Partial Beclin 1 silencing aggravates doxorubicin- and Fas-induced apoptosis in HepG2 cells.
OBJECTIVE
To investigate the role of Beclin 1 on the susceptibility of HepG2 cells to undergo apoptosis after anti-Fas antibody or doxorubicin treatment.


METHODS
Beclin 1 silencing was achieved using RNA interference. DNA ploidy, the percentage of apoptotic cells and the mitochondrial membrane potential were assessed by flow cytometry. Levels of Beclin 1, Bcl-X(L) and cytochrome c, and the cleavage of poly (ADP-ribose) polymerase (PARP) were assayed by using Western blots.


RESULTS
Beclin 1 expression decreased by 75% 72 h after Beclin 1 siRNA transfection. Partial Beclin 1 silencing significantly increased the percentage of subG1 cells 24 and 40 h after treatment with doxorubicin or anti-Fas antibody, respectively, and this potentiation was abrogated by treatment with a pan-caspase inhibitor. Partial Beclin 1 silencing also increased PARP cleavage, mitochondrial membrane depolarization and cytosolic cytochrome c. The pro-apoptotic consequences of partial Beclin 1 silencing were not associated with a decline in Bcl-X(L) expression.


CONCLUSIONS
Partial Beclin 1 silencing aggravates mitochondrial permeabilization and apoptosis in HepG2 cells treated with an anti-Fas antibody or with doxorubicin.
Daniel
Fanny
F
U773 INSERM (equipe 3), Centre de Recherche Bichat Beaujon CRB3, Paris, France. fdaniel@bichat.inserm.fr
Legrand
Agnès
A
Pessayre
Dominique
D
Vadrot
Nathalie
N
Descatoire
Véronique
V
Bernuau
Dominique
D
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D000071186:Beclin-1; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000737:chemistry / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D004317:Doxorubicin / Q000494:pharmacology*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology*; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology; D010539:Permeability; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D034741:RNA, Small Interfering; D014162:Transfection; D051020:bcl-X Protein / Q000032:analysis
D000903:Antibiotics, Antineoplastic; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D004273:DNA, Neoplasm; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C411023:anti-Fas monoclonal antibody; D051020:bcl-X Protein; D004317:Doxorubicin; D045304:Cytochromes c