Cytotoxicity Effect and Morphological Changes of Chrysanthemum Morifolium Methanolic Extract against Chronic Myeloid Leukaemia K-562 Cell Line

Chrysanthemum morifolium, also known as “Bunga kekwa” in Malaysia, has various benefits and widely used in Chinese herbal medicines. The plant extract was reported to have significant biological activities, such as anti-inflammation, anti-tumour, anti-oxidant, and anti-cancer. Nonetheless, its anti-cancer potential on chronic myeloid leukaemia has remained elusive. The main goal of this study is to evaluate the cytotoxic effect of C.morifolium buds and flowers in methanolic extracts on chronic myeloid leukaemia malignancy K-562 cell lines. The bud and flower of C.morifolium were macerated for 72 hours in 100% methanol then were concentrated under reduced pressure using a rotary evaporator and oven-dried to obtain crude extracts. K-562 cells were treated with six different concentrations 400, 200, 100, 50, 25, and 12.5 µg/ml and incubated for 24, 48 and 72 hours. The in vitro cytotoxic activity was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) test and was quantified using a microplate reader at 570 nm. Acridine orange and propidium iodide (AO/PI) staining were used to assess morphological alterations. MTT assays results showed moderate toxicity of both extracts. The lowest maximal half inhibitory concentration (IC50) value were observed at 72 hours of incubation; 182 ± 4.04 ug/ml for BM and 161 ± 7.88 ug/ml for flower extract (FM). However, there was a significantly different IC50 value (p<0.05) between the incubation periods of both treatments where the IC50 value at 24 hours was 301.33 ± 8.51 ug/ml 301 µg/ml in BM, 216 ± 10.79 ug/ml 216 µg/ml in FM and at 48 hours was 227 ± 12.25 ug/ml 227 µg/ml in bud extract (BM), 174 ± 11.92 ug/ml 174 µg/ml in FM. The morphological changes evidence was shown in AO/PI staining by the appearance of a mixed population of cells; early apoptosis, late apoptosis and necrotic cells. These findings suggested that methanolic C.morifolium extracts showed moderate cytotoxic effect on chronic myeloid leukaemia K-562 cells. Further study needed to identify the mode and mechanism of cell death in K-562 cells treated with the C.morifolium extracts.

UVRAG-Knockdown Regulates Mitochondrial Autophagy in Chronic Myeloid Leukaemia Cells by Targeting BNIP3L

Objective: To explore whether UVRAG regulates mitochondrial autophagy via BNIP3L in K562 cellsMaterial and methods: We designed various assays to verify the relation between UVRAG and BNIP3L, we estabilished a mitochondrial autophagy model of K562 cells by CCCP, a mitochondrial autophagy inducer, and regulated the expression of UVRAG by cells transfection. Then we detected the expression of the BINP3L and autophagy-related proteins LC3-II/LC3-Ⅰ and P62 by Western blot. The changes of ROS, mitochondrial mass, and mitochondrial membrane potential (MMP) were detected by flow cytometry technology.Results: We found that CCCP could induce K562 cells mitochondrial autophagy, along with the change of MMP, mitochondrial mass and accumulation of ROS, also our experiment proved that UVRAG-Knockdown could reverse this phenomenon. Investigating the pathway of mitochondrial autophagy revealed UVRAG knockdown was accompanied by a decrease in BNIP3L and LC3 expression, a increase in P62 during mitochondrial autophagy. Conclusion: In our study, the results suggested that UVRAG may regulate mitochondrial autophagy of K562 cells via targeting BINP3L, which may be a potential target for the treatment of CML.