Bcl-x(L) and Myeloid cell leukaemia-1 contribute to apoptosis resistance of colorectal cancer cells.
OBJECTIVE
To explore the role of Bcl-x(L) and Myeloid cell leukaemia (Mcl)-1 for the apoptosis resistance of colorectal carcinoma (CRC) cells towards current treatment modalities.


METHODS
Bcl-x(L) and Mcl-1 mRNA and protein expression were analyzed in CRC cell lines as well as human CRC tissue by Western blot, quantitative PCR and immunohistochemistry. Bcl-x(L) and Mcl-1 protein expression was knocked down or increased in CRC cell lines by applying specific siRNAs or expression plasmids, respectively. After modulation of protein expression, CRC cells were treated with chemotherapeutic agents, an antagonistic epidermal growth factor receptor (EGFR1) antibody, an EGFR1 tyrosine kinase inhibitor, or with the death receptor ligand TRAIL. Apoptosis induction and cell viability were analyzed.


RESULTS
Here we show that in human CRC tissue and various CRC cell lines both Bcl-x(L) and Mcl-1 are expressed. Bcl-x(L) expression was higher in CRC tissue than in surrounding non-malignant tissue, both on protein and mRNA level. Mcl-1 mRNA expression was significantly lower in malignant tissues. However, protein expression was slightly higher. Viability rates of CRC cells were significantly decreased after knock down of Bcl-x(L) expression, and, to a lower extent, after knock down of Mcl-1 expression. Furthermore, cells with reduced Bcl-x(L) or Mcl-1 expression was more sensitive towards oxaliplatin- and irinotecan-induced apoptosis, and in the case of Bcl-x(L) also towards 5-FU-induced apoptosis. On the other hand, upregulation of Bcl-x(L) by transfection of an expression plasmid decreased chemotherapeutic drug-induced apoptosis. EGF treatment clearly induced Bcl-x(L) and Mcl-1 expression in CRC cells. Apoptosis induction upon EGFR1 blockage by cetuximab or PD168393 was increased by inhibiting Mcl-1 and Bcl-x(L) expression. More strikingly, CD95- and TRAIL-induced apoptosis was increased by Bcl-x(L) knock down.


CONCLUSIONS
Our data suggest that Bcl-x(L) and, to a lower extent, Mcl-1, are important anti-apoptotic factors in CRC. Specific downregulation of Bcl-x(L) is a promising approach to sensitize CRC cells towards chemotherapy and targeted therapy.
Schulze-Bergkamen
Henning
H
First Department of Medicine, Johannes-Gutenberg-University Mainz, Mainz, Germany. bergkam@uni-mainz.de
Ehrenberg
Roland
R
Hickmann
Lothar
L
Vick
Binje
B
Urbanik
Toni
T
Schimanski
Christoph-C
CC
Berger
Martin-R
MR
Schad
Arno
A
Weber
Achim
A
Heeger
Steffen
S
Galle
Peter-R
PR
Moehler
Markus
M
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000378:metabolism* / Q000473:pathology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D000077146:Irinotecan; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009944:Organoplatinum Compounds / Q000494:pharmacology; D000077150:Oxaliplatin; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism
D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009944:Organoplatinum Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D051020:bcl-X Protein; D019014:fas Receptor; D000077150:Oxaliplatin; D000077146:Irinotecan; C512478:EGFR protein, human; D066246:ErbB Receptors; D005472:Fluorouracil; D002166:Camptothecin