Lovastatin-induced apoptosis in thyroid cells: involvement of cytochrome c and lamin B.
OBJECTIVE
The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin, induces apoptosis in the thyroid cell line TAD-2 and in proliferating normal human thyroid cells in culture, through a p53-independent mechanism involving caspase-3-like proteases. The combination of lovastatin with other anti-neoplastic drugs potentiates chemotherapy of tumors. This drug has been suggested for the chemotherapy of tumors and is potentially useful in the treatment of thyroid proliferative diseases. Based on this premise, we analyzed in more detail the role of some molecular effectors and the role of the caspase family proteases in the lovastatin-induced apoptotic pathway in TAD-2 cells.


METHODS
TAD-2 cells were treated with lovastatin to induce apoptosis, and expression of p53, Bc1-2, Bcl-XL and Bax was analyzed by Western blot. Caspase activation was evaluated by the assay of enzymatic activity with chromogenic peptides and Western blot. Nuclear, cytosolic and mitochondrial fractions were prepared by differential centrifugation and the presence of cytochrome c and lamin B was evaluated by Western blot.


RESULTS
p53, Bc1-2, Bcl-XL and Bax protein expression were unchanged during apoptosis. Cytochrome c was released from mitochondria into the cytosol, a pivotal event in the activation of caspase-3. Caspase-3 and -6 but not caspase-2 were activated, and proteolysis of PARP and lamin B, a caspase-6 substrate located in the inner nuclear membrane, was demonstrated by Western blot. The nuclear localization of lamin B was also inhibited by lovastatin.


CONCLUSIONS
These data demonstrate that, in TAD-2 thyroid cells, lovastatin induces lamin B proteolysis and inhibits its nuclear localization and induces cytochrome c release from mitochondria into the cytosol.
Di Matola
T
T
Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita Federico II, Via S Pansini 5, 80131, Naples, Italy.
D'Ascoli
F
F
Luongo
C
C
Bifulco
M
M
Rossi
G
G
Fenzi
G
G
Vitale
M
M
D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053143:Caspase 2; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D019254:Genes, bcl-2 / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D034921:Lamin Type B; D034882:Lamins; D008148:Lovastatin / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D013961:Thyroid Gland / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D015854:Up-Regulation / Q000187:drug effects; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D000911:Antibodies, Monoclonal; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D003574:Cytochrome c Group; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D008148:Lovastatin; C505407:CASP3 protein, human; C505514:CASP6 protein, human; D053143:Caspase 2; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases