The effect of proteasome inhibitor Carfilzomib on pyroptosis cell death pathway in multiple myeloma cells

Öz

Objective: Multiple myeloma (MM), characterized by excessive accumulation of monoclonal antibody-secreting abnormal plasma cells in the bone marrow, is a B-cell malignancy. In clinical practice, the second-generation proteasome inhibitor Carfilzomib (CFZ) is used in the treatment regimens of relapsed or treatment-resistant patients. However, the mechanisms of CFZ-induced cell death in MM cells have not been fully elucidated. The aim of this study is to investigate the effect of CFZ on pyroptosis, which is one of the regulated non-apoptotic cell death pathways in MM cells. Methods: Human NCI H929, RPMI 8226, and U266 MM cell lines were treated with IC50 doses of CFZ for 48 hours. In treated cells, the differences in mRNA levels of GSDMD and GSDME, which are important substrates of pyroptosis, and in mRNA levels of Bax as well as in mRNA levels of Bcl-2 were determined by quantitative real-time PCR (qPCR) method. Results: CFZ treatment led to a significant increase in GSDMD and GSDME mRNA levels in NCI H929 and RPMI 8226 cells, while a remarkable increase was detected only in GSDME mRNA levels in U266 cells (p<0.05). Furthermore, CFZ treatment in MM cells showed a trend toward a significant increase in Bax mRNA expression, and this increase was statistically significant only in RPMI 8226 cells. In MM cells treated with CFZ, Bcl-2 mRNA levels were found to be unchanged. Conclusion: This study for the first time determined that CFZ might show an anti-myeloma effect by triggering the pyroptosis in MM cells.

Anahtar Kelimeler

• Multiple Myeloma
• Proteasome Inhibitors
• Carfilzomib
• Pyroptosis
• Gasdermin E

Proteozom inhibitörü Carfilzomib’in multipl miyelom hücrelerinde piroptozis hücre ölüm yolağı üzerine olan etkisi

Öz

Amaç: Multipl miyelom (MM), monoklonal antikor salgılayan anormal plazma hücrelerinin kemik iliğinde aşırı birikimi ile karakterize bir B hücre malignitesidir. Klinik uygulamalarda ikinci nesil proteozom inhibitörü carfilzomib (CFZ), relaps veya tedaviye dirençli hastaların tedavi rejimlerinde kullanılmaktadır. Ancak, MM hücrelerinde CFZ’in tetiklediği hücre ölümü mekanizmaları tam olarak aydınlatılamamıştır. Bu çalışmanın amacı, MM hücrelerinde CFZ’in apoptotik olmayan düzenli hücre ölüm yolaklarından biri olan piroptozis üzerine olan etkisinin araştırılmasıdır. Yöntem: İnsan RPMI 8226, U266 ve NCI H929 MM hücre hatları, CFZ’in IC50 dozları ile 48 saat süre boyunca muamele edildi. Muamele edilen hücrelerde piroptozisin önemli substratları olan GSDMD ve GSDME ile Bax ve Bcl-2 genlerinin mRNA düzeylerindeki farklılıklar kantitatif eş zamanlı PCR (qPCR) yöntemiyle belirlendi. Bulgular: NCI H929 ve RPMI 8226 hücrelerinde CFZ uygulamasının hem GSDMD hem de GSDME mRNA düzeylerinde anlamlı artışa neden olduğu belirlenirken, U266 hücrelerinde ise sadece GSDME mRNA seviyesinde anlamlı bir artış tespit edildi (p<0.05). MM hücrelerinde CFZ uygulaması Bax mRNA ifadesinde genel bir artışa neden olurken sadece RPMI 8226 hücrelerinde bu artış istatistiksel olarak anlamlı bulundu. CFZ ile muamele edilen MM hücrelerinde Bcl-2 mRNA seviyesinde ise bir değişiklik saptanmadı. Sonuç: Bu çalışmada, ilk kez CFZ’in MM hücrelerinde piroptozis ölüm yolağını da tetikleyerek anti-miyelom özellik gösterebileceği belirlenmiştir.

Anahtar Kelimeler

• Multipl Miyelom
• Proteazom İnhibitörü
• Carfilzomib
• Piroptozis
• Gasdermin E

