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ΔNp63 expression in four carcinoma cell lines and the effect on radioresistance--a siRNA knockdown model
Maximilian Moergel1,2, Jutta Goldschmitt3, Marcus Stockinger4
1Department of Oral and Maxillofacial Surgery, University of Mainz, Medical Center, Augustusplatz 2, 55131, Mainz, Germany. maximilian.moergel@unimedizin-mainz.de.
Objectives:
This study investigated the expression of ΔNp63α in carcinoma cell lines of the upper aerodigestive tract and their potential influence on radioresistance using a small interfering RNA (siRNA) knockdown approach.
Materials And Methods:
Four carcinoma cell lines were investigated for the expression of the ΔNp63 isoform by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (0, 24, 48 h) with and without single dose irradiation of 6 Gy. Furthermore, all cell lines were transfected with siRNA against the ΔNp63α isoform over 24 h. Knockdown effectiveness was controlled by qRT-PCR and Western blot. Apoptotic events were evaluated by terminal transferase dUTP nick end labeling (TUNEL) assay and cross-checked by a test for cell viability (WST-1, Roche) over 48 h.
Results:
All cell lines presented varying expression of the ΔNp63α isoform with and without irradiation. A sufficient knockdown rate was established by siRNA transfection. Knockdown of the ΔNp63 isoform showed an effect on radiation sensitivity proven by an increase of apoptotic events detectable by immunofluorescence (TUNEL assay) and likewise a significant reduction of formazan production (WST-1 test) in three cell lines.
Conclusions:
We found overexpression of ΔNp63α with and without irradiation in three cell lines, and the knockdown of ΔNp63α led to increased apoptotic events and fewer viable cells. Thus, the overexpression of ΔNp63α might protect carcinoma cells against irradiation effects.
Clinical Relevance:
The present work supports the hypothesis that protein 63 might serve as a negative predictor for irradiation response and survival in a clinical setting and may be a target for future therapeutic strategies.
Insights
Overexpression of delta Np63 alpha (ΔNp63α) in upper aerodigestive tract cancers may protect cells from radiation. Reducing ΔNp63α levels increased cell death, suggesting it as a therapeutic target.
Area of Science:
- Oncology
- Molecular Biology
- Radiation Oncology
Background:
- Delta Np63 alpha (ΔNp63α) is a key isoform of the p63 protein family.
- p63 isoforms play critical roles in epithelial development and cancer.
- The role of ΔNp63α in radioresistance of upper aerodigestive tract (UAT) cancers remains to be fully elucidated.
Purpose of the Study:
- To investigate the expression of ΔNp63α in UAT carcinoma cell lines.
- To determine the influence of ΔNp63α on radioresistance.
- To evaluate ΔNp63α as a potential therapeutic target for UAT cancers.
Main Methods:
- Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to measure ΔNp63α expression.
- Small interfering RNA (siRNA) to knockdown ΔNp63α expression.
- Western blot to confirm knockdown effectiveness.
- Terminal transferase dUTP nick end labeling (TUNEL) assay and WST-1 assay to assess apoptosis and cell viability post-irradiation.
Main Results:
- All investigated UAT carcinoma cell lines exhibited varying levels of ΔNp63α expression.
- Successful knockdown of ΔNp63α was achieved using siRNA.
- Knockdown of ΔNp63α significantly increased apoptotic events and reduced cell viability in three out of four cell lines after irradiation.
Conclusions:
- Overexpression of ΔNp63α confers radioresistance in UAT carcinoma cells.
- Targeting ΔNp63α could enhance the efficacy of radiotherapy.
- ΔNp63α may serve as a predictive biomarker for radiation response and a therapeutic target in UAT cancers.
