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Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells

  • Authors:
    • Satsuki Kishita
    • Naoki Umemura
    • Hiromi Miyazaki
    • Makoto Adachi
    • Hideki Yagi
    • Emika Ohkoshi
  • View Affiliations / Copyright

    Affiliations: Division of Immunobiology, Graduate School of Pharmaceutical Sciences, International University of Health and Welfare, Ohtawara, Tochigi 324‑8501, Japan, Department of Oral Biochemistry, Asahi University School of Dentistry, Mizuho, Gifu 501‑0296, Japan, Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama, Saitama 359‑8513, Japan, Department Oral and Maxillofacial Surgery, Nagoya Tokushukai General Hospital, Kasugai, Aichi 487‑0016, Japan, Division of Pharmacognosy, Graduate School of Pharmaceutical Sciences, International University of Health and Welfare, Ohtawara, Tochigi 324‑8501, Japan
    Copyright: © Kishita et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY 4.0].
  • Article Number: 163
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    Published online on: September 24, 2025
       https://doi.org/10.3892/or.2025.8996
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Abstract

In recent years, immune checkpoint inhibitors such as nivolumab have been used to treat recurrent or metastatic head and neck cancer. However, some patients do not respond to nivolumab, and the treatment options for these patients are limited. Therefore, identifying compounds for developing new therapeutic strategies for intractable cancer is important. The acquired multidrug‑resistant metastatic head and neck squamous cell carcinoma cell line, R HSC‑3, expresses refractory cancer‑specific proteins such as the drug excretion transporter, ATP binding cassette subfamily G member 2, the cancer stem cell markers, CD44, SRY‑box transcription factor 9 and Notch, and the poor prognosis factor, fibroblast growth factor 9, and is a useful in vitro model for acquired multidrug resistance. In the present study, compounds that may be more effective than conventional chemotherapeutic drugs in R HSC‑3 cells were searched and the cell death mechanism was investigated. The results showed that naphthoquinones inhibited the viability of R HSC‑3 cells at low concentrations and induced necroptotic cell death. Naphthoquinone‑induced necroptotic cell death in R HSC‑3 cells induced the expression of calreticulin, an immunogenic marker. It was further found that mitochondrial‑derived reactive oxygen species mediated the oxidative stress damage by naphthoquinone‑induced necroptotic cell death in these cells. Moreover, necroptotic cell death by shikonin, a naphthoquinone, may generate immunogenic signals from multidrug‑resistant cancer cells. The present study revealed that naphthoquinones may not only induce necroptosis in refractory head and neck cancer cells but also induce tumor immunity. Therefore, naphthoquinones may represent a new avenue for the development of new therapeutic agents targeting multidrug‑resistant head and neck cancer.
View Figures

Figure 1

Induction of RIP1 by naphthoquinones
in drug-resistant head and neck squamous cell carcinoma cells. (A)
Chemical structures of naphthoquinones. (B) Western blot analysis
of RIP1 in R HSC-3 cells treated with naphthoquinones for the
indicated times. RIP1, receptor interacting protein 1 kinase.

Figure 2

Effect of oxidative stress on
naphthoquinone-induced cell death. (A) The effect of
naphthoquinones on the viability of R HSC-3 cells. (B) The effect
of NAC pretreatment on the cell viability of naphthoquinone-treated
R HSC-3 cells. *P<0.05. Data are presented as the mean ± SD
(n≥3). NAC, N-acetyl cysteine.

Figure 3

Evaluation of the mitochondrial
membrane potential by JC-1 staining of naphthoquinone-treated R
HSC-3 cells. R HSC-3 cells treated as indicated were stained with
JC-1 (red and green) and Hoechst 33342 (blue). Hoechst33342 was
used to stain nuclei and facilitate visualization of all cells.
JC-1 staining pattern indicates loss of the mitochondrial membrane
potential in treated R HSC-3 cells. Scale bar, 50 µm.

