MATERIALS AND METHODS: Activity was assessed using broth microdilution, time kill viability, microtiter plate, scanning electron microscope (SEM) and Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR).
RESULTS: The susceptibility tests revealed promising antibacterial activities of all honeys against both bacteria. The MICs of JH, KMH, GH, and AH ranged from 20% to 25% compared to MH (12.5%) against both bacteria. The MBCs of JH, KMH, GH, and AH ranged from 20% to 50% compared to MH (20%) against both bacteria. Treatment of both bacteria with 2× MIC (Minimum inhibitory concentration) of MH, JH, KMH, GH, and AH for 9 hours resulted in reduction in colony-forming unit (CFU/ml). SEM images showed that the morphological changes, cell destruction, cell lysis and biofilm disruption in both bacteria after exposure to all honeys. RT-qPCR analysis revealed that the expression of all genes in both bacteria were downregulated following treatment with all honeys. Among the all-tested honeys, MH showed the highest total antibacterial and antivirulence activities.
CONCLUSION: Our results indicate that all honeys activity included inhibition of both bacteria due to a decrease in expression of essential genes associated with both bacteria, suggesting that all honeys could potentially be used as an alternative therapeutic agent against certain microorganisms particularly against P. aeruginosa and S. pyogenes.
MATERIALS AND METHODS: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting.
RESULTS: The IC50 for imatinib on K562 was 362 nM compared to 3,952 nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down- regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562.
CONCLUSIONS: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.
MATERIALS AND METHODS: Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting.
RESULTS: P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells.
CONCLUSIONS: These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.