Trastuzumab down-regulates Bcl-2 expression and potentiates apoptosis induction by Bcl-2/Bcl-XL bispecific antisense oligonucleotides in HER-2 gene--amplified breast cancer cells.
OBJECTIVE
To investigate the possible existence of an antiapoptotic cross-talk between HER-2 and antiapoptotic Bcl-2 family members.


METHODS
Bcl-2 and Bcl-XL expression and apoptosis induction were analyzed in HER-2 gene-amplified (BT474) and nonamplified (ZR 75-1) breast cancer cell lines exposed to trastuzumab, alone or in combination with either Bcl-2/Bcl-XL bispecific antisense oligonucleotides (AS-4625) or the small-molecule Bcl-2 antagonist HA14-1.


RESULTS
In addition to HER-2 and epidermal growth factor receptor, trastuzumab down-regulated Bcl-2, but not Bcl-XL, protein, and mRNA expression in BT474 cells. Interestingly, trastuzumab-induced down-regulation of HER-2 and Bcl-2 was also observed in three of five and two of three breast cancer patients undergoing trastuzumab treatment, respectively. Despite Bcl-2 down-regulation, however, trastuzumab only marginally increased the rate of apoptosis (7.3 +/- 3.5%). We therefore investigated whether a combination of AS-4625 and trastuzumab might increase proapoptotic efficiency. AS-4625 treatment of BT474 cells decreased both Bcl-2 and Bcl-XL expression, resulting in a 21 +/- 7% net apoptosis induction; the combination of AS-4625 followed by trastuzumab resulted in a significantly stronger induction of apoptosis (37 +/- 6%, P <0.01) that was not observed with the reverse treatment sequence (trastuzumab followed by AS-4625). Similar results were obtained with the Bcl-2 antagonist HA14-1; indeed, exposure of BT474 cells to HA14-1 followed by trastuzumab resulted in a striking proapoptotic synergism (combination index=0.58 +/- 0.18), as assessed by isobologram analysis.


CONCLUSIONS
Altogether our findings suggest that combined targeting of HER-2 and Bcl-2 may represent a novel, rational approach to more effective breast cancer therapy.
Milella
Michele
M
Division of Medical Oncology A, Laboratory of Experimental Preclinical Chemotherapy, Laboratory B, Division of Pathology, and Division of Clinical Pathology, Regina Elena National Cancer Institute, Rome, Italy. milella@ifo.it
Trisciuoglio
Daniela
D
Bruno
Tiziana
T
Ciuffreda
Ludovica
L
Mottolese
Marcella
M
Cianciulli
Anna
A
Cognetti
Francesco
F
Zangemeister-Wittke
Uwe
U
Del Bufalo
Donatella
D
Zupi
Gabriella
G
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001578:Benzopyrans / Q000494:pharmacology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology; D045744:Cell Line, Tumor; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D009570:Nitriles / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D000068878:Trastuzumab; D015854:Up-Regulation; D051020:bcl-X Protein
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009570:Nitriles; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051020:bcl-X Protein; C456800:ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; D000068878:Trastuzumab; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one