**7.1 Tocotrienols in breast cancer**

Tocotrienols were found to possess anti-proliferative and pro-apoptotic activities in breast cancer cells. Tocotrienols had particularly been shown to reduce the levels of cyclin D1 and Cyclin-Dependent Kinases (CDK) 2, 4 and 6 and to enhance the expression of CDK inhibitors [24]. Tocotrienols are also implicated in the suppression of the PI3K/Akt/mTOR and the Ras/Raf/MEK/ERK signalling pathways and to decrease c-Myc levels [70]. Tocotrienols were able to induce intrinsic apoptosis in breast cancer cells, accompanied by cytochrome *c* release, mitochondrial membrane depolarization, caspase activation, DNA fragmentation and poly(ADP-ribose) polymerase cleavage [72, 77]. In breast cancer cells, tocotrienols were also able to trigger the extrinsic apoptotic pathway by activation of caspase-8 [78].

Tocotrienols were also shown to exert potent anticarcinogenic effects in mammary carcinomas through reduction of proliferation by means of downregulation of the HMG-CoA reductase activity and inhibition of cholesterol synthesis, and also through the induction of oxidative stress-related mitochondrial apoptosis [68, 73].

The Human Epidermal Growth Factor Receptor 2 (HER-2) is a receptor tyrosine-protein kinase that induces breast cell proliferation. It is encoded by the erbB-2 oncogene. Overexpression of this oncogene is found in 30% of all breast tumours, thus representing an important biomarker and target for breast cancer management [118]. A previous study had shown that HER-2 receptors and tocotrienols specifically accumulate in breast cancer cell lipid raft microdomains. Furthermore, it had been discovered that tocotrienols markedly transform the composition of the lipid rafts, with subsequent disruption of their integrity, and the consequent inactivation of the associated HER-2 receptors and the downstream signalling pathways [67].


#### *Palm Oil Tocotrienols in Cancer Chemoprevention and Treatment DOI: http://dx.doi.org/10.5772/intechopen.98199*



*Abbreviations:* ↑*- Upregulation;* ↓*- Downregulation; AAF - 2-acetylaminofluorene; Bax - BCL2-Associated X; Bcl-2 - B- cell lymphoma 2; CDK - cyclin-dependent kinase; DEN - Diethylnitrosamine; DHC - Dihydroceramide; DHS - Dihydrosphingosine; DMBA - 7,12 Dimethylbenz(a)anthracene; DR - death-receptor; EGFR - epidermal growth factor receptor; ER - Endoplasmic reticulum; ER-*β *-Estrogen receptor beta; ErbB- erb-b2 receptor tyrosine kinase; ERK - extracellular signal–regulated kinase; GGT-gamma-glutamyltranspeptidase; GSSG-Rx-glutathione reductase; GSH-Px-glutathione peroxidase; GST- glutathione S-transferase; HER2 - Human epidermal growth factor receptor 2; HIF-Hypoxia- inducible factor; HMGR - HMG-CoA reductase; IL - Interleukin; JNK - Jun amino-terminal kinases; KEAP - Kelch Like ECH Associated Protein; MAPK - Mitogen-activated protein kinases; MMP - Matrix metalloproteinases; mTOR - mechanistic target of rapamycin; NF-*κ*B - nuclear factor kappa-lightchain-enhancer of activated B cells; NQO2-NAD(P)H dehydrogenase quinone 2; pAkt - phosphorylated Akt; PARP - Poly (ADP-ribose) polymerase; PDK - 3-phosphoinositide dependent protein kinase; PI3K-Phosphatidylinositol-4,5-bisphosphate 3-kinase; Ras - rat sarcoma virus; pRb- phosphorylated retinoblastoma protein; SREBP-2 -sterolregulatory-element-binding protein isoform 2; STAT3 - Signal transducer and activator of transcription 3; TGF-*β *- Transforming growth factor beta; TG - triglyceride; UDP-glucuronyltransferase; VEGF-Vascular endothelial growth factor; VPS - Vacuolar protein sorting-associated protein. a Alpha-tocotrienol. b beta-tocotrienol.*

*c gamma-tocotrienol.*

*d delta-tocotrienol.*

*e Tocotrienol-rich fraction (TRF).*

*f Rice bran tocotrienol.*

*g 6-O-carboxypropyl-alpha-tocotrienol.*

*h Oxazine derivatives of gamma- & delta-tocotrienol.*

*i BALB/c mice.*

*j athymic nude mice.* *k Sprague–Dawley rats. l xenografts mice. mOrthotopic mice. n C3H/HeN mice. o ddY mice. p Rattus norvegicus. q Mutant mice.*

#### **Table 1.**

*The Summary of Tocotrienols Mechanism of Action Against Different Cancers.*

Almost 70% of human breast cancers are oestrogen-dependent and oestrogen receptor-positive. Tocotrienols have been postulated to facilitate the nuclear translocation of the anti-proliferative Oestrogen Receptor (ER) β and to decrease the tumorigenic ERα expression in breast cancer cells [74]. Additionally, a previous study had suggested that the delta isomers of the tocotrienols were able to induce cytotoxic effects in breast cancer cells independently of their ER status [68].

