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.

COX Inhibitory and Cytotoxic Naphthoketal-Bearing Polyketides from Sparticola junci

Axenic fermentation on solid rice of the saprobic fungus Sparticola junci afforded two new highly oxidized naphthalenoid polyketide derivatives, sparticatechol A (1) and sparticolin H (2) along with sparticolin A (3). The structures of 1 and 2 were elucidated on the basis of their NMR and HR-ESIMS spectroscopic data. Assignment of absolute configurations was performed using electronic circular dichroism (ECD) experiments and Time-Dependent Density Functional Theory (TDDFT) calculations. Compounds 1–3 were evaluated for COX inhibitory, antiproliferative, cytotoxic and antimicrobial activities. Compounds 1 and 2 exhibited strong inhibitory activities against COX-1 and COX-2. Molecular docking analysis of 1 conferred favorable binding against COX-2. Sparticolin H (2) and A (3) showed a moderate antiproliferative effect against myelogenous leukemia K-562 cells and weak cytotoxicity against HeLa and mouse fibroblast cells.

Improved gene delivery to K-562 leukemia cells by lipoic acid modified block copolymer micelles

Although there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]101-b-[n-(butyl methacrylate)124-co-(lipoic acid methacrylate)22]} (P(DMAEMA101-b-[nBMA124-co-LAMA22])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.

Chemical Analysis and in Vitro Antiproliferative Potential of Eugenia uniflora L. (Myrtaceae)

Natural products and especially medicinal plants, have been extensively studied and have exhibited antiproliferative effects. The species Eugenia uniflora L. (Myrtaceae) is native to Brazil and distributed throughout Australia, East Asia, and the Americas. The leaves are commonly used for the treatment of diarrhea, fever, and hypertension. However, the chemical properties and antiproliferative potential of the extracts remain to be elucidated. In this work, the antiproliferative effects of hydroethanolic (HEE) and dichloromethane (DEE) extracts of leaves from E. uniflora against a panel of human tumor cell lines in vitro were evaluated. Quercetin was identified using an HPLC, and the chemical component β-sitosterol was isolated. The HEE was an inactive extract, whereas the DEE extract showed antiproliferative activity against OVCAR-3 ovarian cells. In a bioassay-guided process, more pronounced antiproliferative effects were found for β-sitosterol isolated from DEE, which demonstrated potent antiproliferative effects against glioblastoma (U-251 cells) and leukemia (K-562 cells). This study contributes to the knowledge of E. uniflora and suggests that β-sitosterol is a potential chemotherapeutic compound.

Quercetin Promotes Cell Cycle Arrest and Apoptosis and Attenuates the Proliferation of Human Chronic Myeloid Leukemia Cell Line-K562 Through Interaction with HSPs (70 and 90), MAT2A and FOXM1

Background: Chronic Myeloid Leukaemia (CML) starts in certain blood-forming cells of the bone marrow when cells acquire Philadelphia chromosome. Nowadays, scientists attempt to find novel and safe therapeutic agents and approaches for CML therapy using Tyrosine Kinase Inhibitors (TKIs), CML conventional treatment agents, has some restrictions and also adverse effects. Recently, it has been proposed that phytochemicals, such as flavonoids due to their low side effects and notable safety have the potential to be used for CML therapy. Materials and Methods: K-562 cells were exposed with three concentrations of the querectin (10, 40 and 80µM) for 12, 24 and 48 hours. After that, these cells apoptosis rate was estimated using Annexin-V/PI staining and flowcytometry analysis, and their proliferation rate was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT). Finally, the expression of the 70 and 90 kilodalton heat shock proteins (HSP70 and 90), methionine adenosyltransferase 2A (MAT2A), Forkhead box protein M1 (FOXM1), caspase-3 and -8, Bcl-X(L) and Bax involved in leukemic cells survival and proliferation was assessed using Real-Time PCR within 12, 24 and 48 hours after exposure with quercetin 40 and 80µM. Results: Considering consequences, querecetin induced apoptosis in K-562 cells, and also abrogated these cells proliferation. On the other hand, RT-PCR results showed a reduction in some of the candidate genes expression, especially HSP70, Bcl-X(L) and FOXM1, when cells were treated with quercetin 40 and 80µM. Also, Bax, caspase-3 and caspase-8 expression was significantly improved in K-562 cells upon quercetin exposure. Conclusion: We concluded that CML therapy by querecetin due to its anti-proliferative and anti-survival potentials could lead to the promising therapeutic outcome through targeting major survival and proliferation involved genes expression.

