - 17 tocopherol, -carotene, -carotene, and lycopene compared to subjects administered placebo (55, 56). 18 These molecules are transported by lipoproteins; therefore, a reduction in their plasma levels might be 19 attributable to the reduced capacity of the plasma carriers (57, 58). Thus, their plasma levels are generally 20 standardized for total cholesterol (55, 56). After correction, only the plasma carotenoid concentrations 21 were eventually found lower after phytosterol intake (56). However, the observed levels of fat vitamins 22 were maintained within the normal ranges, suggesting that it may have no health impact. 23 3. Cancer prevention properties of dietary phytosterols 24 Several studies have shown a direct relationship between dietary habits and the incidence of cancer (5-9). 25 Representative epidemiologic data support the results that the prevalence of cancer is frequently lower in 26 representative vegetarians (e.g., Adventists) than in the general population (10, 11). In this regard, the 27 estimated dietary intake of phytosterols of the Adventists is reportedly higher than that of the general 28 population (59). Similarly, the naturally-occurring dietary intake of phytosterols in a Spanish free-living 29 population has been also estimated to be higher than that of people living in other non-Mediterranean 30 European countries (60), suggesting that this could be a part of the Mediterranean diet. A randomized, -6- 1 clinical trial involving a Mediterranean diet intervention showed a reduction in the breast cancer risk of 2 the intervention group (61). Although the specific contribution of the main component(s) responsible for 3 the favorable anti-cancer effect of plant-based diets remains undefined, it can be supposed that the cancer 4 protection provided by these diets can be attributed, at least in part, to their increased phytosterol content 5 (13, 62). Probably the most convincing experimental data supporting this hypothesis is that from the 6 efficacy analysis of this class of molecules on cancer protection in animal models of different types of 7 cancers (4). 8 In the last years, a growing body of experimental evidence has supported the hypothesis of the protective 9 effects of phytosterol consumption against tumor growth and tumor cell metastasis (Figure 1). Most 10 studies have been conducted on cancer cell lines in vitro (63). In contrast, relatively fewer in vivo studies 11 on the antitumor effect of phytosterols have been conducted, most of them using different rodent models 12 of chemically-induced cancers, xenographed with cancer cells, (3, 4) or genetic models of breast and 13 prostate cancer (64, 65). 14 The anti-proliferative and anti-metastatic effects of phytosterols (i.e., either -sitosterol or other 15 phytosterol mixtures) support the epidemiological studies that suggest a protective role of phytosterols 16 against the development of various cancers. 17 4. Potential cellular mechanisms of anticancer action 18 The exact mechanisms involved in the phytosterol-mediated protection against cancer are still poorly 19 defined. Different potential anticancer mechanisms have been proposed, including those involving the 20 inhibition of carcinogen production, cancer cell proliferation, invasion, and metastasis, as well as the 21 induction of cell cycle arrest and apoptosis (reviewed in (3, 4, 22, 66), thus conferring cancer 22 chemopreventive and therapeutic potential. Apart from these, other mechanisms including the reduction 23 of angiogenesis, invasion, and adhesion of cancer cells have been also suggested (Figure 1). 24 4.1. Effect on cell cycle 25 Cell cycle dysregulation may be mainly attributed to the alterations in certain control proteins. 26 Phytosterols have been reported to reduce cell cycle kinetics in different cultured cancer cell lines (4, 66). 27 These compounds may potentially act as weak cell cycle inhibitors of cancer cells. Compelling 28 experimental evidence from different studies suggests that exposure to phytosterols (i.e., -sitosterol) may 29 either slow down cancer cell growth, as revealed by cell cycle arrest at different phases, depending on the 30 study (i.e., G2/M and G1/S transitions, G0/G1, or G2 phases) or promote cell accumulation of the sub-G1 -7- 1 apoptotic population (4, 67-72). In this regard, pretreatment of a rat model of renal carcinogenesis with - 2 sitosterol has proven effective against renal cancer, partly owing to the inhibition of cellular proliferation; 3 hence, a significant reduction in the gene expression of proliferative markers (i.e., cyclin D and 4 Proliferating Cell Nuclear Antigen [PCNA]) has been concomitantly observed in renal biopsies (73). The 5 mechanisms involved in cell arrest triggered by phytosterols are still poorly defined. 