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An optimization experiment was performed to determine the route and the dose of fucoidan administration. A subcutaneous xenograft tumour was established in 14 female BALB/c nu/nu athymic mice (see Section 2.9.5) and fucoidan was delivered with different doses (high and low) and routes (IP and IV) once tumours was appeared. The best route and dose were selected for the rest of the experiments in chapters 5 and 6 of the thesis.

To examine the anti-tumour effects of fucoidan and its synergy with ATO, 28 mice (n=7/group) were randomly divided into four treatment groups; the control, fucoidan, ATO and fucoidan+ATO. A subcutaneous xenograft tumour was established by injecting NB4 cells in the right flank of mice (see Section 2.9.5). Treatment was commenced following tumour appearance at the following doses: 100 µ g/g body weight (b.w.) fucoidan, 2.5 µg/g b.w. ATO or in combination, daily. The control group was treated with IP injection of vehicle (sterile PBS). Mouse weight and tumour volume were measured daily. Once tumour volume reached 1000 mm3, this time point was considered as the end point and the mice were humanely euthanised with inhalation of CO2 (see Section 2.9.7). Tumour volume doubling time was calculated for each mouse.

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5.3 Results

5.3.1 Fucoidan Increases the Anti-proliferative Activity of ATO in APL Cells

in Vitro.

The acute promyelocytic leukaemia NB4 cell line was treated with fucoidan plus clinical and sub-clinical doses of ATO for 48 hours, and cell proliferation and apoptosis were evaluated using different assays.

NB4 cell proliferation was significantly decreased when cells were co-treated with fucoidan and ATO, compared to single treatment with either agents (Figures 5.1, 5.2). The decreased proliferation was observed in both low (0.25, 0.5 µM) and clinical doses of ATO (1 µM). At lowest dose of 0.25 µM ATO, combination of 10 µg/mL fucoidan with ATO resulted in decreased cell proliferation to approximately 80%. Whereas combination of 20 µg/mL of fucoidan with ATO reduced the cell proliferation to near 60%. According to the results obtained, fucoidan at the concentration of 20 µg/mL was used for the rest of the apoptosis experiments.

In the clinic, ATO is only used for treatment of acute promyelocytic leukaemia and not for other sub-types of AML as it is not effective in treatment of other subtypes (286). To examine whether the ATO activity observed was specific to APL cells, the non-APL t(8;21) positive AML Kasumi-1 cell line was similarly treated with ATO plus fucoidan. Neither combined fucoidan+ATO nor single treatment with fucoidan and ATO decreased the proliferation of Kasumi-1 cells and the percentage of viable cells in all groups remained at >80% for up to 96 hours (Figure 5.3).

To investigate whether the reduced cell proliferation in NB4 cells was due to cell death or cell cycle arrest, the DNA content of the NB4 cells treated with fucoidan+ATO was analysed using flow cytometry. The amount of the sub-G0/G1 population, which represents dead cells, significantly increased when cells were treated with fucoidan+ATO compared to ATO alone at both therapeutic and sub-therapeutic doses (p≤0.001) (Figure 5.4). Table 5.1 displays distribution of all cell cycle phases in NB4 cells.

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Figure 5.1. Inhibitory effects of ATO plus 10 µg/mL fucoidan on APL cell proliferation. NB4 cells were treated with low to high doses of ATO with or without fucoidan for 48 hours and cell proliferation was evaluated using the WST-8 assay. Mean ± SEM of at least three replicates is shown. Statistical significance was determined by two-way ANOVA, followed

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Figure 5.2. Inhibitory effects of ATO plus 20 µg/mL fucoidan on APL cell proliferation. NB4 cells were treated with low to high doses of ATO with or without fucoidan for 48 hours, and cell proliferation was evaluated using the WST-8 assay. Mean ± SEM of at least three replicates is shown. Statistical significance was determined by two-way ANOVA, followed

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Figure 5.3. Inhibitory effects of ATO combined with fucoidan on non-APL Kasumi-1 cell proliferation. Kasumi-1 cells were treated with low and high doses of ATO with or without 20 µg/mL fucoidan, and cell proliferation was evaluated using WST-8 assay after 96

hours. Mean ± SEM of at least three replicates is shown. Statistical significance was determined by ANOVA, followed by Tukey’s multiple comparison test.

