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Biomarker Expression Profiling in Cervix Carcinoma Biopsies Unravels WT1 as a Target of Artesunate
Mohamed E M Saeed1, Candela Cives-Losada1,2, Thomas Efferth3
1Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Mainz, Germany.
Background/Aim:
Artemisinin and its derivatives are not only approved antimalarial drugs but also exert strong anticancer activity. Based on the clinical activity of artesunate (ART) that has been previously reported in cervix carcinoma, we investigated a panel of 12 different biomarkers and identified the Wilms Tumor 1 (WT1) protein as a potential target of ART.
Patients And Methods:
Matched biopsies of cervical carcinoma before, during, and after therapy from patients treated with ART were investigated for induction of apoptosis (TUNEL assay) and expression of Wilms Tumor protein 1 (WT1), 14-3-3 ζ, cluster of differentiation markers (CD4, CD8, CD56), ATP-binding cassette transporter B5 (ABCB5), glutathione S-transferase P1 (GSTP1), inducible nitric oxide synthase (iNOS), translationally controlled tumor protein (TCTP), eukaryotic elongation factor 3 (eIF3), and ADP/ATP translocase by immunohistochemistry. WT1 has been selected for more detailed analyses using molecular docking in silico, microscale thermophoresis using recombinant WT1, and cytotoxicity testing (resazurin assay) using HEK293 cells transfected with four different WT1 splice variants.
Results:
The fraction of apoptotic cells and the expression of WT1, 14-3-3 ζ, and CD4 increased upon ART treatment in tumors of patients. ART was bound in silico to a domain located at the DNA-binding site of WT1, while dihydroartemisinin (DHA) was bound with low affinity to a different site of WT1 not related to DNA-binding. The results were verified using microscale thermophoresis, where ART but not DHA bound to recombinant WT1. Transfectants overexpressing different WT1 splice variants exerted low but significant resistance to ART (≈2-fold).
Conclusion:
WT1 may represent a novel target of ART in cancer cells that contribute to the response of tumor cells to this drug.
Insights
Artesunate (ART) shows anticancer effects by targeting Wilms Tumor 1 (WT1) protein in cervical cancer. This study identifies WT1 as a novel therapeutic target for ART in cancer treatment.
Area of Science:
- Oncology
- Pharmacology
- Molecular Biology
Background:
- Artemisinin derivatives, like artesunate (ART), possess known antimalarial and potent anticancer activities.
- Previous clinical observations highlighted ART's efficacy in cervix carcinoma, prompting further investigation into its molecular targets.
Purpose of the Study:
- To identify potential molecular targets of ART in cervical cancer.
- To investigate the role of Wilms Tumor 1 (WT1) protein as a target for ART.
Main Methods:
- Immunohistochemistry was used to analyze apoptosis and biomarker expression (including WT1) in cervical cancer biopsies before, during, and after ART treatment.
- In silico molecular docking, microscale thermophoresis, and cytotoxicity assays were employed to confirm the interaction between ART and WT1.
Main Results:
- ART treatment increased apoptosis and the expression of WT1, 14-3-3 ζ, and CD4 in patient tumors.
- Molecular docking and microscale thermophoresis demonstrated that ART directly binds to WT1, specifically at its DNA-binding domain.
- Cells overexpressing WT1 splice variants showed a modest but significant resistance to ART.
Conclusions:
- Wilms Tumor 1 (WT1) is identified as a novel molecular target of artesunate (ART) in cancer cells.
- WT1 may play a role in mediating tumor cell response to ART, suggesting its potential as a therapeutic target.