6 4.2. Effect on apoptosis 7 The most accepted hypothesis is that phytosterol supplementation at physiological concentrations 8 ultimately inhibits cancer cell/tumor growth through apoptosis stimulation [4]. Several researches have 9 shown that -sitosterol promotes apoptosis in many different types of cancers (4, 68-71, 74-79). While 10 the mechanisms involved in the apoptotic action of phytosterols are not yet fully understood, some 11 progress has been made in this field. In this context, -sitosterol exposure to breast MCF-7 and MDA- 12 MB-231 adenocarcinoma cells has been found to increase First apoptosis signal (Fas) levels and caspase- 13 8 activity that are involved in the extrinsic apoptotic pathway (77, 79). The increase in the caspase-8 14 activity and apoptosis in these cells is accompanied by an enhanced deposition of -sitosterol in their cell 15 membranes (79). This finding suggests a potential mechanism by which -sitosterol might induce 16 apoptosis, at least in part, by changes to the membrane sterol content and the Fas apoptotic pathway (79). 17 In addition, the activity of other caspases (i.e., caspase-3 and -9) also increases in colon cancer cells, 18 following incubation with -sitosterol (76). The latter has been associated with the down-regulation of the 19 anti-apoptotic protein Bcl-2 (80) and up-regulation of the pro-apoptotic Bax, an activator for ceramide- 20 mediated apoptosis (81), as well as with a release of cytochrome C from the mitochondria that is involved 21 in the intrinsic apoptotic pathway. In a similar manner, -sitosterol modulated Bax/Bcl-2 proteins and 22 caspase-3 and caspase-9 in vitro in leukemic cells (70) and stomach cancer cells (68) and in vivo in a rat 23 model of renal carcinogenesis (73). Similar effects are produced by 7α-hydroxy--sitosterol, a rare 24 phytosterol oxide isolated from Chisocheton tomentosus with the ability to dysregulate the Bax/Bcl-2 25 ratio (82) or guggulsterone that is found in the resin of Commiphora mukul and induces apoptosis through 26 the intrinsic mitochondrial pathway in hepatic carcinoma (83) and cholangiocarcinoma cells (84). Both 27 apoptotic pathways (extrinsic as well as intrinsic) are also involved in the induction of apoptosis by - 28 sitosterol in fibrosarcoma cells (75). In this case, -sitosterol reduced the cellular levels of Bcl-2 and the 29 inhibitor of the apoptosis protein (IAP) family and increased Bax, p53, and p21. It is interesting to note 30 that this phytosterol also reduced the activity of known anti-apoptotic molecules (i.e., PI3K and Akt) and -8- 1 increased the activation of pro-apoptotic signal transduction enzymes, including the extracellular signal- 2 regulating kinase (ERK) and p38 mitogen activated protein kinase that are activated (via phosphorylation) 3 due to extracellular signals and are involved in the activation of caspases (75). 4 4.3. Effect on metastasis 5 Metastasis is one of the most threatening aspects of cancer (85). Despite major advances in cancer 6 treatment, there are still no specific treatments to prevent metastasis. This is because metastasis is a 7 multistep process that comprises certain processes, including tumor cell migration, invasion, adhesion, 8 and growth. 9 Dietary phytosterols (i.e., -sitosterol) have been reported to exert a protective effect against prostate and 10 breast cancer metastasis in immunodeficient (SCID) mice (71, 86). Mice (male) inoculated with cultured 11 prostate cancer cells (PC-3 type) and fed with phytosterols exhibited (37%) about one-half the rate of 12 metastasis than cholesterol-fed mice (73%), with fewer cases of metastases to the lymph nodes and the 13 lungs (86). Furthermore, mice treated with phytosterols did not present detectable signs of metastasis in 14 the liver compared to the 18% prevalence detected in the control mice with tumors. In keeping with this, a 15 study based on the same mouse model as the one described above for prostate cancer but using SCID 16 mice (female) inoculated with cultured breast cancer cells (MDA-MB-231 type) and fed phytosterols also 17 exhibited fewer cases of metastases to the lymph nodes and the lungs (52%) compared to the control 18 group (71%). Investigations regarding the metastatic step targeted by phytosterols have found that - 19 sitosterol offered protection from breast cancer metastasis by inhibiting cell invasion and adhesion to the 20 basement membrane proteins (87), potentially by directly influencing the expression of integrin receptors 21 on the tumor cells. It is noteworthy that, in the same study, campesterol (another phytosterol examined in 22 the study) failed to protect against metastasis. 