In ERβ positive breast cancer cells, tocotrienols potentially activated the ERβ signalling pathway and increased the expression of the estrogen-responsive genes such as MIC-1. This will subsequently trigger the caspase-dependent apoptosis pathway [75]. Past research has also shown that tocotrienols induce apoptosis in breast cancer cells by increasing endoplasmic reticulum (ER) stress and autophagy [69, 119]. Moreover, tocotrienols also induced apoptosis *in vitro* by inhibiting NF-κB, PI3K/Akt/mTOR, and subsequently downregulate FLIP, increase Bax/Bcl-2 ratio and PARP cleavage [63, 72].

Another finding revealed that the apoptotic property of gamma-tocotrienols in breast cancer cells is mediated by *de novo* ceramide synthesis-dependent activation of JNK/CHOP/DR5 signalling. The anti-angiogenic effect of delta-tocotrienol against breast cancer was also mediated by its ability to reduce the synthesis of pro-angiogenic markers such as IL-8 and VEGF [63]. Apart from these findings from cellular experiments, a number of studies performed in animals have also confirmed the anti-cancer activities of tocotrienols against breast cancer. For example, it has been strongly suggested that tocotrienols increased tumour latency and reduced the tumour incidence and number in 7,12 dimethylbenz(a)anthracene-induced breast cell carcinomas in rats [120]. A recent study had suggested that gamma-tocotrienol could potentially acts as proteasomes inhibitor which was able to conquer the deficiencies in growth-inhibitory or pro-apoptotic molecules in breast cancer cells. The inhibition of proteasome proteins was postulated to induce apoptosis in breast cancer cells [121].

Apart from the various tocotrienol isoforms, other formulations of tocotrienols have also been shown to be effective in the treatment and prevention of breast cancer. This is exemplified by tocotrienol oxazine derivatives, which is effective in suppressing breast carcinoma progression by inactivating the pAkt, NF-κB, COX-2, cyclin D1, CDK-2, −4, −6 pathways, and also by amplifying the levels of cell-cycle arrest proteins (p21 and p27) [71].

A relatively new formulation of tocotrienols i.e., the annatto tocotrienols (comprising 90% delta-tocotrienol and 10% gamma-tocotrienol), which is derived from the annatto fruit, has been found to suppress the progression of breast carcinoma by accelerating the process of apoptosis and senescent-like growth arrest in HER-2/ neu transgenic mice [122].

The tocotrienol rich fraction derived from palm oil (TRF) has also been shown to be effective in experimental breast cancer management. Treatment of MCF7 injected athymic mice with tocotrienol-rich fraction (TRF) resulted in significant downregulation of c-Myc and CD59 glycoprotein precursor gene involved in the immune system, which positively contributed towards its anticarcinogenic effects [76]. Another tocotrienol product derived from palm oil i.e., tocomin, which

#### *Palm Oil Tocotrienols in Cancer Chemoprevention and Treatment DOI: http://dx.doi.org/10.5772/intechopen.98199*

consists of a mixture of naturally occurring tocotrienols and tocopherols, induced apoptosis in breast cancer cells [119].

A small-scale clinical trial was conducted in female patients with early breast cancer in order to investigate the efficacy of tocotrienols administration as a potential adjuvant with tamoxifen [123]. These patients were diagnosed with either stage I or II oestrogen receptor-positive breast cancer, and were divided into two groups. The treatment group was administered 400 mg/day tocotrienol-rich fraction (TRF) derived from palm oil, in addition to conventional tamoxifen chemotherapy. The control group was administered a placebo, together with tamoxifen. The result of the experiment indicated that the 5-year breast cancer-specific survival was 98.3% in the treatment group and 95% in the control group, while the 5-year disease-free survival was 86.7% and 83.3%, respectively. The mortality risk was 60% lower in the TRF group versus controls, however, it was not statistically significant. This was probably due to the small sample size of the experiment. Accordingly, these studies comprehensively summarize the possible benefits of tocotrienols (in particular the TRF) in the prevention and management of breast cancer.
