Tocotrienols Modulate a Life or Death Decision in Cancers

Malignancy often arises from sophisticated defects in the intricate molecular mechanisms of cells, rendering a complicated molecular ground to effectively target cancers. Resistance toward cell death and enhancement of cell survival are the common adaptations in cancer due to its infinite proliferative capacity. Existing cancer treatment strategies that target a single molecular pathway or cancer hallmark fail to fully resolve the problem. Hence, multitargeted anticancer agents that can concurrently target cell death and survival pathways are seen as a promising alternative to treat cancer. Tocotrienols, a minor constituent of the vitamin E family that have previously been reported to induce various cell death mechanisms and target several key survival pathways, could be an effective anticancer agent. This review puts forward the potential application of tocotrienols as an anticancer treatment from a perspective of influencing the life or death decision of cancer cells. The cell death mechanisms elicited by tocotrienols, particularly apoptosis and autophagy, are highlighted. The influences of several cell survival signaling pathways in shaping cancer cell death, particularly NF-κB, PI3K/Akt, MAPK, and Wnt, are also reviewed. This review may stimulate further mechanistic researches and foster clinical applications of tocotrienols via rational drug designs.


Introduction
Cancer incidence rate has been increasing steadily every year, imposing significant social and economic impacts globally. In 2018, it was estimated that new cancer cases and mortality would increase to 18.1 million and 9.6 million, respectively, for the world population. In other words, 1 in 5 men and 1 in 6 women may develop cancer in their lifetime, while 1 in 8 men and 1 in 11 women may die from cancer [1]. In the United States (US) alone, it was predicted that there would be about 1.74 million new cancer cases, and approximately 600,000 people would eventually die from this disease in 2018. The estimated national expenditure for cancer care in the US was $147.3 billion in 2017. This figure is expected to increase continuously due to a high prevalence of cancer cases in aged population [2]. Extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway begins outside of a cell and involves the binding of extracellular death ligands to their respective transmembrane death receptors. The activation of tumor necrosis factor (TNF) receptor superfamily, such as first apoptosis signal (Fas), takes place, leading to activation of caspase-8 (initiator caspase) and recruitment of the Fas-associated protein with death domain (FADD) adapter molecule. It transduces a downstream signaling cascade to the effector caspase (caspase-8), leading to the proteolytic activation of caspase-3. In fact, the extrinsic pathway can cause amplification of cascade via intrinsic mitochondrial pathway whereby caspase-8 cleaves Bid to promote mitochondrial outer membrane permeabilization (MOMP) [32]. The intrinsic pathway is initiated by internal stimuli, such as genetic damage, growth factor deprivation, hypoxia, oxidative stress, and flux of calcium ions (Ca 2+ ) [33]. The stimuli then perceive cell death signals via the mitochondrion, which represents the metabolic status of a cell. The MOMP is often regarded as the primary step required for activation of caspases. Proapoptotic and antiapoptotic B-cell lymphoma 2 (Bcl-2) family proteins are involved in regulating the permeability of outer mitochondrial membrane [34,35]. Upon apoptotic stimulus, MOMP takes place, leading to the release of cytochrome c from the intermembrane space. In the cytoplasm, cytochrome c engages apoptotic protease activating factor 1 (Apaf-1) and eventually leads to the activation of caspase-9 (initiator caspase). Following that, caspase-9 activates executioner caspases, such as caspase-3, -6, and -7, which subsequently cause the downstream biochemical events, leading to apoptosis [36].
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 29 Figure 2. The process of autophagy. During autophagy, phagophore (cup-shaped, double-membrane sac) emerges in cytoplasm, driven by unc-51-like kinase 1 (ULK1) complex and vacuolar protein sorting (Vps) 34 complex. The expansion of phagophore is facilitated by Atg5-12/Atg16L complex to uptake cargos from the cytoplasm into a double-membrane autophagosome. The loaded autophagosome then fuses with lysosome to allow the degradation of cargo by lysosomal proteases, while microtubule-associated protein light chain 3 (LC3-I) will be recycled back to cytosol. The endogenous LC3-I, present in the cytoplasm, is processed to LC3-II and bound to the autophagosome during autophagy. Therefore, the ratio of LC3-I (water soluble) and LC3-II (lipidated) is often used as a marker to assess autophagy. Then, the lysosomal permeases and transporters export amino acids and other by-products of degradation back to the cytoplasm, where they can be reused for building macromolecules and for metabolism [37]. Abbreviations: Atg, autophagy-related protein; FIP200, focal adhesion kinase family interacting protein of 200 kDa.

