Background: 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. Methods: We 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. Results: PBTDs-mediated suppression of K562 cell proliferation was induced by apoptosis characterized by the appearance of DNA fragmentation and was associated with the poly(ADPribose) 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 microM 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/BcI-2. Furthermore, Bcl-2 overexpressing cells exhibited less cytocrome c release during PBTDs-induced apoptosis. Conclusion: These 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.
PYRROLO[1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) induce apoptosis in K562 cells / Marfè, G.; DI STEFANO, C.; Silvestri, Romano; Abruzzese, E.; Catalano, G.; DI RENZO, Livia Maria; Filomeni, G.; Giorda, E.; LA REGINA, Giuseppe; Morgante, E.; Ciriolo, M. R.; Russo, M. A.; Amadori, S.; SINIBALDI SALIMEI, P.. - In: BMC CANCER. - ISSN 1471-2407. - 7:(2007), p. 207. [10.1186/1471-2407-7-207]
PYRROLO[1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) induce apoptosis in K562 cells.
SILVESTRI, Romano;DI RENZO, Livia Maria;LA REGINA, GIUSEPPE;
2007
Abstract
Background: 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. Methods: We 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. Results: PBTDs-mediated suppression of K562 cell proliferation was induced by apoptosis characterized by the appearance of DNA fragmentation and was associated with the poly(ADPribose) 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 microM 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/BcI-2. Furthermore, Bcl-2 overexpressing cells exhibited less cytocrome c release during PBTDs-induced apoptosis. Conclusion: These 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
