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Goniothalamin as anti-leukemic agent in WEHI-3B myelomonocyte leukaemia animal model in vitro and in vivo
xiv,150 leaves : ill. (some col.) ; 30 cm.
Goniothalamin (GTN) is a styryl-lactone compound, which was extracted and isolated from Goniothalamus macrophyllus belonging to the Annonacae family. Goniothalamin was reported to be cytotoxic towards several tumor cell lines such as pancreas carcinoma, gastric carcinoma, breast carcinoma and leukemia cell lines. In this study, the in vitro cytotoxic activity toward Mouse myelomoncytic leukemia cell line (WEHI 3B) and in vivo anti leukemic activity of GTN were carried out. The Cytotoxic effects of fifty percent (IC50) for GTN-treated WEHI 3B cells were determined using microtetrazolium (MTT) assay; it was 1.7±O.2 ug/ml, The compound had no effect on the normal peripheral blood lymphocyte and normal mouse fibroblast cell line (3T3) at the same concentration used in the leukemia cell lines. The cell proliferation rate of GTN treated WEHI 3B leukemia cell line was reduced significantly in a dose and time dependent manner as compared to the untreated control. Treatment of WEHI 3B cells with IC50 and IC25 concentrations of GTN caused a reduction of the cell viability in the dose and time dependent manner. Moreover, MTT Proliferation assay showed that the growth rates for GTN-treated WEHI 3B cells decreased in the dose and time dependent manner. It was confirmed that the mode of cell death in response to GTN treatment on WEHI 3B cell line was by apoptosis. Morphological features of apoptosis were observed by the Phase Contrast Microscopy and Fluorescence Microscopy (Acridine Orange (AO) and Propidium Iodide (PI) staining). The features observed included chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and membrane blebbing without disintegration of the cellular membrane. These were further confirmed with DNA laddering in the agarose gel electrophoresis assay. However, analysis of the cellular DNA content using PI showed that GTN caused an increase in sub-Gl region (apoptosis peaks). Early apoptosis was observed by annexin V flow cytometry method after 6 hrs. The result also showed that GTN induced the DNA damage in WEHI-3B cells and it stimulated caspase 9 and there was no increase in caspase 3/7 and 8. Furthermore, the anti-leukemic activity of GTN was evaluated on myelomoncytic leukemia induced BALB/c mice. The mice groups treated with GTN showed a significant increase, in the survival rate as compared to the mice group without any treatment. Thus, such results indicated that GTN significantly decreased the weights of liver and spleen in the treated mice groups compared to the untreated mice group. The total white blood cell and percentage of blasts cell in the blood, bone marrow and spleen smears were significantly lower (p<O.05) in the treated mice groups than they were in the untreated group. Histopathological studies confirmed haematological results. Based on the results obtained in the present study, the treated mice groups showed similar results as vincristine, which is a commercial drug for leukaemia. In conclusion, Goniothalamin potent antitumor agent and the mode of cell death induced by GTN is apoptosis. Goniothalamin has the ability to kill WEHI 3B leukemia cells in vitro and in vivo.
1. Introduction -- 2. Literature review -- 3. Material and method -- 4. Results -- 5. Discussion -- 6. Conclusion and recommendations
Thesis (Degree of Doctor of Philosophy) - Universiti Sultan Zainal Abidin, 2014
Includes bibliographical references (leaves 115-126)
Faculty of Bioresources and Food Industry