Tocotrienols Induce Mitochondria-Mediated Apoptosis
Mitochondria are tiny organelles in a cell, which exert both vital and lethal functions. In addition to serving as a powerhouse for fueling energy to cells, this organelle also contains homicidal molecules that can subject a cell to death [49]. Tocotrienols exhibit mitochondrial disruption capacity via mitochondrial outer membrane permeabilization (MOMP) induction [50][51][52], culminating in mitochondria-mediated apoptosis. In fact, MOMP is a critical event in the intrinsic apoptotic pathway. It has been reported that the blockade of mitochondrial permeability transition pore (MPTP) with cyclosporine A completely abolished the cytotoxic effects of TRF, β-T3, γ-T3, and δ-T3 in activated rat pancreatic stellate cells, which could support the growth and invasiveness of pancreatic ductal adenocarcinoma [53,54].
Although the actual role of tocotrienols in mitochondria-mediated apoptosis remains elusive, four potential interactions have been proposed ( Figure 3). Several lines of evidence have reported that tocotrienols alter Bcl-2/Bax ratio, rendering depolarization of mitochondria [50,55,56]. A study conducted on neuroblastoma SH-SY5Y cells shed a light on the potential interaction between γ-T3 and B-cell lymphoma 2 (Bcl-2) proteins. This research showed that γ-T3 competes with 8-Anilino-1naphthalenesulfonic acid ammonium salt (ANS) for binding to the hydrophobic groove of Bcl-2. Hence, it was suggested that γ-T3 acts as Bcl-2 homology 3 (BH3) mimetic to displace proapoptotic The process of autophagy. During autophagy, phagophore (cup-shaped, double-membrane sac) emerges in cytoplasm, driven by unc-51-like kinase 1 (ULK1) complex and vacuolar protein sorting (Vps) 34 complex. The expansion of phagophore is facilitated by Atg5-12/Atg16L complex to uptake cargos from the cytoplasm into a double-membrane autophagosome. The loaded autophagosome then fuses with lysosome to allow the degradation of cargo by lysosomal proteases, while microtubule-associated protein light chain 3 (LC3-I) will be recycled back to cytosol. The endogenous LC3-I, present in the cytoplasm, is processed to LC3-II and bound to the autophagosome during autophagy. Therefore, the ratio of LC3-I (water soluble) and LC3-II (lipidated) is often used as a marker to assess autophagy. Then, the lysosomal permeases and transporters export amino acids and other by-products of degradation back to the cytoplasm, where they can be reused for building macromolecules and for metabolism [37]. Abbreviations: Atg, autophagy-related protein; FIP200, focal adhesion kinase family interacting protein of 200 kDa.

Tocotrienols Induce Mitochondria-Mediated Apoptosis
Mitochondria are tiny organelles in a cell, which exert both vital and lethal functions. In addition to serving as a powerhouse for fueling energy to cells, this organelle also contains homicidal molecules that can subject a cell to death [49]. Tocotrienols exhibit mitochondrial disruption capacity via mitochondrial outer membrane permeabilization (MOMP) induction [50][51][52], culminating in mitochondria-mediated apoptosis. In fact, MOMP is a critical event in the intrinsic apoptotic pathway. It has been reported that the blockade of mitochondrial permeability transition pore (MPTP) with cyclosporine A completely abolished the cytotoxic effects of TRF, β-T3, γ-T3, and δ-T3 in activated rat pancreatic stellate cells, which could support the growth and invasiveness of pancreatic ductal adenocarcinoma [53,54].
Although the actual role of tocotrienols in mitochondria-mediated apoptosis remains elusive, four potential interactions have been proposed (Figure 3). Several lines of evidence have reported that tocotrienols alter Bcl-2/Bax ratio, rendering depolarization of mitochondria [50,55,56]. A study conducted on neuroblastoma SH-SY5Y cells shed a light on the potential interaction between γ-T3 and B-cell lymphoma 2 (Bcl-2) proteins. This research showed that γ-T3 competes with 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS) for binding to the hydrophobic groove of Bcl-2. Hence, it was suggested that γ-T3 acts as Bcl-2 homology 3 (BH3) mimetic to displace proapoptotic members from Bcl-2 sequestration. As a result, proapoptotic molecules become available to permeabilize the outer mitochondrial membrane and release cytochrome c to the cytosol, leading to caspase-9-and caspase-3-dependent apoptosis [57]. However, it will be more worthwhile if γ-T3, which is claimed to serve as an inhibitor of antiapoptotic Bcl-2 members, can be further characterized to allow development of derivatives that embrace a greater therapeutic efficacy [57].
survivin in colorectal cancer [59,60]. Of note, cancer displays an elevated IAP profile to promote uncontrollable cell division, which may hinder the effectiveness of treatments that rely on caspase activation. Hence, the ability to block the activity of IAP could be an opportunity to culminate in a complete activation of apoptosis.
Other than indirectly modulating the Bcl-2 family to induce MOMP, γ-T3-treated MDA-MB-231 breast cancer cells have been found to show a direct disruption of mitochondrial membrane potential independent from Bax/Bcl-2 ratio alteration and poly(ADP-ribose) polymerase (PARP) cleavage [50]. The authors suggested that cytochrome c release is not a critical event for apoptosis induction in this scenario. In another study, γ-T3 treatment on human T cell lymphoma Jurkat cells elevated mitochondrial reactive oxidative species (ROS) production [61]. Hence, it is plausible to suggest that γ-T3 could promote mitochondrial dysfunction, probably via ROS generation, leading to metabolic catastrophe and cell death, rather than engaging caspase-dependent intrinsic apoptosis. Additionally, it can also be speculated that γ-T3 induces caspase-independent cell death following the release of soluble intermembrane mitochondrial proteins, such as apoptosis inducing factor (AIF) [50], high temperature requirement A (HtrA), and endonuclease G (EndoG). On the other hand, another study revealed that the elevated Bax expression induced by δ-T3 correlates to the induction of zinc finger transcription factor, known as early growth response protein 1 (EGR1). This transcription factor binds to the Bax gene promoter, leading to the induction of proapoptotic Bax in pancreatic cancer cells [58].
Suppression of inhibitor of apoptosis (IAP) could be another target of tocotrienols in mitochondrial pathway. IAP can block apoptosis by deactivating caspases activity. Interestingly, tocotrienols have been reported to be able to downregulate the IAP level, such as cIAP-1, cIAP2, and survivin in colorectal cancer [59,60]. Of note, cancer displays an elevated IAP profile to promote uncontrollable cell division, which may hinder the effectiveness of treatments that rely on caspase activation. Hence, the ability to block the activity of IAP could be an opportunity to culminate in a complete activation of apoptosis.
Other than indirectly modulating the Bcl-2 family to induce MOMP, γ-T3-treated MDA-MB-231 breast cancer cells have been found to show a direct disruption of mitochondrial membrane potential independent from Bax/Bcl-2 ratio alteration and poly(ADP-ribose) polymerase (PARP) cleavage [50]. The authors suggested that cytochrome c release is not a critical event for apoptosis induction in this scenario. In another study, γ-T3 treatment on human T cell lymphoma Jurkat cells elevated mitochondrial reactive oxidative species (ROS) production [61]. Hence, it is plausible to suggest that γ-T3 could promote mitochondrial dysfunction, probably via ROS generation, leading to metabolic catastrophe and cell death, rather than engaging caspase-dependent intrinsic apoptosis. Additionally, it can also be speculated that γ-T3 induces caspase-independent cell death following the release of soluble intermembrane mitochondrial proteins, such as apoptosis inducing factor (AIF) [50], high temperature requirement A (HtrA), and endonuclease G (EndoG).

