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.

636 Global cardiovascular assessment in patients with PH+ chronic myeloid leukaemia treated with TKI: long-term follow-up

Aims Patients affected by Philadelphia chromosome+ chronic myeloid leukaemia (Ph+CML) undergoing to therapy with tyrosine kinase inhibitors (TKIs) are prone to develop cardiovascular complications, which have relevant prognostic implications. Speckle-tracking echocardiography, allowing strain and myocardial work analyses, can be useful in the early detection of cardiac toxicity. Aim of our study was to assess the cardiotoxic effects of TKIs. Methods We evaluated, at baseline and during FU, 20 patients affected by Ph+ CML (59.7 ± 12.2 years, 13 males), treated Imatinib (52.6%), Nilotinib (36.8%), Ponatinib (5.3%), Dasatinib (5.3%). We measured systolic and diastolic blood pressure (SBP-DBP) and calculated corrected QT interval (QTc). In addition, we analysed echocardiographic parameters including left ventricular ejection fraction (LVEF), global longitudinal strain (GLS), global work index (GWI), global constructive work (GCW), global wasted work (GWW), global work efficiency (GWE), and peak left atrial longitudinal strain (PALS). Cardiovascular (CV) events that we considered were symptomatic or asymptomatic LV dysfunction, acute coronary syndrome (ACS), peripheral artery disease (PAD), and arrhythmias. Results Follow-up (FU) time was 3.4 ± 1 years. Most of patients (63.2%) had cardiovascular risk factors, including arterial hypertension (50%), type2 diabetes mellitus (15%), dyslipidaemia (40%) and cigarette smoking (15%). At the end of FU, SBP was unchanged (128.9 ± 19.6 mmHg vs. 129.1 ± 9.8 mmHg; P=NS) whereas DBP increased (69.4 ± 8.5 mmHg vs. 75 ± 7.7 mmHg; P = 0.004); moreover QTc was longer than baseline (404.4 ± 20.1 ms vs. 424.3 ± 29.8 ms; P &lt; 0.001) and LVEF showed a significant decrease (62.2 ± 3.9% at baseline vs. 59.3 ± 4.8% at FU; P = 0.003); similarly, GCW (2444.1 ± 540mmHg% vs. 2234.7 ± 179.4 mmHg%; P = 0.034), GWI (2158.1 ± 589.6 mmHg% vs. 1923.1 ± 174.5 mmHg%; P = 0.022) and PALS (36.3 ± 17.1% vs. 32.8 ± 9.7%; P = 0.002) decreased during cancer therapy. On the other hand, GLS (−18.6 ± 3.1% vs. −19.4 ± 1.1%; P=NS), GWE (94.3 ± 4.1% vs. 93.6 ± 3.6%; P=NS) and GWW (120.6 ± 94.3 mmHg% vs. 106.3 ± 68.9 mmHg%; P=NS) did not change significantly. CV events were observed in 66.7% of the study population. These were mostly represented by ACS, atrial arrhythmias and symptomatic LV dysfunction (30.7% for each) and, to a lesser extent, PAD (7.6%). By comparing patients with events (group A) with those without events (group B) we found that differently from group B, group A showed during FU a significant increase of DBP (from 66 ± 5.2 mmHg to 71.2 ± 6.1 mmHg, P = 0.010; vs. group B= from 76 ± 12.mmHg to 78 ± 4.1 mmHg, P=NS) and a significant QTc prolongation (from 415.7 ± 16.1 ms to 441 ± 29.8 ms, P &lt; 0.001; vs. group B= from 390.4 ± 19.3 ms to 405.6 ± 23.3 ms, P=NS); as to echocardiographic parameters, we found, in patients with CV events, a significant decrease of: LVEF (from 62.7 ± 4.7% to 58.8 ± 4.3%, P = 0.004; vs. group B from 61.4 ± 2.8% to 60 ± 1.7%, P=NS), GCW (from 2566.2 ± 669.6 mmHg% to 2230.1 ± 199.4 mmHg%, P = 0.021, vs. group B 2194 ± 167.5 mmHg% to 2212.6 ± 160mmHg%, P=NS) and PALS (from 36.1 ± 17% to 29.6 ± 6.6%, P = 0.022 vs. group B from 32.7 ± 8% to 35 ± 8.5%, P = 0.003). Of these parameters, only PALS was significant independent predictor of CV events on logistic regression analysis (OR 0.82 CI 95 0.69–0.98, P = 0.034). Conclusions Advanced echocardiographic parameters, including myocardial work and left atrial strain analysis, are particularly valuable in the early detection of TKI-induced cardiac toxicity. PALS could be an useful tool to predict outcome in these patients.