Overcoming Intrinsic and Acquired Cetuximab Resistance in RAS Wild-Type Colorectal Cancer: An In Vitro Study on the Expression of HER Receptors and the Potential of Afatinib
Abstract
:1. Introduction
2. Results
2.1. Identification of Intrinsically Cetuximab-Resistant CRC Cell Lines and Generation of Acquired Cetuximab-Resistant Cell Lines
2.2. CRC Cell Lines and Patients Show Higher Expression of HER2 and HER3 than EGFR
2.3. Intrinsic and Acquired Cetuximab-Resistant CRC Cell Lines Are Sensitive to Afatinib Treatment
2.4. Treatment of CRC Cell Lines with Afatinib Has Little Influence on the Cell Cycle Distribution and the Induction of Apoptotic Cell Death
2.5. The Combination Treatment of Afatinib with Irinotecan Leads to Additive Effects in CRC Cell Lines
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Cytotoxicity Assays
- Afatinib for 72 h immediately followed by irinotecan for 24 h;
- Irinotecan for 24 h immediately followed by afatinib for 72 h.
4.3. Oxygen Conditions
4.4. Generation of Resistant Cell Clones
4.5. Expression Analysis of HER Family Members
4.6. Assays for Apoptosis and Cell Cycle Distribution
4.7. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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IC50 Afatinib 72 h (μM) | |||
---|---|---|---|
Cell Line | Cetuximab Resistance Status | Normoxia (21% O2) | Hypoxia (1% O2) |
Lim1215 | Sensitive | 0.081 ± 0.021 | 0.178 ± 0.055 |
CaCo2 | Sensitive | 0.341 ± 0.199 | 0.604 ± 0.307 |
SW48 | Intrinsically resistant | 2.379 ± 0.869 | 2.109 ± 0.691 |
HT29 | Intrinsically resistant | 1.805 ± 0.041 | 1.816 ± 0.117 |
Lim1215-PBS | PBS-treated control, sensitive | 0.007 ± 0.002 | 0.010 ± 0.003 |
Lim1215-R | Acquired resistance | 0.174 ± 0.030 | 0.458 ± 0.060 |
CaCo2-PBS | PBS-treated control, sensitive | 0.591 ± 0.384 | 0.260 ± 0.197 |
CaCo2-R | Acquired resistance | 1.570 ± 0.264 | 1.398 ± 0.270 |
Cell Line | Condition | IC50 (μM) | p-Value | CI |
---|---|---|---|---|
Lim1215-PBS | 72 h 0 μM afatinib→24 h irinotecan | 0.28 ± 0.26 | / | / |
72 h 0.001 μM afatinib→24 h irinotecan | 0.36 ± 0.19 | 0.757 | 0.92 ± 0.12 | |
72 h 0.005 μM afatinib→24 h irinotecan | 0.39 ± 0.16 | 0.681 | 0.94 ± 0.08 | |
24 h irinotecan→72 h 0μM afatinib | 0.11 ± 0.04 | / | / | |
24 h irinotecan→72 h 0.001 μM afatinib | 0.09 ± 0.04 | 0.994 | 1.02 ± 0.04 | |
24 h irinotecan→72 h 0.005 μM afatinib | 0.10 ± 0.04 | 0.488 | 1.10 ± 0.07 | |
Lim1215-R | 72 h 0 μM afatinib→24 h irinotecan | 1.48 ± 0.99 | / | / |
72 h 0.02 μM afatinib→24 h irinotecan | 0.64 ± 0.36 | 0.458 | 0.90 ± 0.07 | |
72 h 0.1 μM afatinib→24 h irinotecan | 0.44 ± 0.19 | 0.417 | 0.81 ± 0.10 | |
24 h irinotecan→72 h 0 μM afatinib | 0.09 ± 0.02 | / | / | |
24 h irinotecan→72 h 0.02 μM afatinib | 0.08 ± 0.02 | 0.235 | 0.79 ± 0.10 | |
24 h irinotecan→72 h 0.1 μM afatinib | 0.06 ± 0.02 | 0.116 | 0.72 ± 0.13 | |
CaCo2-PBS | 72 h 0 μM afatinib→24 h irinotecan | ND | / | / |
72 h 0.05 μM afatinib→24 h irinotecan | ND | / | 1.00 ± 0.07 | |
72 h 0.3 μM afatinib→24 h irinotecan | ND | / | 1.03 ± 0.03 | |
24 h irinotecan→72 h 0 μM afatinib | 0.46 ± 0.15 | / | / | |
24 h irinotecan→72 h 0.05 μM afatinib | ND | / | 1.22 ± 0.32 | |
24 h irinotecan→72 h 0.3 μM afatinib | ND | / | 1.22 ± 0.29 | |
CaCo2-R | 72 h 0 μM afatinib→24 h irinotecan | ND | / | / |
72 h 0.75 μM afatinib→24 h irinotecan | ND | / | 0.99 ± 0.08 | |
72 h 1.25 μM afatinib→24 h irinotecan | ND | / | 1.00 ± 0.09 | |
24 h irinotecan→72 h 0 μM afatinib | 0.55 ± 0.28 | / | / | |
24 h irinotecan→72 h 0.75 μM afatinib | ND | / | 1.14 ± 0.20 | |
24 h irinotecan→72 h 1.25 μM afatinib | ND | / | 1.22 ± 0.18 | |
HT29 | 72 h 0 μM afatinib→24 h irinotecan | 40.07 ± 4.59 | / | / |
72 h 2 μM afatinib→24 h irinotecan | 18.96 ± 3.29 | 0.051 | 1.12 ± 0.11 | |
72 h 3 μM afatinib→24 h irinotecan | 14.73 ± 2.77 | 0.023 | 1.10 ± 0.07 | |
24 h irinotecan→72 h 0 μM afatinib | 4.34 ± 1.06 | / | / | |
24 h irinotecan→72 h 2 μM afatinib | 4.74 ± 0.61 | 0.993 | 1.02 ± 0.06 | |
24 h irinotecan→72 h 3 μM afatinib | 5.52 ± 0.69 | 0.566 | 1.10 ± 0.07 |
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De Pauw, I.; Lardon, F.; Van den Bossche, J.; Baysal, H.; Pauwels, P.; Peeters, M.; Vermorken, J.B.; Wouters, A. Overcoming Intrinsic and Acquired Cetuximab Resistance in RAS Wild-Type Colorectal Cancer: An In Vitro Study on the Expression of HER Receptors and the Potential of Afatinib. Cancers 2019, 11, 98. https://doi.org/10.3390/cancers11010098
De Pauw I, Lardon F, Van den Bossche J, Baysal H, Pauwels P, Peeters M, Vermorken JB, Wouters A. Overcoming Intrinsic and Acquired Cetuximab Resistance in RAS Wild-Type Colorectal Cancer: An In Vitro Study on the Expression of HER Receptors and the Potential of Afatinib. Cancers. 2019; 11(1):98. https://doi.org/10.3390/cancers11010098
Chicago/Turabian StyleDe Pauw, Ines, Filip Lardon, Jolien Van den Bossche, Hasan Baysal, Patrick Pauwels, Marc Peeters, Jan Baptist Vermorken, and An Wouters. 2019. "Overcoming Intrinsic and Acquired Cetuximab Resistance in RAS Wild-Type Colorectal Cancer: An In Vitro Study on the Expression of HER Receptors and the Potential of Afatinib" Cancers 11, no. 1: 98. https://doi.org/10.3390/cancers11010098