Tocotrienols Induce Endoplasmic Reticulum Stress
Endoplasmic reticulum (ER) experiences stress during perturbations in Ca 2+ homeostasis, redox imbalance, altered protein glycosylation, or protein folding defects, leading to accumulation of unfolded or misfolded proteins in the ER lumen [62,63]. Eventually, the highly conserved unfolded protein response (UPR) pathway is triggered to alleviate the potentially toxic stress and restore the homeostasis [64]. A persistent and overwhelming ER stress will lead to apoptosis. Several studies have reported that tocotrienols exert ER stress as one of the mechanisms to induce apoptosis [65][66][67]. In this section, the evidences of tocotrienols-induced ER stress are gathered and aligned to the present understanding on apoptosis that is led by ER stress (Figure 4).
The involvement of ER-stress-mediated apoptosis, as triggered by tocotrienols, was first reported by Wali and co-workers [68]. Their work showed that γ-T3 activated protein kinase-like endoplasmic reticulum kinase (PERK) arm of the UPR in neoplastic mouse +SA mammary epithelial cells by phosphorylating (inhibition) eukaryotic translational initiation factor (eIF2α), leading to the increase in activating transcription factor 4 (ATF-4) [68]. Following that, the ER-stress-inducible nuclear protein, CCAAT-enhancer-binding protein homologous protein (CHOP), and tribbles 3 (TRB3) expressions were also increased. Knockdown of CHOP by siRNAs attenuated PARP cleavage induced by γ-T3, suggesting a CHOP-dependent, ER-stress-mediated apoptosis.
Another study conducted on γ-T3-treated MCF-7 and MDA-MB-231 breast cancer cells demonstrated an involvement of ER stress via both the PERK and inositol-requiring enzyme 1 (IRE1) pathways [69], leading to remarkable increment in their downstream targets, such as activating transcription factor 3 (ATF3) and CHOP, in response to γ-T3 treatment [69]. The resultant apoptosis was evident by caspase-7 activation and PARP cleavage. In addition, ATF3 knockdown using siRNA abrogated the apoptosis, suggesting that ATF3 is a putative molecular target in γ-T3-induced apoptosis [69].
In an attempt to study the underlying molecular mechanism, a specific estrogen receptor inhibitor (fulvestrant) employed as a competitive inhibitor to tocotrienols showed a reduction in the expression of proapoptotic genes [70]. Hence, it was hypothesized that γ-T3 may activate the "orphan" receptor (that also binds to fulvestrant), leading to IRE-1 activation and XBP-1 splicing, which finally induce apoptosis that is cognate with the activation of ER stress.
To our knowledge, the involvement of ATF6 pathway was reported as null in at least two studies [69,71]. Differing from PERK and IRE pathways, the activation of ATF6 requires translocation and cleavage in Golgi apparatus, which is often associated with enhanced survival in cancer cells [69,72]. It is possible that tocotrienols-induced ER stress is rapid and favors apoptosis, 7 of 28 leaving behind the prosurvival option. Although a reduction of ATF6α level has been observed in +SA cells, the spatial information in relation to its cleavage has not been studied [68]. Possibly, the ATF6 level reduction is due to cell death but not cleavage in the Golgi apparatus. Hence, the involvement of ATF6 pathway in tocotrienols-induced ER-stress-mediated apoptosis requires further investigation.
The activation of extrinsic apoptotic pathway being coupled to ER stress suggests a close connection between them. For instance, it has been reported that both death receptor 5 and CHOP were upregulated upon treatment of γ-T3 on MDA-MB-231 and MCF-7 breast cancer cells [73]. However, the sequence of activation and the dominance of the respective pathways remain elusive. were upregulated upon treatment of γ-T3 on MDA-MB-231 and MCF-7 breast cancer cells [73]. However, the sequence of activation and the dominance of the respective pathways remain elusive.

