The Value of Tocotrienols in the Prevention and Treatment of Cancer

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1 Review The Value of Tocotrienols in the Prevention and Treatment of Cancer Paul W. Sylvester, PhD, Amal Kaddoumi, PhD, Sami Nazzal, PhD, Khalid A. El Sayed, PhD College of Pharmacy, University of Louisiana at Monroe, Monroe, Louisiana Key words: tocotrienols, breast cancer, intestinal absorption, lipid nanoparticles, tocotrienol analogues Tocopherols and tocotrienols represent the 2 subgroups that make up the vitamin E family of compounds, but only tocotrienols display potent anticancer activity. Although in vitro experimental evidence has been very promising, oral supplementation of tocotrienols in animal and human studies has produced inconsistent results. However, recent studies have now clarified the reasons for these discrepancies observed between in vitro and in vivo studies. Oral absorption of tocotrienols into the circulation is mediated in large part by carrier transporter systems that display saturation and apparently down-regulation when exposed to high concentrations of tocotrienols. To circumvent these limitations in oral absorption of tocotrienols, investigators have developed novel prodrug derivatives and nanoparticle delivery systems that greatly enhance tocotrienol bioavailability and therapeutic responsiveness. Additional studies have also demonstrated that combined treatment of tocotrienols with other traditional chemotherapeutic agents results in a synergistic anticancer response, and this synergistic response was further enhanced when these agents were encapsulated in a nanoparticle delivery system. Taken together, these findings clarify the limitations of oral tocotrienol administration and provide novel alternative drug-delivery systems that circumvent these limitations and greatly enhance the therapeutic effectiveness of tocotrienols in the prevention and treatment of cancer. Key teaching points: N This article describes the anticancer effects resulting from tocotrienol dietary supplementation. N Limits in tocotrienol bioavailability following oral consumption are discussed. N Discussion of novel delivery systems that enhance tocotrienol bioavailability is provided. N Development of semisynthetic water-soluble tocotrienol analogues that greatly enhance intestinal absorption and bioavailability are discussed. INTRODUCTION Vitamin E represents a family of 8 naturally occurring compounds that can be further divided into 2 subgroups called tocopherols and tocotrienols [1,2]. Tocopherols and tocotrienols have the same basic chemical structure characterized by a long aliphatic chain attached at the 2-position of a chromane ring structure. However, tocopherols have a saturated phytyl chain whereas tocotrienols have an unsaturated phytyl chain. Individual tocopherols and tocotrienols include a-, b-, c-, and d-isoforms that differ from each other based on the number and position of methyl groups attached to their chromane ring (Fig. 1). Although tocopherols and tocotrienols are very similar in chemical structure, numerous investigations have clearly demonstrated that individual isoforms within each subgroup display significant differences in their biological activity and potential health benefits [1 4]. Until recently, most vitamin E research focused on the actions of a-tocopherol and very little was known about the therapeutic value of tocotrienols. However, during the past decade, a great deal of information has accumulated regarding the health-related biological properties of tocotrienols. Although cell culture experiments have clearly established that tocotrienol, in contrast to tocopherols, displays potent anticancer activity, Address correspondence to: Paul W. Sylvester, PhD, College of Pharmacy, 700 University Avenue, University of Louisiana at Monroe, Monroe, LA sylvester@ulm.edu Journal of the American College of Nutrition, Vol. 29, No. 3, 324S 333S (2010) Published by the American College of Nutrition 324S

2 Fig. 1. General chemical structure of vitamin E. experimental evidence obtained in various in vivo model systems has produced conflicting results [5 10]. The present review will attempt to clarify the therapeutic value of the dietary intake of palm oil and oral supplementation of tocotrienols in the prevention and treatment of cancer. DIETARY PALM OIL INTAKE AND MAMMARY TUMORIGENESIS The anticancer effects of palm tocotrienols were first discovered in studies investigating the role of high dietary fat intake on carcinogen-induced mammary cancer development and growth in rats [11]. In general, high dietary fat consumption significantly increases carcinogen-induced mammary tumorigenesis in rodents [11]. In these studies, a normalfat diet was