Understanding the proliferative and self-renewal potential of different leukaemic stem cell populations

PhD ThesisAlthough contemporary treatment cures most children with ALL, with survival rates in the region of 90%, treatment outcomes in certain cytogenetic subgroups remain poor. Patients with such abnormalities have a greatly increased risk of relapse and re-treatment responses are poor. Understanding the mechanisms of resistance in these patients is therefore a priority in the search for improvements in chemotherapy. The translocation t(17;19)(q22;p13) is a rare cytogenetic abnormality, which occurs in less than 1% of childhood ALL and is associated with a very poor prognosis and chemotherapy resistance. The translocation results in the fusion of E2A on chromosome 17 with HLF on chromosome 19. It has been commonly assumed that, following relapse, ALL cells will divide more rapidly than at presentation. To test this hypothesis, competitive transplantation studies were performed using paired presentation and relapse samples from a case of t(17;19)-positive ALL in a NSG xenograft mouse model. Surprisingly mice engrafted with the presentation cells survived significantly shorter (p<0.05, logrank test) than those with relapse cells, indicating the aggressiveness and rapid proliferation of presentation cells, while the survival curves of mice engrafted with various proportions of presentation and relapse cells had intermediate levels of survival. Administration of dexamethasone prolonged the survival of mice engrafted with the presentation cells and those with various proportions of cells, with no effect on survival of mice with relapse cells. Flow cytometry analysis showed that a high percentage (70%-95%) of human leukaemic cells engrafted in bone marrow and spleen across all groups tested. RT-PCR amplification identified this t(17;19) case to be a Type 1 E2A/HLF fusion. In order to determine the genes responsible for chemo-insensitivity, whole genome SNP array analysis was performed on the matched presentation and relapse samples which demonstrated deletion of glucocorticoid receptor (NR3C1) gene in relapse cells which is not seen in presentation cells, confirmed by fluorescence in situ hybridisation. This deletion was used to identify the relapse cells (R) in mice engrafted with mixtures of presentation and relapse cells. FISH analysis showed the percentages of presentation cells (P) were higher than the ratios of the cell initially transplanted. In concordance, real-time PCR analysis showed high levels of NR3C1 in all mix cells ratios. These results indicated that presentation cells proliferate more rapidly and outgrow relapse cells in competitive clonal repopulation experiments. Dexamethasone treatment reduced the percentages of presentation cells in all mix populations with high significance (p<0.01, t-test) in the 30%-P+70%-R ratio. Levels of NR3C1 in all mix cell populations were significantly depleted (p<0.05, t-test) by dexamethasone. To be able to physically track the proliferation of leukemic cells populations in vivo and to follow progression of the disease, two lentiviruses pSLIEW (expressing firefly luciferase and green fluorescent protein) and pSRLICW (expressing Renilla luciferase and mCherry fluorescent protein) were generated. pSRLICW was just successfully generated but has not yet been tested. Lentivirus pSLIEW functionality was validated by transducing various leukaemic cell lines. By assessing the percentage of green fluorescence (gfp) cells, transduction efficiencies in SEM and RS4;11 (ALL cell lines) were 45% and 5% respectively, whereas transduction efficiencies in Kasumi-1 and SKNO-1 (AML cell lines) were 72% and 98% respectively, higher than the ALL cell lines. Transduced SEM and Kasumi-1 lines were sorted for higher gfp populations for xenograft purposes. Real time bioluminescence imaging on mice xenografted with sorted transduced SEM cells showed rapid progression of the disease in the systemic while Kasumi-1 cells xenografted in mice produced localised tumours and progressed much slower. These lentiviral-transduced cell lines xenografts have proven the in vivo monitoring capability by real time luminescence imaging. The information gained from this project study provides novel insights into the mechanisms of relapse in childhood ALL. The growth characteristics of the leukaemic blasts should be considered in assigning patients to different therapeutic options.The National University of Malaysia and Malaysian Ministry of Higher Educatio

Effects of epigallocatechin-3-gallate (EGCG) on cell cycle distribution and DNA integrity of K562 cells, a human chronic myeloid leukemia

Epigallocatechin-3-gallate (EGCG) is a naturally derived compound from green tea with high antioxidant activity and various anti-cancer properties. EGCG has been widely investigated worldwide. However, effects of EGCG on cell cycle of K562 have not been clearly stated elsewhere. This study was conducted with the aim to investigate the antiproliferative effect of EGCG on K562 human leukemic cells and its underlying mechanism of action on the cells. MTT assay was conducted to determine cytotoxicity effect of EGCG on the K562 cells. Meanwhile, cell cycle analysis and DNA damage on the cells were determined by Flow cytometry and Comet assay respectively. K562 cells were treated with EGCG at concentrations ranging from 0 to 100μg/ml for 48 hours. The results showed that EGCG effectively decreased the percentage of cell viability in a dose dependent manner. The IC10, IC25 and IC50 of EGCG on K562 cell lines were 5 ± 2.44 μg/mL, 10 ± 5.93 μg/mL and 50 ± 1.93 μg/mL, respectively. In cell cycle assay, EGCG has shown no significant effect (p>0.05) on the cell cycle of K562 cell line as compared to negative control, whereas Imatinib mesylate as the positive control showed cell cycle arrest at S phase in this cell line. Hence, EGCG can be verified as a non-cell cycle specific compound. In addition, EGCG was found to cause a significant increase (p<0.05) in tail moment value and percentage of DNA tail in K562 cell line, suggesting DNA damage as an early signal of EGCG induced cell cytotoxicity. In conclusion, by decreasing the cell viability and inducing DNA damage, EGCG showed promising potential as an alternative treatment for leukemia through non-cell cycle specific pathway and further investigation on other mechanisms of action of EGCG on the cells is recommended

