Deoxypodophyllotoxin inhibited the growth of malignant pleural mesothelioma by inducing necroptosis and mitotic catastrophe

Abstract

Background:

Malignant pleural mesothelioma (MPM) is an extremely aggressive cancer with a poor prognosis and limited effective treatment options. However, recent studies have shown that targeting microtubule regulation is a viable approach for treating MPM.

Purpose:

This study aimed to assess the antitumor behavior of deoxypodophyllotoxin (DPT) on MPM in vitro and in vivo and to identify its underlying mechanisms.

Study Design:

The study employed in vitro and in vivo models to evaluate the efficacy and mechanisms of DPT against MPM. We used cell-culture techniques, molecular-biology assays, and a xenograft mice module to thoroughly study the effects of DPT.

Methods:

Three MPM cell lines (H2452, H28, and 211H) and a xenograft mice module were used to assess the antitumor effects of DPT. The cell-cycle and cell-death rates were assessed by flow cytometry to study DPT-induced mitotic cell death. Moreover, the role of necroptosis in the antitumor effect of DPT was determined through transmission electron microscopy and western blot analysis, with further validation being done via RIP1 inhibition by Necrostatin-1 (Nec-1), a RIPK1 inhibitor, and MLKL silencing by siRNAs.

Results:

DPT was found to inhibit MPM cell growth in a dose-dependent manner in vitro and in vivo. Specifically, transmission electron microscopy showed plasma-membrane rupture with the preserved nuclear integrity of MPM cells after DPT treatment, indicating necroptosis in DPT-treated MPM cells. Moreover, a western blot revealed further proof of tumor necrosis factor alpha (TNF-$\alpha$)-associated necroptosis-pathway activation, as revealed by the phosphorylation of the key proteins receptor-interacting protein kinase 1 (RIP1), receptor-interacting protein kinase 3 (RIP3), and mixed-lineage kinase domain-like pseudokinase (MLKL). Additional experiments with TNF-$\alpha$ receptor TNFR1 silencing, RIP1 inhibitors and MLKL silencing reinforced the influential role of TNF-$\alpha$- RIP1-RIP3-MLKL activation in DPT-induced necroptosis. Also, DPT triggered mitotic catastrophe, observable by a defective spindle assembly, multinucleation, and micronucleation. Pretreatment with the S-phase arrest inducer thymidine was reduced both DPT-induced cell death and RIP1 phosphorylation, suggesting an interplay between necroptosis and mitotic arrest.

Conclusion:

DPT may offer a novel therapeutic option for MPM, with drug-induced necroptosis and mitotic catastrophe being key underlying mechanisms.