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Measuring cell metabolic activity with EZ4U - MTT assay
Our EZ4U (Easy for You) cell proliferation and cytotoxicity MTT assay was applied in a recent study exploring the role of pertuzumab, a monoclonal antibody that targets HER2, in disrupting the transactivation between the epidermal growth factor receptor (EGFR) and HER2 in HER2-positive cancers. The research focuses on understanding how this disruption could predict therapeutic outcomes based on the quantitative analysis of EGFR signaling input (1).
Measuring cell metabolic activity with EZ4U – MTT assay
EZ4U Cell Viability & Cytotoxicity Assay (cat.no. BI-5000)
EASY FOR YOU (EZ4U)
- Non-radioactive & non-toxic
- Convenient single-step incubation – for use on living cells
- Widely cited in more than 280 publications
- Disrupting EGFR-HER2 Transactivation by Pertuzumab in HER2-Positive Cancer: Quantitative Analysis Reveals EGFR Signal Input as Potential Predictor of Therapeutic Outcome. Ujlaky-Nagy L, Szöllősi J, Vereb G. Int J Mol Sci. 2024 May 29;25(11):5978. doi: 10.3390/ijms25115978. PMID: 38892166.
Abstract
Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.
Related publications
Targeted therapeutic options and future perspectives for HER2-positive breast cancer. Wang J, Xu B. Signal Transduct Target Ther. 2019 Sep 13;4:34. doi: 10.1038/s41392-019-0069-2. PMID: 31637013.