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Published on: May 15, 2019
Cinobufagin overcomes bortezomib resistance in multiple myeloma strains by targeting SEC62/TRPM4-mediated NECSO
Zhao Yin1, Guangchao Li2, Qi Zhong1
1The Affiliated Guangdong Second Provincial General Hospital of Jinan University, Guangzhou, Guangdong Province 510317, China.
Background:
Necrosis by sodium overload (NECSO) is a poorly understood, novel form of cell death implicated in cancer. TRPM4 is the only protein currently linked to NECSO and is downregulated in bortezomib-resistant multiple myeloma (MM) patients and cell lines.
Purpose:
To investigate the potential of cinobufagin to overcome bortezomib resistance in MM and elucidate its underlying mechanism of action, particularly regarding TRPM4 and NECSO induction.
Study Design:
This study combined in vitro investigations using bortezomib-resistant MM cell lines (8226-BTZR, KMS-11-BTZR) and in vivo xenograft mouse models with comprehensive molecular interaction studies.
Methods:
Cell proliferation assays, xenograft tumor growth monitoring, and immunohistochemistry (CD138/Ki67) were used to assess cinobufagin's effects. NECSO induction was evaluated mechanistically. TRPM4 expression was analyzed. The interaction between cinobufagin and its target was identified using LiP-MS, molecular docking, and molecular dynamics simulations, and validated by MST and CETSA assays. Protein-protein interactions (SEC62/TRPM4), ubiquitination status, and proteasomal degradation of TRPM4 were assessed by SPR, molecular dynamics simulations and immunoprecipitation assay. The role of SEC62 was confirmed using knockdown experiments.
Results:
Cinobufagin effectively inhibited proliferation of bortezomib-resistant MM cells in vitro and suppressed tumor growth while reducing CD138/Ki67 expression in vivo. Mechanistically, cinobufagin induced NECSO. It upregulated TRPM4 expression and was found to directly bind SEC62. SEC62 interacts with TRPM4, promoting its ubiquitination and proteasomal degradation. Cinobufagin disrupts the SEC62/TRPM4 interaction, thereby stabilizing TRPM4 by inhibiting its ubiquitin-proteasome-mediated degradation. SEC62 knockdown attenuated cinobufagin's effects, confirming SEC62's role in mediating the reversal of bortezomib resistance.
Conclusion:
These findings identify TRPM4 as a promising therapeutic target in bortezomib-resistant MM. Cinobufagin overcomes bortezomib resistance by modulating the SEC62-TRPM4 axis, stabilizing TRPM4, and uniquely inducing the previously unexploited NECSO cell death pathway. This highlights cinobufagin's significant therapeutic potential.
Insights
Cinobufagin overcomes bortezomib resistance in multiple myeloma by stabilizing TRPM4 and inducing NECSO cell death. This study identifies the SEC62-TRPM4 axis as a key therapeutic target for resistant cancers.
Area of Science:
- Oncology
- Cell Biology
- Pharmacology
Background:
- Necrosis by sodium overload (NECSO) is a novel cell death pathway implicated in cancer.
- TRPM4 is the sole protein linked to NECSO and is downregulated in bortezomib-resistant multiple myeloma (MM).
Purpose of the Study:
- Investigate cinobufagin's potential to overcome bortezomib resistance in MM.
- Elucidate cinobufagin's mechanism, focusing on TRPM4 and NECSO induction.
Main Methods:
- Utilized in vitro MM cell lines and in vivo xenograft models.
- Assessed cinobufagin's effects on proliferation, tumor growth, TRPM4 expression, and NECSO induction.
- Investigated the SEC62/TRPM4 interaction, ubiquitination, and degradation pathways using advanced molecular techniques.
Main Results:
- Cinobufagin inhibited bortezomib-resistant MM cell proliferation and tumor growth.
- Cinobufagin upregulated TRPM4, induced NECSO, and disrupted the SEC62-TRPM4 interaction.
- Disruption of SEC62-TRPM4 stabilized TRPM4 by preventing its proteasomal degradation, reversing bortezomib resistance.
Conclusions:
- TRPM4 is a viable therapeutic target for bortezomib-resistant MM.
- Cinobufagin overcomes resistance by modulating the SEC62-TRPM4 axis and inducing NECSO.
- Cinobufagin demonstrates significant therapeutic potential for treating resistant multiple myeloma.
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