defined as containing 5% fat/kcal, whereas a highfat diet was defined as containing 20% fat/kcal. After the completions of these studies, it was determined that in general, nearly all of the different high-fat diets were found to stimulate tumor development regardless of whether the diets were formulated with different animal versus vegetable fats or saturated versus unsaturated fats. The notable exception to this finding was the observation that high dietary intake of palm oil suppressed carcinogen-induced mammary tumorigenesis in experimental animals [11]. Palm oil differs from other animal and vegetable fats in that it naturally contains high levels of tocotrienols. These findings were confirmed in subsequent studies that showed carcinogen-induced mammary tumor incidence was lower in rats fed diets high in palm oil as compared with rats fed diets high in other dietary fats [12]. Furthermore, palm oil diets stripped of tocotrienols were found to enhance mammary tumorigenesis in rats [13]. Although these studies strongly suggest that tocotrienols mediate the anticancer effects observed in treatment groups fed high-palm-oil diets, this hypothesis has been difficult to prove. Other investigations examined the effects of dietary supplementation with isolated vitamin E extracted from palm oil, commonly referred to as the tocotrienol-rich fraction (TRF) of palm oil. TRF contained a mixture of a-tocopherol (20%) and JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 325S

3 a- (16%), c- (44%), and d-tocotrienol (15%) and non vitamin E contaminant (5%) [14,15]. Results showed that TRF dietary supplementation in animals fed diets containing other types of fats had little or no effect in suppressing carcinogen-induced mammary tumor growth and development [8]. In this study, tocotrienol treatment caused a significant increase in tumor latency but had no effect on tumor multiplicity induced by the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) [8]. However, when tumors were induced by the direct-acting carcinogen N-nitrosomethylurea (NMU), tocotrienol treatment had no effect on tumor latency but increased tumor multiplicity [8]. These finding indicated that tocotrienol dietary supplementation had no significant impact on mammary-tumor development after tumor induction with either DMBA or NMU [8]. Other studies showed that dietary supplementation with TRF inhibited growth of +SA mammary tumors transplanted in female syngeneic BALB/c mice [16]. In this study, daily ingestion of 1.5 mg or 15 mg TRF caused a significant increase in tumor latency and a corresponding decrease in average tumor size and weight, without causing any apparent adverse or toxic effects to the animals [16]. However, these antitumor effects of dietary TRF supplementation were not displayed in a typical dose-responsive manner. TRF-induced suppression in +SA mammary tumor growth was essentially identical in the 2 groups of mice receiving TRF [16]. This lack of a doseresponsive inhibitory effect of TRF appeared to be related to the magnitude of tocotrienol levels found in the +SA mammary tumors obtained from the different TRF treatment groups. Specifically, dietary supplementation with 1.5 mg TRF was associated with a modest increase in tumor tocotrienol levels, whereas daily supplementation with 15 mg TRF produced only a slight but insignificant increase in tumor tocotrienol levels as compared with the 1.5-mg TRF treatment groups [16]. These findings indicate that the anticancer effectiveness of oral administration of tocotrienols may be limited due to inefficient or saturated uptake/transport mechanisms within the gut and circulation. Nevertheless, these findings strongly suggest that dietary supplementation with tocotrienols may provide potential health benefits in the prevention and/or treatment of breast cancer in women, particularly if a method could be developed to further optimize tocotrienol delivery to the mammary tumors. In contrast to in vivo studies, the anticancer effects of tocotrienols in vitro have been firmly established. TRF (0 120 mmol/l) supplementation of culture media significantly inhibited mammary tumor cell proliferation and induced cell death in a dose-responsive manner [15,17,18]. Since TRF contains a mixture of a-tocopherol and a-, c-, and d- tocotrienol, subsequent studies were conducted to determine if one or all of these vitamin E isoforms were responsible for mediating the antitumor effects of TRF. Direct comparisons between the 2 vitamin E subclasses showed that tocotrienols were significantly more potent in suppressing growth and inducing cell death than tocopherols and that the relative biopotency of specific vitamin E isoforms displayed a consistent relationship corresponding to d-tocotrienol $ c- tocotrienol. a-tocotrienol. d-tocopherol & c- and a- tocopherol [15,19]. These studies also showed that tumor cells were significantly more sensitive to the antiproliferative and apoptotic actions of tocotrienols than normal mammary epithelial cells [14,15,19]. Specifically, cells with the greatest degree of tumor progression or malignancy were found to be the most sensitive, followed by noninvasive neoplastic, preneoplastic, and finally normal mammary epithelial cells, respectively [14,15,19]. These findings were of particular interest because these anticancer effects were observed using treatment doses that had little or no effect on normal mammary epithelial cell growth or viability [14,15,19]. In summary, tocotrienols display potent antiproliferative and apoptotic activity against mammary tumor cells at doses that have no adverse effects on normal cell growth or viability in vitro. Although consumption of palm oil diets rich in tocotrienols has been shown to suppress mammary tumorigenesis in some studies, these findings have not been consistently demonstrated in other types of animal tumor models. INTESTINAL ABSORPTION OF TOCOTRIENOLS For dietary lipids and fat-soluble vitamins to be absorbed from the gastrointestinal tract, they must first be emulsified by bile and packaged into micelles for transport into the circulation. Bile excretion is dependent on the level and type of dietary fat consumed, and studies have shown that tocotrienol absorption is reduced in fasted versus full-fed individuals [20 22]. It is possible that the fatty acid composition present in palm oil may promote adequate bile excretion and micelle formation to bring about optimal tocotrienol absorption, whereas administration of isolated tocotrienols by gavage or gel capsules may lack sufficient fat content to stimulate enough bile excretion into the small intestine that would be necessary to promote tocotrienol absorption. Following oral administration, tocotrienols are absorbed from the intestine and transported to the systemic circulation through the lymphatic pathway [23]. Pharmacokinetic studies in humans showed that c-tocotrienol relative bioavailability increased 3.5-fold when administered with food [21], whereas c-tocotrienol oral bioavailability was found to be only 9% in rats [22]. Likewise, others have shown that in the fasted human, plasma tocotrienol concentration was not significantly 326S VOL. 29, NO. 3

4 increased following tocotrienol supplementation [24]. Although food enhances c-tocotrienol absorption by stimulating excretion of bile and pancreatic enzymes that enhance the formation of mixed micelles, c-tocotrienol absorption remains limited and far from complete [21,22]. These findings suggest that other mechanisms may be involved in c-tocotrienol absorption and/or transport by primary enterocytes. Oral absorption of various natural and synthetic compounds is regulated by specific transporters present in endothelial cells lining the gastrointestinal tract. However, the role of transporter mechanisms in the intestinal absorption of c- tocotrienol has not been extensively investigated. A recent study evaluated c-tocotrienol intestinal uptake and metabolism using the in situ rat intestinal perfusion model [25]. Isolated segments of rat jejunum and ileum were perfused with c- tocotrienol solution, and measurements were made as a function of concentration (5 150 mm). Intestinal permeability (Peff) and metabolism were studied by measuring total compound disappearance and major metabolite, 2,7,8-trimethyl-2-(b-carboxy-ethyl)-6-hydroxychroman (c-cehc), appearance in the intestinal lumen [25]. c-tocotrienol and metabolite levels were also determined in the mesenteric blood. The Peff of c-tocotrienol was similar in both intestinal segments and significantly decreased at concentrations $25 mm in jejunum and ileum (Fig. 2). Results also showed that metabolite formation was minimal and mesenteric blood concentrations of c-tocotrienol and metabolite remained very low [25]. These results indicate that c-tocotrienol intestinal uptake is a saturable carrier-mediated process and metabolism is minimal. Results from subsequent in situ inhibition studies with ezetimibe, a potent and selective inhibitor of Niemann-Pick C1-like 1 (NPC1L1) transporter, indicated that c-tocotrienol intestinal uptake is mediated by NPC1L1. Comparable findings were obtained when MDCK II cells that overexpress endogenous NPC1L1 were incubated with increasing concentrations of c-tocotrienol alone or in combination with increasing concentrations of ezetimibe. These data demonstrate that intestinal absorption of c-tocotrienol is mediated, at least in part, by NPC1L1 [25]. The results of this study demonstrate that intestinal uptake of