Co-Involvement of Endoplasmic Reticulum Stress and Mitochondria-Mediated Apoptosis
The interconnectivity between organelles could play an important role in perceiving stress upon receiving cancer drugs and influences the life or death decision in the cancer cells. In fact, increasing evidences have shown that ER stress may cooperate with mitochondria for stress signal amplification, thus culminating in apoptosis [74]. For instance, mitochondria-mediated apoptosis was evident in δ-T3-treated BLM and A375 human melanoma cell lines by cytochrome c release and augmentation of Bax/Bcl-2 ratio. At the same time, ER-stress-related proteins, such as PERK, p-eIF2α, ATF4, CHOP, IRE1α, and caspase-4, were activated. The application of salubrinal (inhibitor of ER stress) has been found to successfully block the cytotoxicity of δ-T3, signifying the importance of ER stress for apoptosis induction [71]. In fact, ATF/CHOP or IRE-1 ER stress sensors could be the mediators to connect ER stress to mitochondrial pathway of apoptosis [75], potentially via the modulation of Bcl- Overview of tocotrienols-induced ER-stress-mediated apoptosis. Tocotrienols interact with unknown receptor on cell surface to trigger ER stress. BiP dissociates from unfolded protein response (UPR) sensors (i.e., PERK, IRE1, and ATF6) to trigger the respective signaling pathways, leading to apoptosis. Abbreviations: BiP or GRP78, glucose-regulated protein; CHOP, CCAAT-enhancer-binding protein homologous protein; PERK, PKR-like ER-localized eIF2α kinase; IRE1, inositol-requiring enzyme 1; ATF, activating transcription factor; eIF2-α, eukaryotic initiation factor 2 alpha; Cas-, caspase-; PARP, poly(ADP-ribose) polymerase; sXBP1, spliced X-box binding protein 1, TRB3, tribbles 3. "?" indicates more investigations are required.

Co-Involvement of Endoplasmic Reticulum Stress and Mitochondria-Mediated Apoptosis
The interconnectivity between organelles could play an important role in perceiving stress upon receiving cancer drugs and influences the life or death decision in the cancer cells. In fact, increasing evidences have shown that ER stress may cooperate with mitochondria for stress signal amplification, thus culminating in apoptosis [74]. For instance, mitochondria-mediated apoptosis was evident in δ-T3-treated BLM and A375 human melanoma cell lines by cytochrome c release and augmentation of Bax/Bcl-2 ratio. At the same time, ER-stress-related proteins, such as PERK, p-eIF2α, ATF4, CHOP, IRE1α, and caspase-4, were activated. The application of salubrinal (inhibitor of ER stress) has been found to successfully block the cytotoxicity of δ-T3, signifying the importance of ER stress for apoptosis induction [71]. In fact, ATF/CHOP or IRE-1 ER stress sensors could be the mediators to connect ER stress to mitochondrial pathway of apoptosis [75], potentially via the modulation of Bcl-2 family [76]. It is interesting to note that several studies have suggested ATF4 may facilitate the transcriptional upregulation of BH3-only proteins, such as p53 upregulating the modulator of apoptosis (PUMA) and NOXA during ER stress [77][78][79]. On the other hand, CHOP also downregulates Bcl-2 protein, thus incurring higher susceptibility to MOMP [77]. Additionally, it appears that calcium signaling could play an important role in the ER-mitochondrial communication [76]. It has been suggested that the elevated flux of Ca 2+ from ER, subsequently leading to cytochrome c release, could happen independent of MPTP [77]. Thus far, only limited studies have been conducted on understanding the connectivity of tocotrienols-induced ER stress and mitochondria-mediated apoptosis. Time course studies on molecular targets for both pathways can be conducted in order to elucidate the superiority of each pathway in tocotrienols-induced apoptosis, which may offer a better understanding on the mechanism of action and pave an avenue for discovering novel therapeutics for cancers.