23 More recently, orally administered liposomal -sitosterol also prevented lung metastatic colonization of 24 melanoma cells in an experimental metastasis model in vivo (46). In this study, -sitosterol induced IL-18 25 production in the intestinal epithelium. IL-18 modulates the immunological function and enhances natural 26 killer action. Since -sitosterol is absorbed in very small amounts into the plasma, the authors 27 hypothesized that the stimulation of mucosal immunity rather than a direct action on the tumor cells 28 provided protection against tumor metastasis. 29 4.4. Effect on angiogenesis -9- 1 Angiogenesis is required for invasive tumor growth and metastasis and is critical in the control of cancer 2 progression (88). The angiogenic response involves many molecules and signaling pathways that 3 coordinate multiple cellular processes, such as endothelial proliferation (i.e., vascular endothelial growth 4 factor [VEGF] or its receptor(s), basic fibroblast growth factor [bFGF], etc.), and basement membrane 5 remodeling (i.e., matrix metalloproteinases [MMP], such as MMP-2 and MMP-9) (66). Tumoral 6 microenvironments may also be extremely hypoxic; the activation of hypoxia-dependent signaling 7 pathways, especially hypoxia-inducible factor (HIF)-1, further contribute to tumor angiogenesis (89). In 8 this context, the inhibition of cancer angiogenesis may be a valuable, new approach to cancer therapy. 9 The anti-angiogenic potential of phytosterols remains controversial. Although data first suggested that - 10 sitosterol possesses good angiogenic activity both ex vivo, using a chick embryo chorioallantoic 11 membrane (CAM) assay, and in vivo using a model of ischemia/reperfusion of vascular damage (90, 91), 12 some recent evidences suggest the mechanism by which phytosterols potently inhibit tumor 13 neovascularization (3). For instance, oral pretreatment of -sitosterol has been reported to reverse the 14 VEGF gene expression in a rat model of renal carcinogenesis (73). The administration of another main 15 phytosterol, campesterol, has been shown to prevent bFGF-induced endothelial cell proliferation and 16 capillary-like tube formation as well as neovascularization in fertilized eggs (92). Guggulsterone has been 17 recently reported to suppress capillary tube formation and the migration of endothelial vascular cells as 18 well as matrix metalloproteinases release from the colon cancer cells (66). The anti-angiogenic activity of 19 phytosterols, as one of the components of a dietary phytochemical conjugate, has been also demonstrated 20 in vivo using a murine Lewis lung cancer model (93). Further, it is important to note that the reduction of 21 tumor-associated angiogenesis and growth observed in conjugate-feeding mice were not exclusively 22 attributed to the phytosterol content (i.e., soy sterols) because other plant bioactive components with 23 antitumoral potency were also present in the conjugate formula (i.e., lipoic acid and ferulic acid) (94, 95). 24 However, these effects were similar to the reported effects of these compounds and support the usefulness 25 of combined dietary approaches in cancer prevention. 26 4.5. Effect on immunomodulation 27 Selective modulation of the different components of the immune system has received considerable 28 attention because it forms the treatment bases for several pathological conditions, including cancer (96). It 29 is generally accepted that immune surveillance mechanisms are vital for preventing the development of 30 solid tumors. Such mechanisms depend heavily on a well-balanced immune response favoring a cellular - 10 - 1 outcome (T helper 1 [TH1] mediated) rather than a humoral response (TH2 type) (97). The 2 immunoregulatory role of phytosterols is deeply reviewed in another article of this special issue. 3 5. Potential biochemical mechanisms 4 5.1. Cholesterol-lowering properties 5 Elevated plasma cholesterol levels lead to their accumulation in the cell membranes where they 6 concentrate into particular cell membrane structures termed as lipid rafts. Increased cholesterol content 7 has been associated with increased survival and reduced apoptosis of cancer cells (98). Further, high 8 intakes and elevated blood levels of cholesterol are believed to be associated with elevated cancer risk 9 (99-101). According to this, the use of cholesterol-lowering strategies, including phytosterols, could 10 reduce the risk of cancer. In fact, the use of statins, another cholesterol-lowering therapy, has been 11 experimentally demonstrated to have an anti-cancer effect (102). Consistently, most existing 12 epidemiologic evidence supports a survival benefit of statins in oncological patients (103-105). Thus, it is 13 not completely accurate to believe that lowering the plasma cholesterol levels would result in phytosterol- 14 induced depletion in the cholesterol in the lipid rafts of cancer cells, and thereby induce cell death by 15 regulating signal transduction (3). This might further suggest a potential raft-modulating action that 16 involves the incorporation of phytosterols into the lipid rafts and a potential use for these compounds as 17 anti-cancer agents. 