Theses (PHD)

Tocotrienols, isoforms of vitamin E, are not only known for their antioxidant, lipid-lowering properties and as anti-proliferating agents but also for their inhibitory effects on the growth of human breast cancer cells in vitro and in vivo. In this study, the effects of tocotrienol-rich fraction (TRF) from palm oil and its isomers (α, δ and γ-tocotrienol) were examined in 4T1 mouse mammary cancer cells. The 4T1 cells were cultured and grown in RPMI medium supplemented with different concentrations of tocotrienols. Cell numbers were determined at the end of twelve days of culture. Results showed that TRF and the individual fractions of palm tocotrienols inhibited the growth of 4T1 cells in vitro at lower concentrations (6-20 μg/ml) compared to tocopherols (> 20 μg/ml). Deltatocotrienol was found to be most inhibitory followed by γ-tocotrienol. Complete inhibition of 4T1 proliferation was achieved with 6 μg/ml δ-tocotrienol and 10 μg/ml γ-tocotrienol. The effects of TRF and tocotrienol isomers were investigated for proapoptotic activity by expression of FasL gene. This study showed that γ- and δ- tocotrienol (8 μg/ml) treated 4T1 cells expressed significantly (P < 0.01) higher levels of FasL compared to untreated control cells. The TRF showed pro-apoptotic effects at higher concentrations from 20 – 80 μg/ml). Tumourigenesis was examined and compared against control in both nude and BALB/c mice models. The mice were injected with MDA-MB-231 and 4T1 cells respectively for the different models, and were fed with TRF by oral gavage. Tumour incidence was reduced by 33% and 57% in nude mice and BALB/c mice respectively. In addition, the tumour load was significantly (P < 0.05) reduced by 72% and 93.6% respectively in tocotrienol-supplemented nude and BALB/c mice, when compared to the vehicle-fed control group. This study shows that palm tocotrienols have strong inhibitory effects on the growth of both MDA-MB-231 and 4T1 cells both in vitro and in vivo. The immune modulatory effects of tocotrienols were investigated and it was found that TRF enhanced production of NK cells (P < 0.05) as well as IFN-γ (P < 0.05), which in turn regulate the immune protection against cancer cells. These observations were recorded in both mice models. In this study, adipose tissue from control (vehicle-fed) and experimental (TRF-fed) animals were analysed for the presence of tocopherols and tocotrienols using HPLC. All the tocotrienol isomers increased significantly (P < 0.05) in the TRF supplemented mice compared to control whilst only α-T and γ-T showed significant increase in the supplemented nude mice and BALB/c mice. The anti-angiogenic activity of TRF and two tocotrienol isomers (γ and δ) were investigated using the human umbilical vein endothelial cells (HUVEC). Results showed that the level of Interleukin-8 (IL-8), an angiogenic activator was reduced in the HUVEC treated with TRF (P < 0.05) and the two tocotrienol isomers compared to α-tocopherol and control cells. The level of IL-8 was lowest in the δ-tocotrienol treated cells followed by γ-tocotrienol and TRF. In addition, TRF also down-regulated the expression of various angiogenic activators such as VEGF-C, KDR and FLT1 in tumours from mice fed with TRF as compared to control mice. Moreover, immunohistochemical expression of pro-angiogenic such as VEGF, VEGF-R2 and CD31 were found to be reduced in tumour and lung tissues of mice supplemented with TRF. This study shows that tocotrienols exhibited anti-angiogenic activity, which may play a key role in inhibiting the growth and metastasis of tumour. Microarray analysis was carried out on 4T1 cells treated with TRF, δ- tocotrienol, α-tocopherol and differential gene expression compared to untreated control cells were investigated. The outcome from this study revealed the identity of several interesting genes including Interleukin-24 (IL-24), death-associated protein 3 (Dap3), rat sarcoma oncogene (Rras) and platelet-derived growth factor- B (Pdgfb) that possess important functional role in cancer mechanisms. In this study, the microarray analysis was further subjected to investigate the effect of TRF on regulation of genes from tumour tissues of mice that were supplemented with TRF compared to that of control mice. This analysis again revealed the up-regulation of IL-24 in vivo with TRF-supplementation. In this study, we also found that tocotrienols up-regulated (P < 0.05) the expression of a novel-cytokine, IL-24 in the tumour and 4T1 cells. The IL-24 cytokine has been reported to play a significant role in tumour regression. In addition, while tocotrienols up-regulated the expression of IL-24, they also downregulated pro-angiogenic markers; VEGF and IL-8 at the same time. Studies have previously shown that IL-24 possess anti-angiogenic properties. Thus, the tumour protective mechanism exhibited by tocotrienols may be explained via the IL-24 mechanistic pathway.

