Abstract
The tumor suppressor p53 is a key mediator of cellular stress and DNA damage response cascades and is activated after exposure to ionizing radiation. Amplifying wild-type p53 expression by targeting negative regulators such as MDM2 in combination with external beam radiotherapy (EBRT) may result in increased therapeutic effects. The novel stapled peptide PM2 prevents MDM2 from suppressing wild-type p53, and is thus a promising agent for therapeutic combination with EBRT. Effects of PM2 and potential PM2-induced radiosensitivity were assessed in a panel of cancer cell lines using 2D cell viability assays. Western Blot and flow cytometric analyses were used to investigate the mechanisms behind the observed effects in samples treated with PM2 and EBRT. Finally, PM2-treatment combined with EBRT was evaluated in an in vitro 3D spheroid model. PM2-therapy decreased cell viability in wild-type p53, HPV-negative cell lines. Western Blotting and flow cytometry confirmed upregulation of p53, as well as initiation of p53-mediated apoptosis measured by increased cleaved caspase-3 and Noxa activity. Furthermore, 3D in vitro tumor spheroid experiments confirmed the superior effects of the combination, as the only treatment regime resulting in growth inhibition and complete spheroid disintegration. We conclude that PM2 induces antitumorigenic effects in wt p53 HPV-negative cancer cells and potentiates the effects of EBRT, ultimately resulting in tumor eradication in a 3D spheroid model. This strategy shows great potential as a new wt p53 specific tumor-targeting compound, and the combination of PM2 and EBRT could be a promising strategy to increase therapeutic effects and decrease adverse effects from radiotherapy.
Highlights
Today up to 50% of all cancer patients receive at least one dose of external beam radiotherapy (EBRT) [1,2,3,4]
The NanoBIT assay further verified the specificity of the antagonistic properties of PM2 to murine double-minute 2 (MDM2), with no effect obtained by the scrambled peptide PM2SCRAM (Supplementary Figure 1A)
Previous studies have demonstrated that stabilization of wt p53 may offer great potential to boost the therapeutic effects of radiotherapy [28, 35, 36]
Summary
Today up to 50% of all cancer patients receive at least one dose of external beam radiotherapy (EBRT) [1,2,3,4]. Improvements to EBRTs such as intensity modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT) are undergoing continuous development, exemplifying that EBRT as a field still has room for improvement [5, 7,8,9]. One such improvement is the use PM2 Radiosensitizes Wild-Type p53 Cancer Cells of radiosensitizers, where sensitizing tumor cells to radiation damage may further increase the radiotherapy success rate. Tumor cell radiosensitization could allow reductions in radiation dose, lowering the risk of adverse effects [10,11,12]. In the event of apoptosis, it is linked to transcription of pro-apoptotic proteins such as Phorbol-12-myristate-13-acetateinduced protein 1 (Noxa) [14, 17, 18]
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