18 5.2. Modulation of cell signaling 19 5.2.1 Liver X receptor (LXR) 20 LXRs also act as oxysterol sensors in the body and their potential therapeutic applications have been 21 examined in several types of cancers (106). Phytosterols have been shown to activate the LXR signaling 22 in vivo (4, 107). However, the mechanisms involved in the LXR-mediated anti-cancer effect by 23 phytosterols are still poorly defined. The activation of LXR enhances cholesterol removal from the tissues 24 (108), and it has been proposed that this may directly contribute to further enhancement in cholesterol 25 removal from the cancer cells. In addition to its potential direct impact on cellular cholesterol, its 26 activation by ligands (i.e., T0901317) has been also shown to exert anti-proliferative actions in multiple 27 cancer-related study systems (106, 109-112), potentially by promoting cell cycle arrest at the G1 phase 28 (112). In this context, the overexpression of LXR further sensitizes the cancer cells to the effect of 29 T0901317 (113). The activation of LXR with other agonists has been also reported to have a protective 30 effect against proliferation (i.e., GW3965) and progression of either prostate or colorectal cancer in tumor - 11 - 1 xenograft models in vivo (110, 112). Supporting this notion, the treatment with LXR ligands induces the 2 degradation of the low-density lipoprotein receptor (LDLR), a regulator of tumor cell survival, and 3 increases apoptosis in glioblastoma cells (106). Further, LXR-mediated elevation in apolipoprotein E 4 gene (Apoe), another known LXR target, promotes metastasis suppression in melanoma models (106). 5 These findings might further highlight the role for phytosterols in the inducing APOE-mediated 6 mechanisms in cancer therapy. 7 Phytosterol oxidative derivatives, called oxyphytosterols, may also act as LXR activators (107), and they 8 exert significant antiproliferative effects on cancer cells (114). Although it is still unclear whether 9 oxyphytosterols are formed in the relevant concentrations in vivo (107), significant amounts of these 10 compounds are present in foods (30, 41) and may be detected in the plasma of healthy subjects (115). 11 Taken together, this evidence strongly supports a link between LXR function and cancer. However, it is 12 still uncertain whether the consumption of natural dietary phytosterols might eventually lead to the 13 activation of LXR in vivo. Deeper investigation (i.e., loss-of-function experiments) is needed in order to 14 confirm the role of LXRs for the chemopreventive actions of phytosterols in cancer. 15 5.2.2. Sphingomyelin cycle 16 One of the anti-cancer mechanisms of phytosterols that has received the maximum attention is the 17 induction of the sphingomyelin-based signaling pathway (116, 117). Ceramides as well as other 18 sphingolipid metabolites derived from sphingomyelin can modulate cell growth, survival, and death (118, 19 119). A significant induction of cellular apoptosis has been observed in MDA-MB-231 breast 20 adenocarcinoma cells, LNCaP prostate adenocarcinoma cells, and colon cancer cells exposed to - 21 sitosterol (71, 78, 116, 117). It is important to note that the phytosterol-mediated promotion of apoptosis 22 seems to be selective for cancer cells because exposition to -sitosterol does not promote apoptosis in 23 normal cells. 24 5.3. Role of phytosterols in oxidative stress 25 Oxidative stress is closely related to cancer (120). Cancer cells require a high amount of ATP for aberrant 26 proliferation. This high demand of energy is uncontrolled and leads to the accumulation of reactive 27 oxygen species (ROS); this needs to be counteracted by antioxidant mechanisms to ensure cell survival, 28 even in cancer cells. Current data support the hypothesis that low to moderate levels of ROS may 29 contribute to tumor formation either by activating the signaling pathways or by promoting the mutation of 30 genomic DNA (120). By contrast, at high levels, ROS promote cell death and severe cellular damage. - 12 - 1 Thus, cancer cells need to develop powerful antioxidant mechanisms to counteract the exacerbated levels 2 of oxidative stress produced at the early stages of tumor development and during the metastatic phases of 3 the disease. 