c-tocotrienol is inversely proportional to the concentration of c-tocotrienol present in the intestinal lumen. Studies showed that the elevation in the concentration of the intestinal perfusate resulted in a corresponding reduction in transport into the enterocytes, which strongly suggests that transport of c-tocotrienol across the intestinal membrane involved a carrier-mediated process [25]. These data also indicate that the carrier-mediating intestinal absorption becomes saturated and apparently undergoes down-regulation after exposure to increasingly higher doses of tocotrienols in the intestinal lumen [25]. This hypothesis would explain why it is difficult to obtain elevated levels of tocotrienols in the blood and target tissue Fig. 2. Effective permeability (Peff) of c-tocotrienol as a function of luminal concentration obtained from in situ perfused rat jejunum, n The error bars present the SEM. * p, 0.05 compared with permeability from the jejunum at luminal concentrations of.25 mm. Selective information obtained from reference 25. following oral administration and also why increasing the oral dose does not result in a corresponding increase in bioavailability [21,22,24]. In conclusion, recent studies demonstrate a saturable transport mechanism of c-tocotrienol on the luminal side of the rat intestine. Using the in situ rat intestinal perfusion model, investigators demonstrated that the intestinal uptake of c-tocotrienol is concentration dependent and a saturable process. Results from the in situ and in vitro inhibition studies revealed the significant role of NPC1L1 in the uptake and transport of c-tocotrienol across the cell membrane. These findings have provided essential information necessary for understanding the mechanism of tocotrienol intestinal uptake and important insights required for the development of novel delivery systems that are able to improve c-tocotrienol bioavailability and therapeutic effectiveness in animals and humans. NOVEL DELIVERY SYSTEMS TO INCREASE TOCOTRIENOL BIOAVAILABILITY It is now clearly evident that it is very difficult to obtain and/or sustain therapeutic levels of c-tocotrienol in the blood and target tissues by simple oral administration because absorption and transport mechanisms within the body are extremely limiting and display significant preference for a- tocopherol, the more common form of vitamin E. Although various tocotrienol-containing products are already commercially available, these products are simply capsules filled with a blend of various tocopherols and tocotrienol oils and sold as JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 327S

5 nutritional supplements for oral consumption. Data presented in the previous section demonstrate that this type of formulation or delivery system displays poor solubility in the fluids of the intestine and that high oral doses of tocotrienols inhibit its own absorption from the gut. Consequently, only relatively low levels of tocotrienol will reach the blood when simply formulated as an oil-filled capsule delivery system. Recent investigations have focused on bypassing the natural limits that exist for c-tocotrienol absorption from the gut, transport in the blood, and metabolic breakdown prior to reaching the target tissue. Several novel delivery systems for tocotrienols include microemulsion (oral delivery) and nanoparticle (injectable delivery) formulations. These novel delivery systems not only greatly enhance therapeutic response but also greatly reduce the dose of tocotrienol that is required for treatment. Although the oral formulation of c-tocotrienol is best suited for use as a cancer prevention product to provide daily supplementation, to effectively treat and eliminate existing cancers, higher concentrations of c-tocotrienol need to be delivered to the cancerous tissue. To accomplish this goal, tocotrienol has been encapsulated into vesicle and nanoparticle delivery systems that can be used as an injectable alternative method of delivery. By directly injecting these nanoparticles into the systemic circulation of the patient, intestinal absorption no longer limits bioavailability, and ultimately, large concentrations of tocotrienols can be delivered through the blood. However, to avoid unwanted adverse side effects, it is desirable that these nanoparticles have a targeting moiety on their surface. The purpose of these targeting moieties is to specifically focus nanoparticle delivery to the cancerous tissue. Therefore, when these nanoparticles are injected into the patient, they will move freely in blood and/or other body fluids without metabolic breakdown or accumulation in remote regions of the body and ultimately concentrate in the target cancer tissue. Once at their target site, they