Evaluation of the potential therapeutic effects of curcumin and lycopene in adult malignant brain tumour cells in vitro

Doctoral thesis English OPEN
Abdullah Thani, Noor Azela

Glioblastoma multiforme are the most challenging of cancers to treat and even with recent advances in therapeutic approaches, the prognosis following diagnosis remains poor. Novel therapeutic approaches able to target such tumours without resulting in significant toxicity are needed. Micronutrients have been shown to exert potential therapeutic effects in cell culture and animal models which include anti-cancer activity and other functional properties such as anti-microbial and anti-oxidative effects. The aim of this research was to investigate the anticancer properties of curcumin (77% pure, derived from the ground rhizome of turmeric) and LycoRed powder (containing 10% lycopene from tomato extract) on four primary brain tumour-biopsy derived cell cultures (glioblastoma multiforme malignant cells) using a normal brain cell line as control (CC2565; passage 10 and above).\ud \ud The morphological appearance of the normal brain cell line and glioblastoma cell cultures was compared under phase contrast microscopy. The cytotoxicity of curcumin and Lycored was determined using a DRAQ 7 cell viability assay. Expression of antigens linked to invasive, angiogenic and apoptotic potential was studied using immunocytochemistry and flow cytometry. Induction of apoptosis was investigated using flow cytometry via Annexin-V staining. Anti-invasive and anti-angiogenic potentials were studied using a FluoroBlok invasion and angiogenesis assay (co-culture method) in vitro, respectively.\ud \ud Brain tumour-biopsy derived cell cultures incubated for 24h with increasing concentrations of curcumin showed significant decreased in cell viability (IC75,50,25 values 14.2 to 19.5μg/ml). LycoRed was not found to affect the cell viability of tumour cells ad no IC values could be established; only curcumin was therefore investigated in subsequent assays (using IC75 concentration). Neither curcumin nor LycoRed showed toxicity to normal brain cells.\ud \ud No significant induction of apoptosis by curcumin was observed in the primary brain tumour cells studied. However, three of the primary brain tumour cell cultures investigated were high in invasive capacity and curcumin treatment at IC75 concentration reduced the percentage of invasion after only 24 hours of incubation. In addition, curcumin was found to have antiangiogenic properties in vitro. In the normal brain cell culture, each parameter (number of nodes, junctions and branches) was maintained when treated with increasing concentrations of curcumin (0 to 40μg/ml). When all glioblastoma cell cultures and normal brain cells showed strong positivity for GFAP confirming that they are astrocytic in origin, expression of other antigens studied (Beta-1-integrin, CD44, VEGF, MMP-14, NG2, GD3) varied between the primary brain tumour cell cultures. This suggests that the invasive or angiogenic capacity of the gliobalstoma cell cultures investigated may not be attributable to one common factor as has been reported before, but perhaps an integration of many factors with overlapping functions.\ud \ud In conclusion, the results indicate that curcumin may have anti cancer properties through inhibition of invasion and angiogenesis in these malignant glioblastomas. Further studies are necessary to establish whether LycoRed possesses therapeutic potential.
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    ........................................................................................................................................................... ..15 Figure 4: Chemical structures of curcumininoids; A) Curcumin, B) Demethoxycurcumin C) Bisdemethoxycurcumin and D) Cyclocurcumin

    ............................................................................................................................................................. 33 Figure 6: Metabolic pathways of curcumin in rodents and in vitro culture (rat and human hepatocytes). Taken from: Marczylo et al., 2007

    ............................................................................................................................................................. 39 Figure 13: A histogram shows a negative control in the flow cytometry analysis in which the primary antibody was omitted leaving only secondary antibody (with biotin conjugated) being incubated. M2 will represent the positive stained cells while M1 is negative in staining.

    ............................................................................................................................................................. 68 Figure 14: A quadrant in the Annexin V apoptosis. Each region in the quadrant i.e. lower right, lower left, upper right and upper left represents the population of cells. Axis with FL3-H indicates PI and axis with FL4-H indicates Annexin V.

    ............................................................................................................................................................. 71 Figure 15: The tubule network traced with Paint software (A and B) before analysed using Image J software (C). With the image J software, each colour represents a parameter measured in the angiogenesis. The four parameters are total length of the tubules, node and junction (red in blue circle) and branches (green)

    ............................................................................................................................................................. 74 Figure 16: Phase contrast micrographs of the normal brain cell line; A) CC2565-passage 10 and primary brain tumour cell cultures (GB biopsy-derived); (B) MUBS-passage 6 (C) MUBP-passage 9 (D) MUTC-passage 8 and (E) MUP-passage 8. The different morphology of cells was shown by the colour coded arrows which include spindle (red), small star (green), large star (blue) and broad, straight and blunt end (purple) in shapes. All cell cultures were viewed and compared under phase contrast microscopy (20X magnification)

    ............................................................................................................................................................. 77 Figure 21: Antigen expression of CC2565 cell culture following staining with different antibodies via immunocytochemistry. A) GFAP B) GD3 C) NG2 D) CD44 E) Beta-1 integrin. F) Negative control.

    ............................................................................................................................................................. 92 Figure 22: Antigen expression of MUBS cell culture following staining with different antibodies via immunocytochemistry. A) GFAP B) GD3 C) NG2 D) CD44 E) Beta-1 integrin. F) Negative control.

    ............................................................................................................................................................. 92 Figure 32: Four representative diagrams from Annexin V apoptosis assay of MUBP cells at different hour of incubation (following curcumin treatment); 24h (upper left box), 48h (upper right box), 72h (lower left box) and 96h (lower right box). In a diagram, each quadrant represents viable cells (lower left), early apoptosis cells (lower right), late apoptotic cells (upper right) and necrotic cells (upper left). Percentage of cell gated in the quadrant is made by CellQuest software ........................................................................................................................................................... 111 Figure 33: MUBP cells population from each quadrant (viable, early apoptotic, late apoptotic, necrotic) of every incubation time (following curcumin treatment) are represented in a bar graph.

    Error bars show standard error of mean. Data represents the mean ±standard error of mean for n=3 experiments with each experiment being carried out in duplicate. Percentage of cell gated was followed by statistical analysis (one way ANOVA-Fisher comparison) as to obtain the p value (*p value<0.05) and tabulated as below.

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