Co-Involvement of Extrinsic and Intrinsic Pathways
In addition to tocotrienols inducing apoptosis via intrinsic mitochondrial pathway, a few studies have shown that both intrinsic and extrinsic pathways are activated concurrently. Although death-receptor-mediated extrinsic pathway and mitochondria-mediated intrinsic pathway are triggered by different stimuli, the two pathways are converged on executioner caspases (e.g., caspase-3, -7). γ-T3 has been reported to engage both intrinsic and extrinsic pathways in Hep3B human hepatoma cells, as demonstrated by elevated activities of caspase-8, -9, -3 and accompanied by upregulation of truncated Bid and Bax, resulting in PARP cleavage [45]. In addition, co-elicitation of intrinsic pathway by inducing Ca 2+ release, loss of mitochondrial membrane potential, and increase in Bax/Bcl-2 ratio, and extrinsic pathway via upregulating surface expression of Fas and FasL in γ-T3-treated human T-cell lymphoma (Jurkat cells) has been reported. Eventually, apoptosis was induced via activation of caspase-8, -9, and -3 and PARP cleavage [61]. Moreover, the involvement of Bid, Bax, cytochrome c, disruption of mitochondrial membrane permeabilization-dependent caspase-8 activation induced by α-T3, γ-T3, and δ-T3 have also been reported in A549 lung adenocarcinoma and U87MG brain glioblastoma [40]. In addition, activation of both intrinsic and extrinsic pathways has been reported in leukemic cells, involving upregulation of genes in Bcl-2, caspases, and death receptor families [80]. These evidences therefore suggest a convergence of extrinsic and intrinsic pathways at mitochondria, which potentially serve as an apoptosis signal amplification center. The cooperative interaction by simultaneous activation of intrinsic and extrinsic pathways can potentially lead to enhanced cell death through functional complementation as mutation of more than one pathway is common in cancer [65,81]. However, the benefit of this cooperative action induced by tocotrienols may be more remarkable in type II cancer cells, which requires mitochondrial pathway for death-receptor-induced apoptosis [82].

Interplay between Autophagy and Apoptosis
Autophagy has been well known as a mechanism for cell survival under immense cell stress, whereas apoptosis is a pro-death decision when a cell experiences an unrecoverable damage. Therefore, inhibition of autophagy is often seen as an approach to promote apoptosis. For instance, a study showed that application of autophagy inhibitor 3-methyladenine (3-MA) potentiated apoptosis induced by dietary tocotrienols in breast cancer cells [83], suggesting an antagonistic role of autophagy to apoptosis.
On the contrary, accumulating evidences have shown that autophagy and apoptosis act together to induce cell death in cancer. However, the connections at molecular level are multifaceted and poorly understood [84]. We attempt to establish a connection between tocotrienols-induced apoptosis and autophagy based on currently available literature ( Figure 5). Mitochondria and ER are proposed as the target organelles to drive the connection between the two pathways.  Figure 5. Schematic representation of potential interactions between tocotrienols-induced apoptosis and autophagy. Tocotrienols bind to Bcl-2 protein to displace proapoptotic protein (i.e., Bax) and proautophagic protein (Beclin-1) to initiate apoptosis and autophagy, respectively. Bax initiates MOMP to induce apoptosis, leading to generation of ROS, which serves as a second messenger to trigger the expression of autophagy-related protein (Atg). Tocotrienols upregulate de novo synthesis of ceramides, which can activate JNK signaling by phosphorylating Bcl-2 family proteins and induce Ca 2+ release by ER stress, eventually resulting in apoptosis and autophagy. "?" indicates more investigations are required.