would release free tocotrienols at a therapeutic level. Such a study was recently published characterizing the anticancer effects of transferrin-bearing vesicles encapsulating TRF [26]. These investigators hypothesized that because transferrin receptors are highly expressed on many types of human cancer cells, encapsulation of tocotrienol within transferrin-containing vesicles should result in selective targeting and delivery of tocotrienols to tumors following intravenous administration, and this would result in enhanced therapeutic responsiveness. Initial in vitro studies showed that TRF-containing vesicles induced a 3-fold higher uptake of TRF and more than a 100-fold improvement in cytotoxicity in A431 epidermoid carcinoma, T98G glioblastoma, and A2780 ovarian carcinoma cell lines as compared with treatment with free TRF [26]. Furthermore, intravenous injection of TRFcontaining vesicles into tumor-bearing animals resulted in Fig. 3. Antiproliferative effects of tocotrienol-rich fraction (TRF) loaded nanostructured lipid carriers (NLCs) on neoplastic +SA mammary epithelial cells. Cells were initially plated at a density of cells/well (6 wells/group) in 24-well plates and exposed to formulation-supplemented media for a 4-day treatment period. Viable cell number was determined using the MTT colorimetric assay. Vertical bars indicate the mean cell count 6 SEM (n 5 6). NCLs were made by the hot o/w microemulsion technique at a surfactant-to-lipid ratio of 0.5 to 1 using one of the following as the primary core lipid: free TRF represents TRF added to cultures media in the absence of NLCs, CET represents cetyl palmitate used as core lipid, and COMT represents Compritol 888 ATO used as core lipid. Selective information obtained from reference 37. significant tumor regression and improvement in animal survival in a mouse xenograft model as compared with similar treatment with placebo vesicles [26]. These results demonstrate that encapsulating TRF in transferrin-containing vesicles greatly enhances the anticancer therapeutic responsiveness in tumor-bearing animals without causing any visible toxicity or adverse effects. Other investigations were conducted to develop and optimize a systemic delivery of TRF using colloidal drug carriers classified as nanostructured lipid carriers (NLCs) [27,28]. NLCs are composed of a blend of lipids in which lowmelting-point lipids are entrapped in the form of oily nanocompartments within a solid matrix [29,30]. Additional studies were performed to evaluate the long-term stability of the TRF-NLCs with respect to particle size and to assess the antiproliferative effects of TRF containing NLCs against the highly malignant +SA mammary epithelial cells grown in culture [27,28]. Results demonstrated that entrapping TRF within NLCs resulted in a significantly enhanced antiproliferative activity as compared with equal treatment doses of free TRF dissolved in the treatment media [27,28]. Although the exact reason why packaging TRF in NLCs greatly enhanced the anticancer potency has not yet been determined, it most likely results from increased cellular uptake of TRF into the mammary tumor cells (Fig. 3). 328S VOL. 29, NO. 3

6 SYNERGISTIC ANTICANCER EFFECTS RESULTING FROM COMBINED TOCOTRIENOL AND STATIN THERAPY Both statins and c-tocotrienol are potent inhibitors of 3- hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase and display anticancer activity [31 35]. However, the use of statins in the treatment of cancer has been greatly limited because of their high-dose toxicity characterized by severe myopathy [36]. Statins act to directly inhibit HMG CoA reductase enzymatic activity, whereas tocotrienols have also been shown to induce a posttranscriptional down-regulation in HMG CoA reductase [31,37]. Since the mechanism of action by which c-tocotrienol and statins suppress HMG CoA reductase activity is different, it was hypothesized that combined treatment with these agents should provide a synergistic anticancer response. In recent reports, the effect of statin and c- tocotrienol treatment on highly neoplastic mouse +SA mammary epithelial cells was investigated [33 35]. +SA cells were subjected to treatment with individual statins, c- tocotrienol, or a combination of selected statins with c- tocotrienol. Data showed that treatment with 3 4 mm c-tocotrienol or 2 8 mm of the individual statins simvastatin, lovastatin, and mevastatin resulted in a significant decrease of +SA cell growth, whereas treatment with mm pravastatin had no effect on cancer cell growth [33]. When combined treatment of subeffective doses (0.25 or 10 mm) of individual statins with mm c-tocotrienol was examined, results demonstrated a dose-responsive synergistic inhibition in +SA cell proliferation of