Anti-proliferative Effects of Chromones: Potent Derivatives Affecting Cell Growth and Apoptosis in Breast, Bone-marrow and Cervical Cancer Cells

Background: Previously, we have identified 3,3′–carbonyl–bis(chromones) (1a-h, 5e) and 3–(5–(benzylideneamino)thiozol–3–yl)–2H–chromen–2–ones (7a-j) as potent inhibitors of tissue non-specific alkaline phosphatase (TNAP). The present study was designed to investigate the cytotoxic and pro-apoptotic effect of the said derivatives. Methods: The anti-proliferative effect of the derivatives was investigated in three cancer cell lines i.e., MCF-7, K-562, HeLa and normal BHK21 cells using MTT assay. The pro-apoptotic effect of the most potent derivatives was investigated by using flow cytometry, DAPI and PI staining and DNA binding studies. Results: Among all the screened compounds, 1f, 1d, 1c (from 3,3′–carbonyl–bis(chromones), 7c, 7h and 7i (from 3–(5–(benzylideneamino)thiozol–3–yl)–2H–chromen–2–ones) exhibited remarkable growth inhibitory effects. Compounds 1f and 7c were found to be the most potent cytotoxic derivatives against MCF-7; 1d and 7h inhibited most of the proliferation of K-562 cells, whereas 1c and 7i showed maximum growth inhibition in HeLa cells. The identified compounds exerted lower micromolar potency against the respective cell line with significant selectivity over the normal cells (BHK–21). The identified compounds also induced either G2 or S-phase arrest within the respective cancer cells, chromatin condensation and nuclear fragmentation, as well as maximum interaction with DNA. Conclusions: These results provide evidence that the characteristic chemical features of attached groups are the key factors for their anticancer effects and play a useful role in revealing the mechanisms of action in relation to the known compounds in future research programs.

Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al., 2011). Here, we report the results of those experiments. We found treatment of MLL-fusion leukaemia cells (MV4;11 cell line) with the BET bromodomain inhibitor I-BET151 resulted in selective growth inhibition, whereas treatment of leukaemia cells harboring a different oncogenic driver (K-562 cell line) did not result in selective growth inhibition; this is similar to the findings reported in the original study (Figure 2A and Supplementary Figure 11A,B; Dawson et al., 2011). Further, I-BET151 resulted in a statistically significant decrease in BCL2 expression in MV4;11 cells, but not in K-562 cells; again this is similar to the findings reported in the original study (Figure 3D; Dawson et al., 2011). We did not find a statistically significant difference in survival when testing I-BET151 efficacy in a disseminated xenograft MLL mouse model, whereas the original study reported increased survival in I-BET151 treated mice compared to vehicle control (Figure 4B,D; Dawson et al., 2011). Differences between the original study and this replication attempt, such as different conditioning regimens and I-BET151 doses, are factors that might have influenced the outcome. We also found I-BET151 treatment resulted in a lower median disease burden compared to vehicle control in all tissues analyzed, similar to the example reported in the original study (Supplementary Figure 16A; Dawson et al., 2011). Finally, we report meta-analyses for each result.

Lpxn Regulates Proliferation, Adhesion and Invasion of Acute Myeloid Leukemic Cells

Leupaxin (LPXN) belongs to the focal adhesion-associated adaptor protein family, participating in regulating cell adhesion and metastasis. LPXN is a recurrent fusion partner in translocations identified in AML, namely t(11;12) and t(11;21), highlights its potential leukemogenic role in acute myeloid leukemia (AML). However, the precise mechanisms by which LPXN participate in the pathogenesis and progression of AML remains unknown. In the present study, we found that LPXN had a wide expression among a variety of hematological malignancies, including AML. Stable overexpression of LPXN in K-562 cells highlighted the proliferation advantage induced by this gene observed via colony-forming-assay and nude mice models. Proliferation was believed to be induced by LPXN-driven upregulation of ERK1/2 and pERK1/2 in K562-LPXN cells. Moreover, LPXN-expressing K-562 cells were less inhibited by arsenic trioxide, as compared with control cells, which might due to downregulation of BAX, FAS and upregulation of BCL-2. Adhesion to fibronectin and invasion across matrigel were modulated by overexpression and knockdown of LPXN, respectively. Similarly, activity of MMP-9 and MMP-2, and expression of integrin α4, α5 and β1, correlated with overexpression or silencing of LPXN, respectively. Expression of pP38 also showed a positive correlation with LPXN. In general, our data suggests that LPXN promotes proliferation in vitro and in vivo via the MAPK pathway, as well as enhances adhesion and invasion of AML cells through modulating expression of integrin α4, α5, β1 and MMP-9/MMP-2. Disclosures No relevant conflicts of interest to declare.