Tocotrienols Target Prosurvival Signaling Pathways
Targeting only the cell death pathways may not be sufficient to eliminate cancer cells. Cell death signaling can be overcome by stimulation of cell survival via alternative modes, leading to a more complicated problem, such as development of drug-resistant cancer cells. Tocotrienols have been previously reported to concurrently activate cell death programs and downregulate prosurvival signaling pathways, such as nuclear factor kappa B (NF-κB), phosphoinositide 3-kinase (PI3K)/Akt, Wingless and INT-1 (Wnt), and mitogen-activated protein kinases (MAPKs) ( Figure 6). Therefore, the following section describes the capability of tocotrienols in targeting cell survival and deliberating implications in the cell death mechanisms. Figure 5. Schematic representation of potential interactions between tocotrienols-induced apoptosis and autophagy. Tocotrienols bind to Bcl-2 protein to displace proapoptotic protein (i.e., Bax) and proautophagic protein (Beclin-1) to initiate apoptosis and autophagy, respectively. Bax initiates MOMP to induce apoptosis, leading to generation of ROS, which serves as a second messenger to trigger the expression of autophagy-related protein (Atg). Tocotrienols upregulate de novo synthesis of ceramides, which can activate JNK signaling by phosphorylating Bcl-2 family proteins and induce Ca 2+ release by ER stress, eventually resulting in apoptosis and autophagy. "?" indicates more investigations are required.
The activated pancreatic stellate cells treated with TRF exhibit the ability of tocotrienols to co-induce mitochondria-mediated apoptosis and autophagy [53]. In the study, TRF was reported to cause apoptosis, as indicated by depolarized mitochondrial membrane, cytochrome c release, increased DNA fragmentation, and caspase activation. Concurrently, autophagy was also apparent by the formation of autophagic vacuoles and LC3-II accumulation. An attempt to block apoptosis with zVAD-fmk caspase inhibitor showed a converse enhancement in autophagy, suggesting autophagy could serve as a backup plan to execute the cells when apoptosis fails. In this case, autophagy is engaged in an immediate and active process, rather than as a passive "stress adaptive" function. It has been proposed that tocotrienols interconnect apoptosis and autophagy at mitochondria because the blockade of MPTP with cyclosporine A completely abolishes apoptosis and autophagic response [85].
The actual mechanism has not been fully elucidated; however, it has been suggested that tocotrienols may initiate intrinsic mitochondrial pathway by displacing proapoptotic (Bax, Bak, BH3-only proteins) and proautophagic (Beclin-1) proteins from BH3 docking sites of Bcl-2 or Bcl-xL proteins, resulting in both apoptosis and autophagy [85]. In addition, ROS generated from compromised mitochondria by TRF treatment has been proposed to connect two pathways because ROS is essential to activate autophagic enzymes, such as Atg4 [85], as well as to amplify apoptotic signal. Although this study was not developed from a cancer model, the dual pathways activation shed a light on the novel function of tocotrienols. Later, concurrent activation of apoptosis and autophagy was correspondingly reported in other cancer cell lines, such as breast and prostate [86,87].
In γ-T3-treated mouse (+SA) mammary tumor cells and human (MCF-7 and MDA-MD-231) breast cancer cell lines, proautophagic proteins, such as LC3-II/LC3-I ratio and Beclin-1 levels, and the corresponding increased levels of apoptotic markers including Bax/Bcl-2 ratio, cleaved caspase-3, and cleaved PARP, demonstrated the concurrent induction of autophagy and apoptosis [88]. In the following study, the authors showed autophagy inhibitor surrogating the cytotoxicity [89], further confirming the presence of cytotoxic autophagic cell death. In prostate cancer cells, γ-T3 induced elevated intracellular ceramides (dihydrosphingosine and dihydroceramide), which appear to be potent mediators of apoptosis and autophagy [87]. Ceramides have been well reported as a key player in apoptosis by inducing both intrinsic and extrinsic apoptotic pathways [90]. In comparison, the connection of ceramides with autophagy is emerging; the ceramides may activate c-Jun N-terminal 1 (JNK1) to phosphorylate Bcl-2 (inactivation), eventually leading to the dissociation of Beclin-1 from Bcl-2 [90].

Tocotrienols Target Prosurvival Signaling Pathways
Targeting only the cell death pathways may not be sufficient to eliminate cancer cells. Cell death signaling can be overcome by stimulation of cell survival via alternative modes, leading to a more complicated problem, such as development of drug-resistant cancer cells. Tocotrienols have been previously reported to concurrently activate cell death programs and downregulate prosurvival signaling pathways, such as nuclear factor kappa B (NF-κB), phosphoinositide 3-kinase (PI3K)/Akt, Wingless and INT-1 (Wnt), and mitogen-activated protein kinases (MAPKs) ( Figure 6). Therefore, the following section describes the capability of tocotrienols in targeting cell survival and deliberating implications in the cell death mechanisms.

PI3K/Akt Signaling
The PI3K pathway plays a determining role in regulating cell survival. The activation of Akt phosphorylates and inhibits the proapoptotic Bcl-2 family members, such as Bad [101]. The

PI3K/Akt Signaling
The PI3K pathway plays a determining role in regulating cell survival. The activation of Akt phosphorylates and inhibits the proapoptotic Bcl-2 family members, such as Bad [101].
The antiproliferative effect of γ-T3 in neoplastic +SA mouse mammary epithelial cells is mediated by a reduction in PI3K/PDK-1/Akt mitogenic signaling [102]. Suppression in this signaling pathway further leads to death-receptor-independent caspase-8 activation and reduction of intracellular FLIP expression [103,104]. In pancreatic cancer cell lines (MIA PaCa-2 and Panc-28), γ-T3 and δ-T3 could effectively prevent Akt activation. A further study revealed that several downstream targets of Akt were affected by γ-T3, such as mTOR, S6 kinase (Ser 240/244), GSK-3β, and FOXO3 [105]. These results collectively suggest that the two tocotrienol isoforms can induce apoptosis in pancreatic cancer cells through the suppression of vital cell survival and proliferative signaling pathways [105].