Kesan suplementasi tokotrienol ke atas perubahan hormon dan katekolamin otak tikus aruhan stres

Stres merupakan sebarang gangguan, cabaran atau ancaman kepada seseorang individu yang merangsang perubahan respon fizikal, mental dan emosi. Hormon stres dan katekolamin dirembeskan di dalam otak sebagai respon terhadap stres. Kajian ini mengenal pasti kesan tokotrienol tulen dan vitamin E sawit ke atas perubahan hormon stres dan katekolamin pada tikus yang diaruhkan stres imobilisasi rendaman air. Sebanyak 32 ekor tikus Wistar jantan telah dibahagikan secara rawak kepada empat kumpulan kajian. Dua kumpulan kawalan; kumpulan tanpa stres (NS) dan kumpulan tanpa stres (CS) serta dua kumpulan rawatan yang diberikan tokotrienol (TTS) atau vitamin E sawit (TFS) secara oral paksaan pada dos 60 mg/kg berat badan selama 28 hari. Setelah tamat tempoh rawatan, tikus daripada kumpulan CS, TTS dan TFS telah didedahkan kepada stres imobilisasi rendaman air selama tiga setengan jam. Kemudian, otak tikus diambil untuk pengukuran hormon pelepasan kotikotrofin (CRH), hormon adenokortikotropik (ACTH), norepinefrin, dopamin dan serotonin. Keputusan kajian mendapati peningkatan kandungan CRH, ACTH, norepinefrin dan dopamine di dalam otak tikus yang diaruh stres berbanding kumpulan kawalan tanpa stres. Kandungan serotonin pula menurun akibat stres. Walau bagaimanapun, tikus yang diberi suplementasi tokotrienol dan vitamin E sawit menunjukkan penurunan signifikan kandungan CRH, ACTH, norepinefrin dan dopamin serta peningkatan kandungan dan serotonin sehingga hampir ke aras normal. Tiada perbezaan antara pemberian tokotrienol dan vitamin E sawit. Sebagai kesimpulan, tokotrienol dan vitamin E sawit berkesan dalam mengawal perubahan hormon stres serta katekolamin pada otak tikus yang teraruh stres

The Role of Hibiscus sabdariffa

Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0–1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P<0.05) of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1+ cells (HSCs) at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P<0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs

Medicinal plants with anti-leukemic effects: A review

Leukemia is a leukocyte cancer that is characterized by anarchic growth of immature immune cells in the bone marrow, blood and spleen. There are many forms of leukemia, and the best course of therapy and the chance of a patient’s survival depend on the type of leukemic disease. Different forms of drugs have been used to treat leukemia. Due to the adverse effects associated with such therapies and drug resistance, the search for safer and more effective drugs remains one of the most challenging areas of research. Thus, new therapeutic approaches are important to improving outcomes. Almost half of the drugs utilized nowadays in treating cancer are from natural products and their derivatives. Medicinal plants have proven to be an effective natural source of anti-leukemic drugs. The cytotoxicity and the mechanisms underlying the toxicity of these plants to leukemic cells and their isolated compounds were investigated. Effort has been made throughout this comprehensive review to highlight the recent developments and milestones achieved in leukemia therapies using plant-derived compounds and the crude extracts from various medicinal plants. Furthermore, the mechanisms of action of these plants are discussed

Medicinal Plants with Anti-Leukemic Effects: A Review

Leukemia is a leukocyte cancer that is characterized by anarchic growth of immature immune cells in the bone marrow, blood and spleen. There are many forms of leukemia, and the best course of therapy and the chance of a patient’s survival depend on the type of leukemic disease. Different forms of drugs have been used to treat leukemia. Due to the adverse effects associated with such therapies and drug resistance, the search for safer and more effective drugs remains one of the most challenging areas of research. Thus, new therapeutic approaches are important to improving outcomes. Almost half of the drugs utilized nowadays in treating cancer are from natural products and their derivatives. Medicinal plants have proven to be an effective natural source of anti-leukemic drugs. The cytotoxicity and the mechanisms underlying the toxicity of these plants to leukemic cells and their isolated compounds were investigated. Effort has been made throughout this comprehensive review to highlight the recent developments and milestones achieved in leukemia therapies using plant-derived compounds and the crude extracts from various medicinal plants. Furthermore, the mechanisms of action of these plants are discussed