Figure 4

Induction of cell death by
naphthoquinones under oxidative phosphorylation-dominant culture
conditions. (A) Naphthoquinones induced mitochondria dysfunction in
R HSC-3 cells under galactose-containing oxidative conditions. (B)
Effects of NAC on naphthoquinone-treated R HSC-3 cells were
examined. (C) Expression of gasdermin D in R HSC-3 cells treated
with shikonin. Data are presented as the mean ± SD (n≥3). NAC,
N-acetyl cysteine.

Figure 5

Effect of naphthoquinone-induced ROS
production in combination with necroptosis inhibitors on R HSC-3
cells. ROS assay of R HSC-3 cells pretreated with RIP1 inhibitor
(50 µM, 7-O-Cl-Nec1) and MLKL inhibitor (10 µM, necrosulfonamide)
before naphthoquinone treatment (20 µM shikonin or 30 µM acetyl
shikonin, 6 h) and cultured in OXPHOS conditions (culture medium
containing 10 nM 2DG) for 24 h. Naphthoquinones (20 µM shikonin or
30 µM acetyl shikonin) were applied to necroptosis inhibitor
pretreated R HSC-3 cells for 6 h, and ROS production was measured.
Data are presented as the mean ± SD (n=3). **P<0.01,
***P<0.0005. ROS, reactive oxygen species; RIP1, receptor
interacting protein 1 kinase; MLKL, mixed lineage kinase-domain
like; OXPHOS, oxidative phosphorylation; 2DG, deoxy-D-glucose; Glc,
glucose; NSA, necrosulfonamide; RLU, relative fluorescence
units.

Figure 6

Expression of calreticulin in R HSC-3
cells treated with shikonin. (A) Western blot analysis of
calreticulin was performed in R HSC-3 cells treated as indicated.
(B) Fluorescence microscopy imaging of calreticulin in R HSC-3
cells infected with lentivirus expressing RFP-calreticulin at 15,
30 and 60 min after the addition of shikonin. Scale bar, 50 µm.

Figure 7

Induction of immunogenic cell death
by naphthoquinones in R HSC-3 cells. Naphthoquinones cause cell
death by direct or indirect mechanisms that induce mitochondrial
dysfunction. RIP, receptor interacting protein; MLKL, mixed lineage
kinase-domain like; NSA, necrosulfonamide; ROS, reactive oxygen
species; NAC, N-acetyl cysteine.
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Spandidos Publications style
Kishita S, Umemura N, Miyazaki H, Adachi M, Yagi H and Ohkoshi E: Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells. Oncol Rep 54: 163, 2025.
APA
Kishita, S., Umemura, N., Miyazaki, H., Adachi, M., Yagi, H., & Ohkoshi, E. (2025). Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells. Oncology Reports, 54, 163. https://doi.org/10.3892/or.2025.8996
MLA
Kishita, S., Umemura, N., Miyazaki, H., Adachi, M., Yagi, H., Ohkoshi, E."Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells". Oncology Reports 54.6 (2025): 163.
Chicago
Kishita, S., Umemura, N., Miyazaki, H., Adachi, M., Yagi, H., Ohkoshi, E."Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells". Oncology Reports 54, no. 6 (2025): 163. https://doi.org/10.3892/or.2025.8996
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Spandidos Publications style
Kishita S, Umemura N, Miyazaki H, Adachi M, Yagi H and Ohkoshi E: Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells. Oncol Rep 54: 163, 2025.
APA
Kishita, S., Umemura, N., Miyazaki, H., Adachi, M., Yagi, H., & Ohkoshi, E. (2025). Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells. Oncology Reports, 54, 163. https://doi.org/10.3892/or.2025.8996
MLA
Kishita, S., Umemura, N., Miyazaki, H., Adachi, M., Yagi, H., Ohkoshi, E."Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells". Oncology Reports 54.6 (2025): 163.
Chicago
Kishita, S., Umemura, N., Miyazaki, H., Adachi, M., Yagi, H., Ohkoshi, E."Induction of immunogenic necroptosis by shikonin in drug‑resistant head and neck squamous cell carcinoma cells". Oncology Reports 54, no. 6 (2025): 163. https://doi.org/10.3892/or.2025.8996
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