MAP Kinase Signaling
MAPK signaling pathway plays a critical role in the outcome of, and sensitivity to anticancer therapies [106]. There are three important MAPK members for maintenance of cells, namely, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38-MAPKs. ERKs have been found to be important for cell survival, whereas JNKs are deemed stress-responsive and thus involved in apoptosis [107]. On the other hand, p38-MAPK has been reported to exhibit a dual role as cell death and cell survival regulators depending on the types of cell and stimulus received [108]. Of note, it has been proposed that the roles of p38 and JNK regulate the balance of apoptosis and autophagy in response to chemotherapeutic agents, portraying a significant role of MAPK signaling in deciding life or death matter.
γ-T3 and δ-T3 treatments in pancreatic cells have been found to show reductions of ERK activation and its downstream mediator RSK (ribosomal protein S6 kinase), which correlate to the downregulation of HER/ErbB2 expression [105]. On the other hand, a research illustrated that γ-T3 treatment caused suppressions of ERK and p38 MAPK but an activation of ERK in T-cell lymphoma, leading to both intrinsic and extrinsic apoptosis [61]. In contrast, another study reported that γ-T3 elevated death receptors, DR4, and DR5, which are regulated through ERK activation [60]. In fact, the contradictory roles of ERK activation for promoting cell death or cell survival could be delineated by studying the subcellular localization of ERK proteins [109]. For instance, translocation of ERK1/2 to nucleus carries an anticancer role, while positioning to mitochondria may promote cell survival [110]. Collectively, these studies reveal a connection between proliferative signaling and apoptosis, which further strengthens the versatility of tocotrienols in targeting cancers.

Wnt Signaling
Wnt signaling is one of the key cascades for regulating development and stemness; overactivation of this pathway commonly occurs in cancer, predominantly in colorectal cancer [111]. Wnt pathway is commonly divided into canonical (β-catenin-dependent) and noncanonical (β-catenin-independent) signaling [111]. Serving as an important driver in the canonical pathway, the activation of Wnt receptors leads to translocation of β-catenin into nucleus. Subsequently, it forms an active complex with lymphoid enhancer factor (LEF), T-cell factor (TCF), and histone-modifying coactivators to initiate transcriptional activities for multiple processes such as cell proliferation and cell survival, such as c-Myc, cyclin D1, and survivin [111,112]. Additional evidence has shown that activation of Wnt signaling can overcome the apoptosis mediated by Notch in gastric cancer [113], portraying the importance of Wnt signaling in shaping apoptotic cell death.
δ-T3 has been reported to inhibit SW620 colon cancer cells by downregulating the expression levels of Wnt-1, β-catenin, c-Jun, and cyclin D1 in the Wnt signal pathway [114]. In addition, γ-T3 inhibited cell viability through suppression of β-catenin/TCF signaling in human colon carcinoma HT-29 cells [42]. TRF suppressed the growth of human colon cancer xenografts in Balb/c nude mice via the Wnt pathway, by which the expressions of β-catenin, Wnt, and c-Myc proteins in xenografts were significantly downregulated [23]. In breast cancer cells, γ-T3 reversed the epithelial-to-mesenchymal transition in human breast cancer via the inhibition of canonical Wnt signaling [115]. Taken together, the ability of tocotrienols in targeting Wnt pathway suggests a value-added therapy to effectively regulate proliferation, apoptosis, and metastasis simultaneously.

