Radioresistance is a principal culprit for the failure of radiotherapy in hepatocellular carcinoma (HCC). Insights on the regulation genes of radioresistance and underlying mechanisms in HCC are awaiting for profound investigation. In this study, the suppressor of cytokine signaling 2 (SOCS2) were screened out by RNA-seq and bioinformatics analyses as a potential prognosis predictor of HCC radiotherapy and then were determined to promote radiosensitivity in HCC both in vivo or in vitro. Meanwhile, the measurements of ferroptosis negative regulatory proteins of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), intracellular lipid peroxidation and Fe²⁺ concentration suggested that a high level of ferroptosis contributed to the radiosensitization of HCC. Moreover, SOCS2 and SLC7A11 were expressed oppositely in HCC clinical tissues and tumour xenografts with different radiosensitivities. Mechanistically, the N-terminal domain of SLC7A11 was specifically recognized by the SH2-structural domain of SOCS2. While the L162 and C166 of SOCS2-BOX region could bind elongin B/C compound to co-form a SOCS2/elongin B/C complex to recruit ubiquitin molecules. Herein, SOCS2 served as a bridge to transfer the attached ubiquitin to SLC7A11 and promoted K48-linked polyubiquitination degradation of SLC7A11, which ultimately led to the onset of ferroptosis and radiosensitization of HCC. In conclusion, it was demonstrated for the first time that high-expressed SOCS2 was one of the biomarkers predicting radiosensitivity of HCC by advancing the ubiquitination degradation of SLC7A11 and promoting ferroptosis, which indicates that targeting SOCS2 may enhance the efficiency of HCC radiotherapy and improve the prognosis of patients.

The observation of radiation-induced bystander responses, in which cells respond to their neighbors being irradiated, has important implications for understanding mechanisms of radiation action particularly after low-dose exposure. Much of this questions the current dogma of direct DNA damage driving response in irradiated systems. In this study, we have used a charged-particle microbeam to target individual helium ions ( ³ He ²⁺ ) to individual cells within a population of radioresistant glioma cells cultured alone or in coculture with primary human fibroblasts. We found that even when a single cell within the glioma population was precisely traversed through its cytoplasm with one ³ He ²⁺ ion, bystander responses were induced in the neighboring nonirradiated glioma or fibroblasts so that the yield of micronuclei was increased by 36% for the glioma population and 78% for the bystander fibroblast population. Importantly, the yield of bystander-induced micronuclei was independent of whether the cytoplasm or nucleus of a cell was targeted. The bystander responses were fully eliminated when the populations were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide or filipin, which scavenge nitric oxide (NO) and disrupt membrane rafts, respectively. By using the probe 4-amino-5-methylamino-2′,7′-difluorofluorescein, it was found that the NO level in the glioma population was increased by 15% after 1 or 10 cytoplasmic traversals, and this NO production was inhibited by filipin. This finding shows that direct DNA damage is not required for switching on of important cell-signaling mechanisms after low-dose irradiation and that, under these conditions, the whole cell should be considered a sensor of radiation exposure.

The radiation-induced bystander effect (RIBE) increases the probability of cellular response and therefore has important implications for cancer risk assessment following low-dose irradiation and for the likelihood of secondary cancers after radiotherapy. However, our knowledge of bystander signaling factors, especially those having long half-lives, is still limited. The present study found that, when a fraction of cells within a glioblastoma population were individually irradiated with helium ions from a particle microbeam, the yield of micronuclei (MN) in the nontargeted cells was increased, but these bystander MN were eliminated by treating the cells with either aminoguanidine (an inhibitor of inducible nitric oxide (NO) synthase) or anti-transforming growth factor β1 (anti-TGF-β1), indicating that NO and TGF-β1 are involved in the RIBE. Intracellular NO was detected in the bystander cells, and additional TGF-β1 was detected in the medium from irradiated T98G cells, but it was diminished by aminoguanidine. Consistent with this, an NO donor, diethylamine nitric oxide (DEANO), induced TGF-β1 generation in T98G cells. Conversely, treatment of cells with recombinant TGF-β1 could also induce NO and MN in T98G cells. Treatment of T98G cells with anti-TGF-β1 inhibited the NO production when only 1% of cells were targeted, but not when 100% of cells were targeted. Our results indicate that, downstream of radiation-induced NO, TGF-β1 can be released from targeted T98G cells and plays a key role as a signaling factor in the RIBE by further inducing free radicals and DNA damage in the nontargeted bystander cells.

Purpose : To investigate the signal factor and its function in the medium-mediated bystander effect during heavy-ion irradiation of human salivary gland (HSG) neoplastic cells. Materials and methods : Unirradiated recipient HSG cells were co-cultivated with HSG donor cells irradiated with 290 MeV/u carbon beams having different LET values. Cell proliferation and micronucleus (MN) induction in recipient cells with and without treatment of a NO scavenger (PTIO) were measured and the concentration of nitrite in the co-culture medium was detected. As a direct control, the effects of a nitric oxide (NO) generator (sper/NO) on cell proliferation and MN induction were also examined. Results : Increases in cell proliferation and MN induction were found in the recipient HSG cells as a result of co-culturing and cell proliferation was obviously enhanced during a further subculture. In comparison with 13 keV/ μ m, 100 keV/ μ m carbon-ion irradiation was found to be a more efficient inducer of the medium-mediated bystander effect. The treatment of cells by PTIO resulted in elimination of such effects, which supports a role for NO in the medium-mediated bystander effect. As an oxidization product of NO, nitrite was detected in the co-culture medium, and the dose-response for its concentration was similar to that of cell proliferation and MN induction in the recipient cells. When the HSG cells were treated by sper/NO with a concentration of less than 20 μ M, cell proliferation was enhanced, whereas MN increased along with sper/NO concentration. Conclusion : NO participated in the medium-mediated bystander effects on cell proliferation and MN induction, depending on the LET of irradiation.

DNA-damaging treatments such as radiotherapy (RT) have become promising to improve the efficacy of immune checkpoint inhibitors by enhancing tumor immunogenicity. However, accompanying treatment-related detrimental events in normal tissues have posed a major obstacle to radioimmunotherapy and present new challenges to the dose delivery mode of clinical RT. In the present study, ultrahigh dose rate FLASH X-ray irradiation was applied to counteract the intestinal toxicity in the radioimmunotherapy. In the context of programmed cell death ligand-1 (PD-L1) blockade, FLASH X-ray minimized mouse enteritis by alleviating CD8 ⁺ T cell-mediated deleterious immune response compared with conventional dose rate (CONV) irradiation. Mechanistically, FLASH irradiation was less efficient than CONV X-ray in eliciting cytoplasmic double-stranded DNA (dsDNA) and in activating cyclic GMP-AMP synthase (cGAS) in the intestinal crypts, resulting in the suppression of the cascade feedback consisting of CD8 ⁺ T cell chemotaxis and gasdermin E-mediated intestinal pyroptosis in the case of PD-L1 blocking. Meanwhile, FLASH X-ray was as competent as CONV RT in boosting the antitumor immune response initiated by cGAS activation and achieved equal tumor control in metastasis burdens when combined with anti–PD-L1 administration. Together, the present study revealed an encouraging protective effect of FLASH X-ray upon the normal tissue without compromising the systemic antitumor response when combined with immunological checkpoint inhibitors, providing the rationale for testing this combination as a clinical application in radioimmunotherapy.