Chemically modified tetracyclines induce cytotoxic effects against J774 tumour cell line by activating the apoptotic pathway.
Here, we have studied the effects of chemically modified tetracyclines (CMTs) on apoptosis both at the level of the cytoplasmic proteolytic caspase cascade, and on Bcl-2 and c-myc mRNA expression in the J774 macrophage cell line. The results indicate that CMTs induce morphological changes consistent with apoptotic events, as clearly demonstrated both by the acridine orange and ethidium bromide staining, and by TUNEL and fragmentation ELISA assays. Furthermore, the analysis of the cell cycle by flow cytometry shows an evident apoptotic sub-G0G1 peak, without important modifications in the cell cycle distribution. CMTs induce programmed cell death (PCD) in a dose-dependent manner and CMT-8 is the strongest among them. CMT-1 and CMT-8 activate mainly caspase-8 as attested by the inhibitory effects of Z-VAD-fmk and Z-IEDT-fmk on CMT-induced apoptosis. Part of CMT-induced PCD is due to the activation of caspase-9, since it is reduced by the specific caspase-9 inhibitor, Z-LEHD-fmk. Besides, CMTs increase Bcl-2 and c-myc mRNA expression. Collectively, these data indicate that CMTs are potentially anti-tumour agents, since they strongly trigger apoptosis both activating the proteolytic system of the caspase family and modulating genes involved in PCD regulation.
D'Agostino
Pietro
P
Department of Immuno-Haematology and Transfusion, University of Palermo, Palermo, Italy.
Ferlazzo
Viviana
V
Milano
Salvatore
S
La Rosa
Marzia
M
Di Bella
Gloria
G
Caruso
Rosalba
R
Barbera
Caterina
C
Grimaudo
Stefania
S
Tolomeo
Manlio
M
Feo
Salvatore
S
Cillari
Enrico
E
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017737:Macrophages, Peritoneal / Q000187:drug effects; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D013754:Tetracyclines / Q000737:chemistry* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D013754:Tetracyclines; C117153:tetracycline CMT-8; C053450:4-des-dimethylaminotetracycline; D020169:Caspases; D013752:Tetracycline