Carbon-Ion Radiotherapy Drives Ferroptosis in Gastric Cancer via DHODH

Study Background and Research Question

Gastric cancer remains a major global health burden, ranking among the most frequently diagnosed malignancies and leading causes of cancer-related mortality. Conventional therapies, including surgery and photon-based radiotherapy, face significant limitations when treating advanced or metastatic disease due to insufficient local control and the risk of damaging surrounding healthy tissues. Carbon-ion radiotherapy (CIRT), a high linear energy transfer (LET) modality characterized by the Bragg peak effect, offers superior dose distribution and relative biological effectiveness compared to photons or protons. Despite these advantages, the specific mechanisms underlying CIRT’s impact on gastric cancer, and its interplay with tumor cell death and immune modulation, have not been fully elucidated. A key consideration in radiotherapy resistance is the tumor’s metabolic state. Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme involved in pyrimidine biosynthesis and has been implicated in cancer progression and radiosensitivity. Wang and Cai (2025) set out to determine whether CIRT exerts anti-tumor effects in gastric cancer by regulating DHODH, thereby modulating ferroptosis (an iron-dependent, non-apoptotic cell death pathway) and macrophage polarization.

Key Innovation from the Reference Study

The principal innovation of this study lies in demonstrating, for the first time, a direct mechanistic link between CIRT, DHODH downregulation, ferroptosis induction, and M1 macrophage polarization in gastric cancer models. By integrating in vitro and in vivo models, the authors reveal that CIRT not only triggers ferroptosis in tumor cells but also fosters a tumor microenvironment supportive of anti-tumor immunity via macrophage reprogramming. The suppression of DHODH emerges as a pivotal event, highlighting its potential as a radiosensitization target to maximize the therapeutic benefits of CIRT.

Methods and Experimental Design Insights

The research employed a combination of human gastric cancer cell lines (HGC27, AGS) and murine xenograft models to dissect the biological effects of CIRT. Key experimental approaches included:

• Exposure of tumor cells to CIRT at doses of 0 Gy, 2 Gy, and 4 Gy, with assessment of cell viability, migration, and invasiveness using MTT and Transwell assays.
• Measurement of intracellular iron and reactive oxygen species (ROS) accumulation, key indicators of ferroptosis, following irradiation.
• Evaluation of DHODH and ferroptosis-related marker expression (e.g., ACSL4, GPX4) by Western blotting and quantitative RT-PCR.
• Analysis of macrophage polarization using flow cytometry, with macrophages exposed to conditioned medium from irradiated tumor cells to probe immune modulation.
• Establishment of BALB/c nude mouse xenograft models (subcutaneous AGS cell injection), randomized into control, CIRT, and DHODH-overexpressing CIRT groups, to assess tumor growth and in vivo molecular changes.

Protocol Parameters

• CIRT treatment: Administered at 0, 2, and 4 Gy to gastric cancer cell lines; higher doses correlated with greater biological effects.
• Macrophage polarization assay: Tumor-conditioned medium from irradiated cells used to treat macrophages; polarization assessed by CD86+CD206− surface markers.
• Protein detection: Western blotting to quantify DHODH, ACSL4, GPX4, and cytokines associated with M1/M2 phenotypes.
• Tumor xenograft model: AGS cells injected subcutaneously; mice randomized for treatment arms; tumor volume and marker expression tracked post-treatment.

Core Findings and Why They Matter

According to the reference study, CIRT significantly inhibited the viability, migration, and invasion of gastric cancer cells. Mechanistically, CIRT suppressed DHODH expression, which led to increased intracellular iron and ROS levels—hallmarks of ferroptosis. This was corroborated by upregulation of ACSL4 and downregulation of GPX4, two molecular markers of ferroptotic cell death. Importantly, the study discovered that conditioned medium from CIRT-treated tumor cells promoted M1-like polarization of macrophages, as evidenced by increased CD86+CD206− populations and elevated M1-associated cytokines. In vivo, CIRT reduced tumor growth in xenografted mice, with the anti-tumor effect attenuated by forced DHODH overexpression, underscoring DHODH’s functional relevance. These findings position CIRT as a dual-action therapy: directly triggering tumor cell ferroptosis and indirectly enhancing anti-tumor immunity via macrophage reprogramming.

Comparison with Existing Internal Articles

Recent internal resources, such as Elevating Translational Research: ECL Chemiluminescent Kit Insights, emphasize the importance of sensitive and reproducible detection technologies for elucidating complex cell death and immune pathways. The APExBIO ECL Chemiluminescent Substrate Detection Kit, highlighted in these workflow guides, is routinely used for Western blot chemiluminescence detection of proteins such as ACSL4, GPX4, and DHODH. This aligns closely with the protein detection strategies employed by Wang and Cai, underscoring the translational value of robust chemiluminescent substrate kits in cancer radiobiology research. Further, the article ECL Chemiluminescent Substrate Detection Kit: Precision & Workflow Insights offers protocol enhancements and troubleshooting relevant to the detection of low-abundance cell death markers—a challenge frequently encountered in ferroptosis and immuno-oncology research. These internal guides complement the referenced study by providing actionable workflow improvements for protein detection by ECL in similar experimental frameworks.

Limitations and Transferability

Wang and Cai’s findings, while compelling, are subject to certain limitations. The mechanistic insights were derived from established cell lines and immunodeficient mouse models, which may not fully recapitulate the complexity of human gastric tumors or the broader immune context. The impact of CIRT and DHODH modulation on other tumor types and in immunocompetent settings remains to be systematically explored. Additionally, the specificity and safety of targeting DHODH for radiosensitization warrant further preclinical and clinical validation. Transferability of the protocols and findings to other cancer systems is plausible, particularly given the conserved nature of ferroptosis pathways and macrophage polarization. However, tissue-specific microenvironmental factors and differences in CIRT accessibility across clinical centers represent important real-world constraints.

Research Support Resources

For researchers seeking to reproduce or extend these findings, reliable detection of ferroptosis and immune markers is essential. The ECL Chemiluminescent Substrate Detection Kit (SKU K1129) from APExBIO is widely adopted for chemiluminescent immunoassay and Western blot applications, enabling sensitive detection of HRP-conjugated antibodies targeting proteins such as DHODH, ACSL4, and GPX4. When incorporated into workflows similar to those described in Wang and Cai (2025), this chemiluminescent substrate kit supports accurate protein quantification and enhances reproducibility in protein detection by ECL. Researchers are encouraged to consult in-depth protocols and troubleshooting guides referenced above for best practices in Western blot chemiluminescence detection and chemiluminescent immunoassay, ensuring optimal sensitivity and reliability in translational cancer research.