Abstract
BackgroundThe objective of this study was to gain insight into the molecular mechanism of induced cell death (apoptosis) by PYRROLO [1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) series compounds, using human (K562) cells as a model.MethodsWe focused our attention on some members of the PBTDs family to test their potential apoptotic activity in K562 cells. Important apoptotic activity was demonstrated, as evidenced by the concentration and percentage of cell death quantified by measuring PI-uptake by flow cytometry, and DNA fragmentation analyzed by agarose gel electrophoresis, generating a characteristic ladder pattern of discontinuous DNA fragments. The expression of Bcl-2 family was tested using western blotting and transfection method.ResultsPBTDs-mediated suppression of K562 cell proliferation was induced by apoptosis characterized by the appearance of DNA fragmentation and was associated with the poly(ADP-ribose)polymerase (PARP) cleavage. PBTD-1 and -3 treatment resulted in caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we used K562/vector and K562/bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with K562/vector, K562/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 10 μM PBTD-1 and -3 for 24 h produced morphological features of apoptosis and DNA fragmentation in K562/vector cells, respectively. In contrast, PBTD-1 and -3-induced caspase-3 activation and apoptosis were inhibited in K562/Bcl-2. Furthermore, Bcl-2 overexpressing cells exhibited less cytocrome c release during PBTDs-induced apoptosis.ConclusionThese results indicate that PBTDs effectively induce apoptosis of K562 leukemia cells through the activation of caspase cascades. In addition, these findings indicate that Bcl-2 inhibits PBTD-1 and -3 induced-apoptosis via a mechanism that interferes with cytocrome c release, and the activity of caspase-3, which is involved in the execution of apoptosis.
Highlights
The objective of this study was to gain insight into the molecular mechanism of induced cell death by PYRROLO [1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) series compounds, using human (K562) cells as a model
PBTD-1 and -3 induced Apoptosis in cancer cells To gain insights into the mechanism of PTBD-1 and -3 suppression of K562 cell proliferation, we determined its apoptotic effect by flow cytometry following staining with propidium iodide (Table 2, 3)
Apoptosis induction by PBTD-1 and 3 was confirmed by analysis of DNA fragmentation and the results are shown in Figure 2A, 2B, Table 2: Percentage of the cell death was quantified measuring propidium iodide (PI)-uptake by flow cytometry
Summary
The objective of this study was to gain insight into the molecular mechanism of induced cell death (apoptosis) by PYRROLO [1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) series compounds, using human (K562) cells as a model. Chronic myeloid leukaemia is an hematopoietic stem cell cancer cytologically characterized by the Philadelphia chromosome, which results from reciprocal translocation between chromosome 9 and chromosome 22 [4,5] This mutation leads to the formation of the chimeric fusion protein Bcr/Abl, which manifests an uncontrolled tyrosine kinase activity. Bcr-Abl kinase stimulates a variety of downstream survival pathways, including the mitogen-activated protein kinase cascade, Akt, the signal transducers and activators of transcription. Activation of these pathways in Bcr-Abl positive cells results in increased expression of several antiapoptotic proteins, such Bcl-xl and Bcl-2, which block the release of cytocrome c from mithocondria, resulting in inactivation of caspases [10,11]
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