J Cancer 2021; 12(5):1520-1530. doi:10.7150/jca.46316

Research Paper

Carbon ion triggered immunogenic necroptosis of nasopharyngeal carcinoma cells involving necroptotic inhibitor BCL-x

Cihang Bao1,3#, Yun Sun2,3#, Bilikere Dwarakanath2,3, Yuanli Dong1,3,4, Yangle Huang1,3,4, Xiaodong Wu2,3, Chandan Guha5, Lin Kong1,3✉, Jiade J. Lu1,3✉

1. Department of Radiation Oncology, Shanghai Proton and Heavy Ion Center, Fudan University Cancer Hospital, Shanghai, China
2. Department of Research and Development, Shanghai Proton and Heavy Ion Center, Shanghai, China
3. Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai, China
4. Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, China
5. Department of Radiation Oncology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, USA
#Both authors (Drs. Cihang Bao and Yun Sun) contributed equally to this article.

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Bao C, Sun Y, Dwarakanath B, Dong Y, Huang Y, Wu X, Guha C, Kong L, Lu JJ. Carbon ion triggered immunogenic necroptosis of nasopharyngeal carcinoma cells involving necroptotic inhibitor BCL-x. J Cancer 2021; 12(5):1520-1530. doi:10.7150/jca.46316. Available from https://www.jcancer.org/v12p1520.htm

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To explore the potential and mechanisms of necroptosis, a form of immunogenic cell death, induced by carbon ion as compared to photon beams in established photon resistant- (PR-) and sensitive nasopharyngeal carcinoma (NPC) cells. MLKL is considered a central executor of necroptosis and phosphorylation of MLKL (p-MLKL) was a critical event of necroptosis. The clonogenic survival and DNA microarray demonstrated that after repeated photon irradiation, radiosensitive NPC cells became apoptosis-resistant but could be effectively inhibited by carbon ion irradiation. The relative biologic effectiveness (RBE) at D10 and D37 were 2.15 and 2.78 for PR-NPC cells. Carbon ion induced delayed DNA damage repair, cell cycle arrest, cytogenetic damage, morphological change and cell necrosis, indicating the possibility of necroptosis in both PR- and sensitive NPC cell types. The lower expression of necroptotic inhibitors (caspase-8 and Bcl-x) and higher level of MLKL in PR-NPC cells showed it was relatively more predisposed to necroptosis compared to the sensitive cells. Subsequent experiments demonstrated the significant upregulation of p-MLKL in the PR-NPC cells treated by carbon ion (4 Gy) compared with photon irradiation at both physical (4 Gy) and RBE (10 Gy) doses (P≤0.0001). Moreover, carbon ion induced a robust (up to 28 folds) p-MLKL in the PR-NPC cells as well as sensitive cells (up to 6-fold) coupled with a lower level of BCL-x expression and increased GM-CSF implicated in resculputure of immune system. These results suggested that carbon ion could induce necroptosis of NPC cells, especially in PR-NPC cells, and its mechanisms involve BCL-x.

Keywords: carbon ion irradiation, necroptosis, immunogenic cell death, nasopharyngeal carcinoma, photon-resistant cells, mixed lineage kinase domain-like pseudokinase