Kaynakça

1. Corre J, Munshi NC, Avet-Loiseau H. Risk factors in multiple myeloma: is it time for a revision? Blood. 2021;137(1): 16-19. https://doi.org/10.1182/blood.2019004309.
2. Groen K, van de Donk NWCJ, Stege C, Zweegman S, Nijhof IS. Carfilzomib for relapsed and refractory multiple myeloma. Cancer Manag Res. 2019;11: 2663-2675. https://doi.org/10.2147/CMAR.S150653.
3. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 2018;25(3): 486-541. https://doi.org/10.1038/s41418-017-0012-4.
4. Robinson N, Ganesan R, Hegeds C, Kovács K, Kufer TA, Virág L. Programmed necrotic cell death of macrophages: Focus on pyroptosis, necroptosis, and parthanatos. Redox Biol. 2019;26: 101239. https://doi.org/10.1016/j.redox.2019.101239.
5. Touzeau C, Maciag P, Amiot M, Moreau P. Targeting Bcl-2 for the treatment of multiple myeloma. Leukemia. 2018;32(9): 1899-1907. https://doi.org/10.1038/s41375-018-0223-9.
6. Shi C-S, Kehrl JH. Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL. Cell Death Discov. 2019; 9;5: 151. https://doi.org/10.1038/s41420-019-0230-2.
7. Maral S, Albayrak M, Pala Ç. Current Treatment Approaches to Multiple Myeloma with New Agents. J Clin Exp Investig. 2018;9(2): 103-112 https://doi.org/10.5799/jcei.433823
8. Ri M. Endoplasmic-reticulum stress pathway-associated mechanisms of action of proteasome inhibitors in multiple myeloma. Int J Hematol. 2016;104(3):.273-80. https://doi.org/10.1007/s12185-016-2016-0.

9. Fadeel B, Orrenius S. Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med. 2005;258(6): 479-517. https://doi.org/10.1111/j.1365-2796.2005.01570.x.
10. Kuhn DJ, Chen Q, Voorhees PM, Strader JS, Shenk KD, Sun CM, et al. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood. 2007;110(9): 3281-90. https://doi.org/10.1182/blood-2007-01-065888.
11. Han B, Yao W, Oh Y-T, Tong J-S, Li S, Deng J, et al. The novel proteasome inhibitor carfilzomib activates and enhances extrinsic apoptosis involving stabilization of death receptor 5. Oncotarget. 2015;6(19): 17532-42. https://doi.org/10.18632/oncotarget.3947.
12. Moriwaki K, Chan FK-M. Regulation of RIPK3- and RHIM-dependent Necroptosis by the Proteasome. J Biol Chem. 2016;291(11): 5948-5959. https://doi.org/10.1074/jbc.M115.700997.
13. Ali M, Mocarski ES. Proteasome inhibition blocks necroptosis by attenuating death complex aggregation. Cell Death Dis. 2018;9(3): 346. https://doi.org/10.1038/s41419-018-0371-x.
14. Jarauta V, Jaime P, Gonzalo O, de Miguel D, Ramírez-Labrada A, Martínez-Lostao L, et al. Inhibition of autophagy with chloroquine potentiates carfilzomib-induced apoptosis in myeloma cells in vitro and in vivo. Cancer Lett. 2016; 382(1):1-10. https://doi.org/10.1016/j.canlet.2016.08.019.
15. Yu P, Zhang X, Liu N, Tang L, Peng C, Chen X. Pyroptosis: mechanisms and diseases. Signal Transduct Target Ther. 2021;6(1): 128. https://doi.org/10.1038/s41392-021-00507-5.
16. Hergueta-Redondo M, Sarrió D, Molina-Crespo Á, Megias D, Mota A, Rojo-Sebastian A, et al. Gasdermin-B Promotes Invasion and Metastasis in Breast Cancer Cells. PLoS One. 2014;9(3): e90099. https://doi.org/10.1371/journal.pone.0090099.
17. Hou J, Zhao R, Xia W, Chang C-W, You Y, Hsu J-M, et al. PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis. Nat Cell Biol. 2020;22(10)1264-1275. https://doi.org/10.1038/s41556-020-0575-z.
18. Miguchi M, Hinoi T, Shimomura M, Adachi T, Saito Y, Niitsu H, et al. Gasdermin C Is Upregulated by Inactivation of Transforming Growth Factor ? Receptor Type II in the Presence of Mutated Apc, Promoting Colorectal Cancer Proliferation. PLoS One. 2016;11(11): e0166422. https://doi.org/10.1371/journal.pone.0166422.
19. Ma Y, Chen Y, Lin C, Hu G. Biological functions and clinical significance of the newly identified long non?coding RNA RP1?85F18.6 in colorectal cancer. Oncol Rep. 2018;40(5): 2648-2658. https://doi.org/10.3892/or.2018.6694.
20. Rogers C, Erkes DA, Nardone A, Aplin AE, Fernandes-Alnemri T, Alnemri ES. Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation. Nat Commun. 2019;10(1): 1689. https://doi.org/10.1038/s41467-019-09397-2.
21. Smalley KSM. Two Worlds Collide: Unraveling the Role of the Immune System in BRAF–MEK Inhibitor Responses. Cancer Discov. 2020;10(2): 176-178. https://doi.org/10.1158/2159-8290.CD-19-1441.
22. Wang Y, Yin B, Li D, Wang G, Han X, Sun X. GSDME mediates caspase-3-dependent pyroptosis in gastric cancer. Biochem Biophys Res Commun. 2018;495(1): 1418-1425. https://doi.org/10.1016/j.bbrc.2017.11.156.
23. Yang W, Liu S, Li Y, Wang Y, Deng Y, Sun W, et al. Pyridoxine induces monocyte-macrophages death as specific treatment of acute myeloid leukemia. Cancer Lett. 2020;492: 96-105. https://doi.org/10.1016/j.canlet.2020.08.018.