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

Cihang Bao; Yun Sun; Bilikere Dwarakanath; Yuanli Dong; Yangle Huang; Xiaodong Wu; Chandan Guha; Lin Kong; Jiade J Lu

doi:10.7150/jca.46316

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

J Cancer. 2021 Jan 1;12(5):1520-1530. doi: 10.7150/jca.46316. eCollection 2021.

Authors

Cihang Bao^{1

2}, Yun Sun^{3

2}, Bilikere Dwarakanath^{3

2}, Yuanli Dong^{1

2

4}, Yangle Huang^{1

2

4}, Xiaodong Wu^{3

2}, Chandan Guha⁵, Lin Kong^{1

2}, Jiade J Lu^{1

2}

Affiliations

¹ Department of Radiation Oncology, Shanghai Proton and Heavy Ion Center, Fudan University Cancer Hospital, Shanghai, China.
² Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai, China.
³ Department of Research and Development, Shanghai Proton and Heavy Ion Center, 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.

Abstract

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; immunogenic cell death; mixed lineage kinase domain-like pseudokinase; nasopharyngeal carcinoma; necroptosis; photon-resistant cells.