Methadone Hydrochloride Induced Apoptosis On Leukaemia Cell Lines
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Date
2018-08
Authors
Kua Vee May Dianne
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Leukaemia is the most common cancer diagnosed in children. It is a type of blood cancer where abnormal blood cells are produced in the bone marrow. Methadone hydrochloride is a synthetic drug that is commonly used in the maintenance treatment for drug addiction. The objective of this research is to determine the cytotoxic activity and apoptotic effects of methadone hydrochloride treatment towards two leukaemia cell lines which are CCRF-CEM and HL-60. CCRF-CEM and HL-60 cells were treated with methadone hydrochloride for 24 and 48 hours to determine the cytotoxic activity. IC50 at 24 hours obtained for CCRF-CEM is 37.63 μg/mL while IC50 for HL-60 cells is 30.07 μg/mL. IC50 obtained for 48 hours of methadone hydrochloride treatment for CCRF-CEM and HL-60 leukaemia cells are 29.21 μg/mL and 27.17 μg/mL, respectively. The cytotoxicity results showed that IC50 values for both cell lines exceeded the standard required for cytotoxic activity of pure compound (≤10 μg/mL) for the compound to be considered active. Results obtained from DNA fragmentation assay showed no characteristic DNA ladder pattern was observed in CCRF-CEM leukaemia cells treated with methadone hydrochloride. However, characteristics DNA ladder pattern was observed in the methadone hydrochloride-treated HL-60 cells. Formation of comets was seen in methadone hydrochloride-treated CCRF-CEM and HL-60 cells with varying degrees of DNA damage. The comets formed by methadone hydrochloride-treated HL-60 cells were more obvious compared to methadone-treated CCRF-CEM cells. The expression of apoptosis-related proteins in methadone-treated CCRF-CEM and HL-60 cells were obtained by incubating the cell lysate with RayBio® Human Apoptosis Antibody Array. Up-regulation of caspase 8 expression and down-regulation of p53 and survivin expression were detected in the cell lysate of methadone hydrochloride treated CCRF-CEM cells when compared to the untreated control. The results obtained suggested that methadone hydrochloride induced apoptosis in CCRF-CEM cells through Type I extrinsic pathway of apoptosis. On the other hand, methadone hydrochloride induced apoptosis in HL-60 cells involved up-regulation of Bid and caspase 8 expressions and down-regulation of Bcl-2, p21, and survivin expression. The results showed that methadone hydrochloride induced apoptosis through Type II extrinsic pathway of apoptosis. Unfortunately, the last objective of this study which is to identify the specific binding protein (s) of methadone in the cell lysate of CCRF-CEM and HL-60 cells using photo cross-linked methadone affinity beads could not be achieved. There is no band of interest observed in the SDS-PAGE gel for methadone conjugated beads incubated with cell lysate of CCRF-CEM and HL-60 cells when compared to the control beads. In summary, this study has provided a better understanding on the effects of methadone hydrochloride treatment on CCRF-CEM and HL-60 cells in term of cell cytotoxicity, the degree of DNA fragmentation, and the expression level of apoptosis-related proteins.
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Keywords
Leukaemia Cell Lines