4 -sitosterol has been reported to elevate the activities of antioxidant enzymes in cultured macrophage 5 cells with phorbol 12-myristate 13-acetate-induced oxidative stress, suggesting that phytosterols can 6 protect cells from damage by ROS (121). -sitosterol has been also reported to decrease free radical 7 generation in vitro (122). Consistent with these findings, the administration of -sitosterol has been 8 recently reported to exhibit chemopreventive potential in 1,2-dimethylhydrazine (DMH)-induced 9 experimental colon cancer in rats by reducing the oxidative stress (123). Although current data supports 10 the theory that phytosterols exert direct antioxidant effects (124, 125), recent data further reveal that the 11 phytosterols may also exert pro-oxidant effects by triggering ROS-dependent apoptosis and DNA damage 12 in multiple myeloma cells (67). This phytosterol-mediated action was attenuated by the ROS scavenger 13 N-acetyl L-cysteine and was associated with the activation of several cancer metabolism-related signaling 14 pathways, such as AMP-activated protein kinase and c-Jun N-terminal kinase. The latter study contradicts 15 the hypothesis regarding the complex role of natural phytochemicals, including phytosterols, and their 16 dual antioxidant or pro-oxidant role in early or metastatic events of chemoprevention and cancer therapy. 17 Lipoprotein oxidation, critical for tumor growth (126, 127), is increased in PyMT mice (128). Data from 18 our group indicate that phytosterols were effective in preventing lipoprotein oxidation in mice fed a high- 19 fat diet; this may explain, at least in part, their anticancer effects observed in these mice (64). In this 20 regard, it is noteworthy that lovastatin, a member of the statin family, inhibits low-density lipoprotein 21 oxidation (129). This property could be, at least in part, involved in the anticancer activity attributed to 22 this category of drugs. 23 6. Potential of phytosterols as anticancer agents 24 Although data on pharmacokinetic and bioavailability has been previously reported (130), few reports 25 have described the -sitosterol dosage forms for their potential application in cancer cell lines. The 26 absorption of dietary phytosterols is only 5%; therefore, the phytosterol concentration used in in vitro 27 studies is generally higher than that actually available to cells in vivo. However, these studies have proven 28 useful for the investigation of the molecular mechanisms involved in the inhibitory effect of phytosterols 29 on tumor growth. - 13 - 1 The anticancer and antiapoptotic potential of phytosterols is currently under investigation. Several trials 2 have suggested that their efficiency may be mainly revealed in combined therapies for cancer treatment 3 (74, 131). These findings seem to indicate that their action may rather depend on the targeted cell type, 4 source, or concentration of these compounds. Consistent with this finding, -sitosterol has shown to 5 become pro-apoptotic in human stomach cancer cells (SGC-7901) (68); however, it has failed to show 6 effectiveness in human intestinal caco-2 cells (132). Moreover, in independent studies, only high 7 concentrations of -sitosterol have shown to induce apoptosis (77, 133). 8 To the best of our knowledge, no clinical trials have been conducted to test the potential efficacy of 9 phytosterols in cancer. In general, epidemiological studies have reported that phytosterol intake is 10 beneficial in reducing the risk of cancer in the lung (134), breast (135), esophagus (136), stomach (137), 11 endometrium (138), and ovaries (139). A recent case-control study has shown that an increased intake of 12 dietary phytosterols, especially -sitosterol, campesterol, and campestanol has been associated with a 13 50% reduction in the risk of colorectal cancer in a Chinese population (140). Although, to our knowledge, 14 there are no clinical studies linking -sitosterol and breast cancer, its administration may enhance the 15 efficacy of tamoxifen, a drug commonly used for the treatment of breast cancer (131). Although 16 phytosterol administration is generally effective against various forms of cancer, some recent studies have 17 reported contradictory results. For example, in a study conducted in the Netherlands, phytosterol intake 18 was not associated with a lower risk of colon and rectal cancer (141). It is noteworthy that there was a 19 positive association of campesterol and stigmasterol with the risk of prostate (142) and colorectal cancer 20 (140). Thus, their value (alone or in combination with other anticancer treatments) in cancer prevention 21 needs to be confirmed in future controlled human trials. 