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Table 5.1. Cell cycle phase distribution. NB4 cells were treated with various doses of ATO with or without fucoidan (20 µg/mL), and DNA content was assessed using flow cytometry

after 48 hours. Data represents mean ± SEM of at least three replicates.

Sub G0/G1 G0/G1 S Mitosis ATO 0.25 µM Control 1.93 ± 0.30 42.46 ± 1.39 22.34 ± 0.06 31.67 ± 1.34 Fucoidan 18.34 ± 1.75 28.66 ± 1.42 22.38 ± 2.70 29.18 ± 3.38 ATO 2.73 ± 1.08 45.10 ± 0.54 20.22 ± 0.41 30.39 ± 0.87 Combination 20.69 ± 1.54 31.30 ± 0.09 25.8 ± 2.68 19.44 ± 2.04 ATO 0.5 µM Control 1.93 ± 0.30 42.46 ± 1.39 22.34 ± 0.06 31.67 ± 1.34 Fucoidan 18.34 ± 1.75 28.66 ± 1.42 22.38 ± 2.70 29.18 ± 3.38 ATO 2.39 ± 1.08 46.10 ± 0.54 20.22 ± 0.41 29.39 ± 0.87 Combination 25.57 ± 0.86 38.42 ± 1.38 15.20 ± 0.34 18.14 ± 1.66 ATO 1.0 µM Control 1.93 ± 0.30 42.46 ± 1.39 22.34 ± 0.06 31.67 ± 1.34 Fucoidan 18.34 ± 1.75 28.66 ± 1.42 22.38 ± 2.70 29.18 ± 3.38 ATO 15.13 ± 0.40 39.81 ± 3.82 16.56 ± 0.80 25.50 ± 2.11 Combination 74.51 ± 2.26 7.29 ± 1.36 9.28 ± 2.28 6.76 ± 0.70

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Figure 5.4. Effects of ATO plus fucoidan on accumulation of sub G0/G1 dead cells. NB4 cells were treated with various doses of ATO with or without fucoidan (20 µg/mL) and cell

cycle was analysed after 48 hours. Sub G0/G1 population representing the dead cells significantly increased when fucoidan was combined with ATO at both clinical and low

doses. Mean ± SEM of at least three replicates is shown. Statistical significance was determined by two-way ANOVA, followed by Bonferroni post-test.

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5.3.2 Fucoidan Synergises with ATO in ATO-Mediated Apoptosis in APL

Cells.

To determine if cell death was due to apoptosis, the annexin V/PI assay was employed. Figure 5.5 shows a representative flow cytometry result for each treatment. As shown, combination of fucoidan with ATO significantly enhanced apoptosis in NB4 cells. At low doses of ATO, the percentage of apoptotic cells (annexin V positive cells) increased from approximately 6% (0.25 µM ATO only) to 32% (fucoidan+0.25 µM ATO) and from 5% (0.5 µM ATO only) to 52% (fucoidan+0.5 µM ATO) (p≤0.001). At the therapeutic dose of 1 µM ATO, the percentage of apoptotic cells increased from approximately 18% (ATO only) to 71% (fucoidan+ATO) (p≤0.001).

Furthermore, the TUNEL assay was employed to measure the amount of DNA fragmentation; which is one of the main features of apoptosis. Consistent with the annexin V/PI assay data, co-treatment of fucoidan with ATO significantly enhanced apoptosis compared to treatment with ATO only (p≤0.001). As shown in Figure 5.6, the mean percentage of cells with fragmented DNA increased to 42.5% (fucoidan+0.25 µM ATO), 60% (fucoidan+0.5 µM ATO), and 79% (fucoidan+1 µM ATO) compared to 1.5%, 1% and 12%, respectively, for the same doses of ATO alone.

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Figure 5.5. Representative annexin V/PI apoptosis assay. NB4 cells were treated with increasing doses of ATO with or without fucoidan (20 µg/mL) and apoptosis was measured

using annexin V/PI apoptosis assay. Results represent flow cytometry histograms of one of three replicates.

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Figure 5.6. DNA fragmentation. The percentage of apoptotic cells with fragmented DNA significantly increased when fucoidan (20 µg/mL) was combined with ATO at both low and

high doses. Mean ± SEM of at least three replicates is shown. Statistical significance was determined by two-way ANOVA, followed by Bonferroni post-test (***: p ≤ 0.001).

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