both treatments. Additional studies showed that combined treatment of c- tocotrienol and statins resulted in a relatively large decrease in intracellular levels of activated MAPK, JNK, p38, and Akt [33]. In addition, the synergistic anticancer effects of combined treatments of low-dose c-tocotrienol and statins have been found to mediate G1 cell cycle arrest in mammary tumor cells [35]. Further studies proved that suppression in HMG CoA reductase activity and mevalonate synthesis is primarily responsible for mediating the synergistic anticancer effects of combined c-tocotrienol and statin treatment [34]. The synergistic effects of these compounds was found to be due to the cooperative action of c-tocotrienol to cause a down-regulation in HMG CoA reductase levels and statins to directly inhibit HMG CoA activity. Since this synergistic anticancer effect was observed using very low doses of each agent, these findings suggest that combined low-dose treatment with c-tocotrienol and statins may have potential value in the treatment of breast cancer without causing the myotoxicity that is associated with high-dose statin monotherapy. ENCAPSULATING TOCOTRIENOLS AND STATINS IN A NANOPARTICLE DELIVERY SYSTEM While tocotrienols and statins can be taken orally, a persistent challenge is to overcome the natural barriers that limit the absorption and delivery of therapeutic concentrations of these drugs to the tumor cells while at the same time minimizing nonspecific adverse effects and toxicities. A recent report provided a novel approach to circumvent such problems by passively targeting simvastatin and tocotrienols coencapsulated in the form of lipid nanoparticles [38]. Previous studies have shown that entrapped drugs within lipid nanoparticles readily penetrate tumors due to a characteristically discontinuous and porous microvasculature existing in most solid tumors [39,40]. Furthermore, tumors also characteristically display poor lymphatic drainage, resulting in a slower clearance of the nanoparticles and subsequent accumulation in tumor cells [41]. These structural features of many solid tumors are commonly referred to as the enhanced vascular permeability and retention effect. Therefore, it was hypothesized that selective extravasation and retention of the nanoparticles in tumors is expected to potentiate the effect of tocotrienol while minimizing the side effects associated with the systemic administration of simvastatin. Studies were also conducted to examine whether the in vitro antiproliferative effects of simvastatin and TRF are retained when they are combined and coencapsulated within the cores of lipid nanoparticles [38]. In these studies, the physicochemical properties of the simvastatin-trf nanoparticles were accessed with regard to particle size, surface morphology, drug entrapment efficiency, in vitro drug release, and stability. In addition, the antiproliferative effects of the simvastatin-trf nanoparticles were determined against the highly malignant neoplastic +SA mammary epithelial cells. Results from these studies showed that the displacement of 50% of the lipid matrix in the nanoparticles with TRF or a- tocopherol resulted in morphological changes in the crystalline structure of the solid matrix and produced imperfections that were sufficient to cause a significant decrease in particle size and a significant increase in statin payload [38]. The formation of a solid solution in which simvastatin was molecularly dispersed in the binary blends of TRF with the lipid matrix was confirmed by differential scanning calorimetry and powder x- ray diffraction studies that showed absence of statin endothermic or crystalline peaks, respectively. When placed in sink conditions, simvastatin NLCs exhibited an initial burst release followed by a more gradual release, as is characteristic of this type of nanoparticle [38]. Furthermore, these nanoparticles induced a significant inhibition of +SA mammary tumor cell growth as compared with cells exposed to blank nanoparticles (controls), as well as cells exposed to the same combined doses of free simvastatin and TRF (not encapsulated in nanoparticle) JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 329S