22 Multidrug resistance is a phenomenon wherein tumors become resistant to chemically unrelated 23 anticancer drugs, posing one of the most important challenges in the field of cancer chemotherapy. One of 24 the most common mechanisms leading to multidrug resistance is the increased efflux of cytotoxic drugs 25 by energy-dependent transporters (ABCs). Recent studies have documented the ability of natural 26 phytochemicals to increase the sensitivity of cancer cells to anticancer drugs (143). In particular, the 27 efficacy of phytosterols has been proven in the treatment of multidrug resistant cancer cells at 28 physiological concentrations (72). It is noteworthy that in the same study, -sitosterol exhibited greater 29 cytotoxicity in the cells with basal expression of ATP-binding cassette, sub-family B, member 1 30 (ABCB1) that is responsible for multidrug resistance phenomena, than in multidrug resistant cells. The - 14 - 1 exact mechanism of phytosterol action in cancer cells is still unclear; however, it has been postulated that 2 phytosterols can exert their action partly by inhibiting the action of ABCB1 (72). This study also 3 suggested that phytosterols can target cancer cells, exhibiting high multidrug resistance potential. 4 7. Concluding remarks 5 As per most experimental studies, phytosterols are potential agents for prevention and, perhaps, for the 6 treatment of cancer. As a natural product, at concentrations present in plant sources or formulas, 7 phytosterols have little or no reported toxicity, making them a promising treatment option for several 8 types of cancer. The major dietary phytosterols are -sitosterol, campesterol, and stigmasterol, and their 9 contents are higher in edible oils, seeds, and nuts. Several experimental and few human clinical studies 10 have also revealed their potential therapeutic value in cancer prevention. In particular, the characterization 11 of phytosterols as LXRs ligands as well as their effects and mechanisms of action in preclinical cancer 12 models may readdress future translational LXR research in cancer therapeutics. Moreover, new leads in 13 the development of new phytosterol derivatives (or analogs) or suitable drug delivery systems for targeted 14 therapy might provide greater efficacy and bioavailability, thus improving their clinical usefulness for 15 human trials. Future studies are therefore warranted to investigate the detailed mechanisms of action of 16 phytosterols in vivo as well as to assess the long-term effects of these compounds on the prevention of 17 tumor initiation, growth, and angiogenesis. 18 19 Abbreviations used: ABCG5/G8, ATP-binding cassette transporters class G type 5 and 8; bFGF, basic 20 fibroblast growth factor; CAM assay, (In Ovo) Chick Chorioallantoic Membrane assay; LXR, liver X 21 receptor; PCNA, proliferating cell nuclear antigen; MMP, metalloproteinases; PyMT, polyoma middle T 22 antigen; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; WHO, World Health 23 Organization. 24 25 Conflict of interest statement 26 The authors declare no conflicts of interest. 27 28 Acknowledgements 29 F.B.-V., L.C., and J.J. have contributed in the editing and revision of the manuscript. This work was 30 partly funded by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (ISCIII) FIS grants - 15 - 1 PI17/00232 (to JJ), PI14/01648 (to FB-V), and FEDER “Una manera de hacer Europa” and by LaMarató 2 2016; grant number 201602.30.31 (to JJ). J.J. is recipient of a Miguel Servet Type 1 contract 3 (CP13/00070; ISCIII). CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is a 4 project of the Instituto de Salud Carlos III. The English grammar and language were corrected by 5 Scribendi Inc. (www.scribendi.com). Institut de Recerca de l'Hospital de la Santa Creu i Sant Pau is 6 accredited by the Generalitat de Catalunya as Centre de Recerca de Catalunya (CERCA). 7 Figure legend 8 Figure 1. Anticancer mechanisms and molecular targets of phytosterols in cancer prevention and therapy. 9 Phytosterols affect the signaling pathways involved in carcinogenesis by acting on different molecular 10 targets; the downward arrows indicate the down-regulation effects, and the upward arrows indicate the 11 up-regulation of the molecular targets or signaling pathways. Bar-headed lines are used to indicate 12 inhibition. 13 Graphical abstract legend: 14 Phytosterols modulate several molecular signaling pathways and cellular mediators that control the 15 survival mechanisms in cancer cells. Phytosterols may promote apoptosis and prevent the proliferation 16 through different pathways that may involve sphingomyelin metabolism, LXR activation, antioxidant 17 mechanisms, and caspases. 18 - 16 - 1 References 2 1. Fitzmaurice, C., Allen, C., Barber, R. M., Barregard, L., Bhutta, Z. A., Brenner, H., Dicker, D. 3 J., Chimed-Orchir, O., Dandona, R., Dandona, L., Fleming, T., Forouzanfar, M. 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