No abstract

Epigenetic silencing of tumor suppressor genes (TSGs) plays an essential role in cancer pathogenesis, including acute myeloid leukemia (AML). All of SHP-1, SOCS-1, and SOCS-3 are TSGs that negatively regulate JAK/STAT signaling. Enhanced re-expression of TSGs through de-methylation represents a therapeutic target in several cancers. Thymoquinone (TQ) is a major component of Nigella sativa seeds with anticancer effects against several cancers. However, the effects of TQ on DNA methylation are not entirely understood. This study aimed to evaluate the ability of TQ to re-express SHP-1, SOCS-1, and SOCS-3 in MV4-11 AML cells through de-methylation. Cytotoxicity, apoptosis, and cell cycle assays were performed using WSTs-8 kit, Annexin V-FITC/PI apoptosis detection kit, and fluorometric-red cell cycle assay kit, respectively. The methylation of SHP-1, SOCS-1, and SOCS-3 was evaluated by pyrosequencing analysis. The expression of SHP-1, SOCS-1, SOCS-3, JAK2, STAT3, STAT5A, STAT5B, FLT3-ITD, DNMT1, DNMT3A, DNMT3B, TET2, and WT1 was assessed by RT-qPCR. The molecular docking of TQ to JAK2, STAT 3, and STAT5 was evaluated. The results revealed that TQ significantly inhibited the growth of MV4-11 cells and induced apoptosis in a dose- and time-dependent manner. Interestingly, the results showed that TQ binds the active pocket of JAK2, STAT3, and STAT5 to inhibit their enzymatic activity and significantly enhances the re-expression of SHP-1 and SOCS-3 through de-methylation. In conclusion, TQ curbs MV4-11 cells by inhibiting the enzymatic activity of JAK/STAT signaling through hypomethylation and re-expression of JAK/STAT negative regulators and could be a promising therapeutic candidate for AML patients.

Leaves of Baeckea frutescens were extracted by methanol, and then were subjected to chemical compound analysis through qualitative High Performance Liquid Chromatography (HPLC) profiling, bioactivity property. The total weight of B. frutescens leaves crude extract obtained was 2.532 g from 10 g of initial weight. The HPLC profiling used three solvents that are, Methanol, Acetonitrile and water. The profile spectrums from HPLC showed a few peaks that represent the chemicals that lie in the methanolic extract especially in methanol. The results from HPLC crude extract profile for leaves extract of B. frutescens showed many peaks at the retention time between 0 to 60 minutes. This showed that a lot of compounds have already been flushed out based on time and polarity range of the solvents. The two types of bioactivity study are antibacterial and cytotoxicity assay. In antibacterial assay, six pathogenic bacteria, were selected and used by using agar well diffusion method. All methanolic extract did not show any antibacterial activity or was not effective at all concentrations of 12.5-100 mg/µL. As a result, methanolic extract was not subjected to broth dilution method for the quantitative measurement of the microbiostatic (inhibitory). In cytotoxic assay the cell line used was HL-60 that were treated with B. frutescens methanolic extract to evaluate the IC 50 value, that is, which concentration of test compounds that cause 50 % inhibition or cell death. It is to compare the cytotoxic effect with 3T3 which acted as a control. B. frutescens methanolic extract is not effective for normal cell line. The methanolic extract of B. frutescens was cytotoxic to HL-60.Their IC 50 of the crude extracts was moderate 21µg/ml.

Gallic acid (GA) comes from benzoic acid or, more specifically, 3,4,5-tryhydroxybenzoic, which derives from the phenolic acid of the non-flavonoid part of the polyphenol compound. It is found ubiquitously in various plants and fruits, such as grapes, gallnuts, pomegranates, and tea leaves [1]. Many scientific journals and articles reported on the pharmacological properties of the photochemical like GA, which has antioxidant properties, antimicrobial, anti-inflammatory, anticancer, cardioprotective, gastroprotective, and neuroprotective activity [2]. Moreover, the anticancer properties of GA have been recognised in several cancers, such as lung cancers, oesophageal cancer cells and leukaemia [3].   The objective of this study is to examine the anti-proliferative and apoptosis inducing effects of GA on HL-60 cell lines. Six concentrations of GA were made using the stock solution of GA compound dissolved in dimethyl sulfide (DMSO). HL-60 cells were treated with concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL and was incubated at three incubation period which were 24, 48, and 72 hours. The quantitative measure was determined with cytotoxicity assay of 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and was read by microplate reader at 570 nm. For qualitative measure was through staining with acridine orang (AO) and propidium iodide (PI) and observed the morphological changes of the treated cells.   Results from the MTT assays show that GA has cytotoxicity effect on HL-60 cells especially for 72 hours incubation period. The maximal half inhibitory concentrations (IC50) value of GA decreases as incubation period increases. The IC50 of GA were 9.03, 6.76, and 3.65 µg/ml for 24-, 48-, and 72-hours incubation, respectively. The IC50 value of GA (p<0.05) was significantly different for different incubation periods. The morphological changes were seen through the AO/PI staining with the appearance of the cell blebbing, early apoptosis, and late apoptosis.   Figure 1 shows early apoptosis (EA), late apoptosis (LA), cell blebbing (CB), membrane loose (MB), nuclear fragmentation (NF), and apoptotic bodies (AB).   These findings show that GA has potential as anti-proliferative and apoptosis inducing effects on HL-60 cells line. More research is needed to determine the pathways of apoptosis in HL-60 treated with GA.