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. 2008 Oct 24:6:8.
doi: 10.1186/1478-811X-6-8.

Gamma-secretase inhibition combined with platinum compounds enhances cell death in a large subset of colorectal cancer cells

Tamara Aleksic  1 , Stephan M Feller
Affiliations

Gamma-secretase inhibition combined with platinum compounds enhances cell death in a large subset of colorectal cancer cells

Tamara Aleksic et al. Cell Commun Signal. .

Abstract

Background: Notch signalling is essential for the development and maintenance of the colonic epithelium. Its inhibition induces a differentiation phenotype in vivo and reduces adenomas in APCmin mice. Whether Notch signals are also required in colorectal cancer (CRC) has remained elusive. Therefore, 64 CRC cell lines were analysed for the occurrence of proteolytically processed, active Notch.

Results: 63 CRC lines contained a fragment with approximately the size of the Notch1 intracellular domain (NICD), which is required for signalling. Subsequent analyses with an antibody that specifically recognises the free Val1744 residue generated by gamma-secretase-mediated cleavage of Notch1 showed that a subset of CRC cells lacks this specific Val1744-NICD. Surprisingly, inhibition of Val1744-NICD signalling with different gamma-secretase inhibitors (GSI) did not lead to substantial effects on CRC cell line growth or survival. However, transient activation of Erk upon GSI treatment was detected. Since cisplatin relies on Erk activation for bioactivity in some cells, platinum compounds were tested together with GSI and enhanced cell killing in a subset of Val1744-NICD-positive CRC cell lines was detected. Erk inhibition ablated this combination effect.

Conclusion: We conclude that gamma-secretase inhibition results in activation of the MAP kinases Erk1/2 and, when used in conjunction, enhances cell death induced by platinum compounds in a large subset of colorectal cancer cell lines.Furthermore the activation of Erk appears to be of particular importance in mediating the enhanced effect seen, as its inhibition abrogates the observed phenomenon. These findings do not only highlight the importance of signalling pathway crosstalk but they may also suggest a new avenue of combination therapy for some colorectal cancers.

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Figures

Figure 1
Figure 1
Expression level and size heterogeneity of Notch fragments detected in CRC cells. A Western blot detection of Notch fragments in total cell RIPA lysates. 100 μg of total cell protein extracts were subjected to SDS-PAGE and immunoblotting to determine the abundance and sizes of C-terminal fragments of Notch1 (NICD). Results from 16 CRC lines are shown. The heterogeneity in the level of NICD expression and the presence of several NICD fragments of variable length is notable. B Comparison of NICD bands obtained with total cell RIPA lysates (R) and lysates obtained by harvesting cells with boiling SDS-PAGE sample buffer (S). 20 CRC lines were analysed in total. Results from 4 CRC lines are shown as an example. Both protein extraction methods result in very similar NICD band patterns. C Size heterogeneity of immunoprecipitated NICD fragments. NICD fragments were precipitated with a polyclonal antibody (sc-6014-R) directed against the C-terminus of Notch1 and, after extended SDS-PAGE on a large 7% gel, immunoblotted with a mouse mAb (N6786) directed against an epitope in the cdc10-NCR region of Notch1.
Figure 2
Figure 2
Variable expression of γ-secretase-cleaved, active Notch1 (Val 1744-NICD) in CRC cells. 100 μg of total cell RIPA lysates were analysed by immunoblotting using anti-Val1744-NICD antibody. Data from 16 cell lines are shown as an example (upper panel). Lysates were also analysed with anti-Hes1 to determine the expression of this well known Notch1 primary target (second panel from top). A short exposure of a western blot with an antibody directed against the C-terminus of Notch1 reveals that the Val1744-NICD fragment is not always the most prominent fragment present in CRC cells (third panel from top; see for example HT55 cells). Actin was analysed as a loading control (bottom panel).
Figure 3
Figure 3
Molecular effects of γ-secretase inhibition on signalling proteins involved in regulating cell growth or death. CCK-81 cells were either left untreated (0), treated with DMSO as a control (DM) or incubated with the γ-secretase inhibitors as indicated. Cells were lysed in RIPA buffer and 50 μg of total protein was subjected to immunoblotting analysis as specified. Both inhibitors result in the down-regulation of Val1744-NICD, which is paralleled by a loss of Hes1 expression, an effect that is evident already after 3 to 6 h. Cleaved PARP, an indicator of cell death, was also analysed and more prominent with L-685,458, which induces cell killing in CCK-81 by an unknown mechanism (Table 1). With both GSI, an increase in pErk (pT202pY204) and pAkt (pS473) is evident. In addition, a downregulation of anti-apoptotic Bcl-2 protein was detectable, to a moderate degree by DBZ and more pronounced with L-685458. Actin was analysed as a loading control.
Figure 4
Figure 4
Combination of Cisplatin with DBZ, a potent GSI, elicits a striking induction of cell death. Cells were treated as indicated and digital images recorded (20× objective). 10 μM Cisplatin (CP) alone leads to a moderate degree of cell death. Note that less cells are present in cultures treated with cisplatin (upper left panel) compared to untreated or DBZ treated cultures (upper right panel). The cells appear flatter and the nuclei larger. A section of the plate was chosen where only few dead cells obstruct the view onto the remaining cells (see Figure 5 for a lower magnification view of CP treated cells). The combination of cisplatin with DBZ leads to massive cell death.
Figure 5
Figure 5
Inhibition of Erk activation suppresses cell death induced by combining cisplatin and GSI. Cells were treated as in Figure 4, or additionally pre-incubated for 1 h with the Mek inhibitor UO126 (30 μM) where indicated and digital images recorded (10× objective). Cells were harvested after 24 h and equal amounts of RIPA lysates (50 μg) analysed for pErk and cleaved PARP as before. UO126 blocks the induction of pErk by DBZ and cisplatin, reduces the level of cleaved PARP and suppresses the cell death induced by the combination of these drugs.
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