title: PYRROLO[1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) induce apoptosis in K562 cells.
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(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 muM 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy. gmarfe1@virgilio.it
authors.forename: Gabriella
authors.initials: G
authors.lastname: Marfe
authors.forename: Carla
authors.initials: C
authors.lastname: Di Stefano
authors.forename: Romano
authors.initials: R
authors.lastname: Silvestri
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Abruzzese
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Catalano
authors.forename: Livia
authors.initials: L
authors.lastname: Di Renzo
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Filomeni
authors.forename: Ezio
authors.initials: E
authors.lastname: Giorda
authors.forename: Giuseppe
authors.initials: G
authors.lastname: La Regina
authors.forename: Emanuela
authors.initials: E
authors.lastname: Morgante
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Ciriolo
authors.forename: Matteo Antonio
authors.initials: MA
authors.lastname: Russo
authors.forename: Sergio
authors.initials: S
authors.lastname: Amadori
authors.forename: Paola
authors.initials: P
authors.lastname: Sinibaldi-Salimei
mesh_terms: D017209:Apoptosis; D001569:Benzodiazepines / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003499:Cyclic S-Oxides / Q000494:pharmacology*; D045304:Cytochromes c / Q000187:drug effects / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D015536:Down-Regulation; D019254:Genes, bcl-2 / Q000502:physiology; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C494826:BAX protein, human; D003499:Cyclic S-Oxides; D019253:Proto-Oncogene Proteins c-bcl-2; C527487:RS 678; C527488:RS 779; D051028:bcl-2-Associated X Protein; D001569:Benzodiazepines; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3