Current and Future Perspectives of Tocotrienols
To date, undoubtedly, promising anticancer effects and molecular targets of tocotrienols in promoting cell death and suppressing survival in cancers have been revealed from numerous in vitro (Table 1) and in vivo (Table 2) studies. These valuable preclinical findings have expectedly warranted further investigations for clinical applications, some recent examples of which are summarized in Table 3. Despite ample evidence of the therapeutic benefits of tocotrienols in various types of cancers, current clinical studies have only embarked on breast, colon, pancreas, lung, and ovary cancers. Other cancer types, such as brain, blood, gastric, and prostate cancers, with positive therapeutic potentials may be lining up in forthcoming clinical trials.
Yet, poor oral bioavailability remains the main hurdle limiting the in vivo therapeutic efficacy of tocotrienols. Saturable uptake in the transport mechanism within intestine and bloodstream, leading to low bioavailability, has therefore greatly compromised the efficacy and potency of tocotrienols via oral intake [116]. Several formulation strategies have been found to enhance the oral absorption of tocotrienols by at least 3 folds, including self-emulsifying delivery systems [117] and nanostructured lipid carriers [118]. In addition, alternative routes of administration have been investigated to circumvent the limitations associated with oral absorption. One such approach is the application of nanoformulation that enables tocotrienols to be administered via intravenous and topical routes. Polymer-conjugated tocotrienols [119] and entrapment of tocotrienols in nanovesicles [120] have been investigated for intravenous injection, while tocotrienol nanoemulsions have been investigated for topical applications [121]. These formulations markedly improved the antiproliferative activities of tocotrienols in vitro and tumor suppression properties in vivo. Hence, further studies on clinical translation of these novel tocotrienol formulations are warranted.
It has been widely reported that administration of high-dose tocotrienols is a futile maneuver due to high metabolic degradation in vivo [43]. Prominently, the hypomethylated forms of tocotrienols, i.e., δ-T3 and γ-T3, show the highest cellular metabolism. In fact, high metabolism of T3s in vivo are associated with the induction of drug metabolizing enzymes, such as cytochrome P450 enzyme (CYP450) and glucuronosyltransferase 1A1 (UGT1A1) as well as the induction of multidrug resistance protein-1 (MDR1) via pregame-X-receptor (PXR) and steroid and xenobiotic receptor (SXR). These enzymes appear to deliver positive cytotoxic effects to cancer cells; however, coadministration of high-dose tocotrienols with other drugs may potentially interfere the metabolism, thereby affecting the therapeutic efficacy of these drugs [122][123][124]. Hence, a synergistic combination of tocotrienols with other anticancer agents at low doses can augment the therapeutic efficacy and potency (both bioactives) as well as reduce the dose-limiting toxicities (i.e., chemotherapeutic agents). In addition, a combined treatment approach could reduce the risk of developing drug resistance in cancer cells. As "one drug one target"-based targeted therapy (e.g., tyrosine kinase inhibitors) is inclined to drug resistance [125], combinatorial application of tocotrienols that concurrently targets multiple signaling pathways can effectively eradicate cancer cell population [126]. In fact, the multitargeted anticancer actions offered by tocotrienols are regarded as a valuable feature that could potentially pave an avenue into polypharmacology, which advocates "one drug multiple targets" [127]. Furthermore, combining existing drugs with tocotrienols could also serve as an immediate and cost-effective solution. So far, combination therapies using tocotrienols with traditional chemotherapeutics, plant bioactives, and targeted inhibitors have shown positive therapeutic responses in vitro and in vivo, as reviewed by Eitsuka et al. [128]. However, metabolism, toxicities, and pharmacokinetics studies of combined treatments in vivo are still lacking, more investigations are therefore necessary to advance effective drug combinations toward a tangible clinical application in the near future.
At present, two completed clinical trials have demonstrated positive benefits of tocotrienols in cancer management. Although statistically insignificant due to limited sample size, the first clinical study conducted on breast cancer patients showed that TRF in combination with tamoxifen improved breast-cancer-specific survival compared to the tamoxifen group (NCT01157026) [38]. The second clinical trial conducted on pancreatic ductal neoplasia patients reported that δ-T3 was well tolerated by patients, while the tumor samples showed an elevated caspase-3 activity, suggesting an enhanced apoptosis due to δ-T3 intervention (NCT00985777). Furthermore, another clinical study investigating the new formulation of γ-δ-T3 showed an enhanced bioavailability in healthy subjects (NCT01571921), undoubtedly heralding a great milestone for accelerating its application for cancer therapy. Of note, a number of clinical trials are currently in progress to study the anticancer effects of tocotrienols and/or in combination with other therapeutic agents (Table 3). Specifically, interventions of tocotrienols are involved as neoadjuvant and adjuvant cancer treatments as well as health supplements [129]. Taken together, these studies could provide a better understanding on clinical applications of tocotrienols with optimal benefits.

Conclusions
Tocotrienols exhibit versatility in inducing cell death by modulating various mechanisms in cancers. Tocotrienols exhibit potent proapoptotic capacities by inducing mitochondria-mediated apoptosis and ER-stress-mediated apoptosis; this co-elicitation is believed to serve as an enhanced cancer-killing strategy. In fact, engagement of both extrinsic and intrinsic apoptotic pathways by tocotrienols could lead to a more effective cancer elimination, particularly in Type II cells, which require mitochondrial pathway in inducing apoptosis. Apart from that, proper modulation of cell survival signaling underscores a pivotal role in shaping the ultimate cancer cellular demise. Of note, autophagy appears to account for a better role in cell death than its classical association in "cell survival" to serve as a backup mechanism in tocotrienols-induced apoptosis. Overall, the pleiotropic effects of tocotrienols in inducing various cell death mechanisms while hampering the prosurvival pathways of cancer cells portray a value-added action in fighting cancers. Yet, the oral bioavailability and metabolic degradation associated with high dose have constrained the clinical applications of tocotrienols. Perhaps the ongoing advancements in nanoformulation and combined treatment approaches could outshine the current cancer regimens using tocotrienols to achieve better therapeutic outcomes.