7 PRODRUG DERIVATIVES OF TOCOTRIENOL Fig. 4. (A) Effects of subeffective doses of c-tocotrienol and simvastatin when given alone or in combination on the growth of neoplastic +SA mammary epithelial cell growth. (B) Antiproliferative effects of nanoparticles loaded with various doses of tocotrienol-rich fraction (TRF) and/or simvastatin on +SA mammary tumor cells. Cells in A and B were initially plated at a density of cells/ well (6 wells/group) in 24-well culture plates and exposed to treatments for a 4-day period. Afterward, the viable cell number was determined using an MTT colorimetric assay. Then, cell count was determined by MTT assay. Vertical bars indicate the mean cell count 6 SEM in each treatment group. * p, 0.05 as compared with vehicle-treated controls. Selective information obtained from references 32 and 41. [38]. These findings showed that not only was the antiproliferative effect of TRF and simvastatin retained, but their anticancer activity was potentiated by nanoparticle encapsulation (Fig. 4). Taken together, these findings demonstrate that tocotrienol entrapped in nanoparticles either alone or in combination with other anticancer agents is an effective method for significantly enhancing drug delivery, bioavailability, and therapeutic responsiveness. It is well established that plasma and tissue concentrations of individual tocopherol and tocotrienol isoforms are limited by the specificity and saturability of these specific transfer proteins and transport mechanisms within the body that display significant preference for a-tocopherol [20,42]. a-tocopherol exhibits significantly greater bioavailability than other vitamin E isoforms due to its selective recognition by specific cellular transporter proteins, such as a-tocopherol transfer and a- tocopherol associated proteins [43 46]. Recent studies were conducted that focused on bypassing the natural limits that exist for c-tocotrienol absorption from the gut, transport in the blood, and metabolic breakdown prior to reaching the target tissue [47]. As a result of these studies, several novel prodrug and polar forms for a-, c-, and d- tocotrienol have been synthesized that are absorbed passively through the gut and bypass the transporter mechanisms normally involved in tocotrienol absorption and metabolic breakdown. Once in the blood and tissue, these prodrug and polar forms of tocotrienol are metabolized to the natural parent compound, which can then act to directly inhibit tumor growth and viability. These novel prodrug and polar derivatives of tocotrienol have also been assayed for anticancer activity, and initial results indicate that they display enhanced tocotrienol delivery and therapeutic responsiveness [47]. Studies have also shown that cancer cells can inactivate various vitamin E family members by rapidly metabolizing them to the corresponding carboxyethyl-hydroxychromans [47]. Semisynthetic modifications of vitamin E isoforms have been shown to produce analogues that are resistant to metabolic degradation, resulting in significant enhancements in bioavailability, potency, and/or solubility in aqueous solutions. One such semisynthetic modification is the esterification and carbamoylation of the C-6 phenolic group [47 49], and amide esters derived from a- and d-tocopheramines and their ester, amide, and ether carboxy analogues have been recently reported to display enhanced anticancer activity [50,51]. The redox-silent analogue of 1,6-O-carboxypropylatocotrienol also showed improved anticancer and anti-invasive activities against A549 lung cancer cells that results from a suppression of hypoxia adaptation by inhibiting hypoxiainduced activation of Src signaling [52]. A recent publication describes the design of semisynthetically C-6 modified redox-silent tocotrienol carbamate and ether analogues with enhanced anticancer effects [47]. The accessible C-6 phenolic group was used to investigate the steric limitations, electronic properties, and hydrogen bond donor and acceptor characters at this position to probe and correlate the surrounding chemical tolerance and biological activity using various aliphatic, olefinic, and aromatic substituents (Fig. 5). Experimental findings showed that 330S VOL. 29, NO. 3

8 Fig. 5. Redox-silent semisynthetic tocotrienol carbamates created from reactions of diverse aliphatic, olefinic, and aromatic isocynates resulting in C- 6 carbamate derivatives. Selective information obtained from reference 47. several new tocotrienol carbamate and ether analogues displayed potent antiproliferative and anti-invasive activities that exceeded that of their parent compounds [47]. In addition, 3-dimensional quantitative structure-activity relationship studies were performed using comparative molecular field and comparative molecular similarity indices analyses to better understand the structural basis for biological activity and provide insights for future design of more potent tocotrienol analogues. CONCLUSION It is now well established that tocotrienols, in contrast to tocopherols, display potent antiproliferative and apoptotic activity against mammary tumor cells at treatment doses that have little or no effect on normal cell growth and function. In addition, numerous studies have demonstrated that combined treatment of c-tocotrienol with other