Carrageenans, the polysaccharide from red algae, especially of the genera Chondrus, Eucheuma and Gigartina, are widely used in food, medicine and as an excipient in cosmetics and skincare products. Carrageenans have been shown to have prospective photoprotective effect against UVB irradiation on immortalised normal human keratinocyte (HaCaT) cells. The primary aim of this study was to assess the potential of iota (ι) and kappa (κ)-carrageenans and their combined effect with vitamin E on UVB-induced extracellular matrix (ECM) and cytoskeletal changes in HaCaT cells, and their antiproliferative and apoptotic effects on epidermoid carcinoma (A431NS) cells. The ECM degradation was assessed through the biological mediators epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), activating protein-1 (AP-1), and matrix metalloproteinases (MMPs) liberated by the UVB-induced cells. The antiproliferative effects on A431NS cells was evaluated based on the cytotoxicity profile, direct antiproliferation effect, DNA synthesis, translationally-controlled tumour protein (TPT1) gene expression and changes in the cytoskeleton structure. Carrageenans did not exhibit cytotoxicity at dosages <200 μg/ml in HaCaT cells. Cells pre-treated with carrageenans exhibited significantly (p<0.05) higher cell viability compared to the cells without treatment after UVB irradiation. Results also showed that the levels of the biological mediators significantly (p<0.05) reduced in carrageenan pre-treated cells, suggesting that carrageenans have the potential in maintaining the skin’s integrity by reducing the damage to ECM upon UVB exposure. The cytoskeletal structure in cells pre-treated with carrageenans were also more intact than the untreated cells. In addition, carrageenans enhanced the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). The percentage of Rat Sarcoma (RAS)-Rapidly Accelerated Fibrosarcoma (RAF) gene mutation was also reduced in treated cells. When tested against epidermoid carcinoma (A431NS) cells, carrageenans exhibited cytotoxicity (CD50<100ug/ml) and antiproliferative effect in a concentration-dependent manner, inhibited DNA synthesis and downregulated the TPT1 gene expression. Also, the cytoskeleton structure was impaired after treatment. The presence of sulphur moieties, gelling effect, polysaccharide nature, vital minerals and ions is postulated to contribute to the photoprotective and anticancer potential of carrageenans. Taken together, the results suggest that carrageenans do possess photoprotective effects against UVB-induced toxicity and ECM damages, in addition to being a prospective anticancer agent. Apart from being just an excipient in cosmetics and skincare products, the presence of carrageenans potentially be of an added value as a photoprotective and anti-cancer agent if its photoprotective mechanism and anti-cancer potential can be established from this study.

Bisphenol A (BPA) is a compound used in the manufacturing of plastics as a hardening agent and is ubiquitous in the environment due to its widespread use. BPA is a known endocrine disruptor and evidence suggests that BPA may influence multiple endocrine-related pathways and increase oxidative stress by disturbing the prooxidant/antioxidant balance of cells. The aim of the present study was to evaluate the toxic effects of BPA in pubertal and prenatal exposed mice and if co- administration of a palm-derived tocotrienol rich fraction (TRF), an antioxidant, can prevent its deleterious effects. In the first study (pubertal exposure), mice (n = 6/group) were randomly assigned into one of four groups (Group I: control; Group II: TRF-fed, Group III: BPA-fed and Group IV: BPA+TRF fed) and orally dosed daily with 50 μg/kg body weight of BPA whilst the TRF mice received 1 mg/day of TRF, starting on post-natal day (PND) 32 until sacrifice (PND50 or PND100). In the second study (prenatal exposure), mice (F0) were randomly distributed into the same groupings (6 mice/group) as the pubertal mice. At time of breeding, the male and female mice in Groups II and IV were dosed daily with TRF at 1 mg/day, and the female pregnant mice were dosed until parturition. The F0 pregnant dams (Groups III and IV) were orally fed with BPA (50 μg/kg BW) once daily, between 1000 and 1200 hours, from gestation day 12 (GD12) till gestation day 20 (GD20). Numerous significant effects were observed in the exposed mice at adulthood including elevation in final body weight, an increase in the length of the oestrous cycle and extended oestrous cycle, simple hyperplasia of the endometrium glands, thickening of the basement membrane in the testis, increased ERα expression in the hypothalamus, an increase in oestradiol levels and a decrease in testosterone levels. There were also changes in the expression of some genes with BPA treatment, including a 2.5 fold increase in Zan and decreases in Scara5 and Cdkn1a genes. Additionally, alterations in non-reproductive behaviours of males and females including anxiety, social interaction, locomotion and activity were observed. The effects of BPA were differentially expressed in the males and females, depending on the type of exposure either pubertal or prenatal. Supplementation with TRF ameliorated the BPA-induced changes to the oestrus cycle, serum oestradiol levels, anxiety and hyperactivity. TRF supplementation also protected against the recorded pathological changes in the uterus and testis and reduced the accumulation of BPA in the liver. In conclusion, TRF can ameliorate the toxic effects of BPA by its cytotoxic and antioxidant mechanism indicating its potential use as a protective antioxidant.