traditional chemotherapies very often results in a synergistic inhibition in cancer cell growth and viability. Although experimental evidence has been very promising, oral supplementation of tocotrienols in clinical studies has produced inconsistent results. It is now clearly evident that the discrepancies between in vitro and in vivo studies are due to inefficient tocotrienol intestinal absorption and delivery to target tissues in the body. Specifically, tocotrienol absorption in the small intestine is primarily dependent on a carrier-mediated transporter mechanism that not only displays saturation but also appears to undergo down-regulation when exposed to high concentrations of tocotrienol in the gut. Since high oral doses of tocotrienols inhibit its own absorption from the gut, oral consumption of high doses of tocotrienol in oil-filled capsules ultimately results in only relatively low levels of tocotrienol reaching the circulation. Recent medicinal chemistry studies have led to the development of novel prodrug tocotrienol derivatives that are highly soluble in aqueous solutions and bypass the natural limits that exist for tocotrienol absorption, transport, and metabolic breakdown prior to reaching the target tissue. Specifically, semisynthetic redox-silent tocotrienol analogues, which contain or do not contain their terminal carboxy group, show great promise as a means of enhancing tocotrienol bioavailability and anticancer therapeutic responsiveness. These prodrug tocotrienol derivatives have the greatest potential as daily oral supplements for use as chemopreventative agents. However, in order to effectively treat and eliminate existing cancers, higher concentrations of c-tocotrienol are needed and require intravenous administration. Recent studies have demonstrated that encapsulated tocotrienols within tumor-targeted vesicles or nanoparticles can be used as an effective injectable method of delivery. These novel drug-delivery systems have been shown to greatly enhance tocotrienol accumulation and retention in tumor cells and greatly potentiate the antiproliferative and apoptotic action of tocotrienols as compared with administration of simple oral administration of tocotrienol oil-filled capsules. Furthermore, nanoparticle formulations containing tocotrienols in combination with other chemotherapeutic agents display synergistic therapeutic responsiveness. Since combination therapy of tocotrienol with other chemotherapeutic agents requires significantly lower treatment doses to suppress cancer cell growth and survival, an additional benefit can also be realized as a corresponding reduction in adverse side effects and toxicity characteristically associated with high-dose chemotherapy. JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 331S

9 In summary, cell culture experiments clearly demonstrate the anticancer effectiveness of tocotrienols, whereas oral supplementation of tocotrienols in animal and human studies has produced disappointing results. Nevertheless, recent studies in the areas of tocotrienol kinetics have provided essential information required for understanding the therapeutic limitations of oral administration of tocotrienols, and the development of prodrug derivatives and novel vesicle and nanoparticle delivery systems shows great potential for optimizing tocotrienol use as a therapeutic agent either alone or in combination with other chemotherapeutic agents in the prevention and treatment of cancer. ACKNOWLEDGMENTS This work was performed at the College of Pharmacy, University of Louisiana at Monroe, Monroe, Louisiana and supported in part by grants from the National Institutes of Health/National Cancer Institute (CA 86833) and First Tech International Ltd. (Hong Kong). The authors would like to thank First Tech International Ltd. for generously providing TRF and isolated tocotrienols for use in these experiments. REFERENCES 1. Sylvester PW, Theriault A: Role of tocotrienols in the prevention of cardiovascular disease and breast cancer. Curr Top Nutraceutical Res 1: , Sylvester PW, Shah SJ: Mechanisms mediating the antiproliferative and apoptotic effects of vitamin E in mammary cancer cells. Front Biosci 10: , Sylvester PW: Vitamin E and apoptosis. Vitam Horm 76: , Sylvester PW: Antiproliferative and apoptotic effects of tocotrienols on normal and neoplastic mammary epithelial cells. In: Watson RR, Preedy VR (eds): Tocotrienols: Vitamin E beyond Tocopherols. Boca Raton,, FL: CRC Press, pp , Mensink RP, van Houwelingen AC, Kromhout D, Hornstra G: A vitamin E concentrate rich in tocotrienols had no effect on serum lipids, lipoproteins, or platelet function in men with mildly elevated serum lipid concentrations. 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