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. 2026 Mar 18;26:526. doi: 10.1186/s12885-026-15865-y

CS2164 combined with radiation suppressed tumor growth via γ-H2AX persistence and ROS accumulation

Shun-li Peng 1,#, Yu-ling Zhou 2,#, Xuexia Liang 1,#, Ren-wei Jiang 1, Shuai Yang 1, Qiao-dan Liu 1,3,✉, Xiao-mou Peng 3,✉
PMCID: PMC13112730  PMID: 41845287

Abstract

Background

CS2164 is an oral multi-target inhibitor that targets angiogenesis-related kinases, mitosis-related kinases, and chronic inflammation-related kinases. However, the added clinical benefit of CS2164 combined with radiation remains unknown.

Methods

The anti-tumor effect of CS2164 combined with radiation were evaluated by CCK-8, colony formation assays and xenograft radiation models (n = 5/per group), based on the hepatocellular carcinoma cells (Huh-7) and cervical cancer cells (HeLa). Meanwhile, the γ-H2AX expression, cell cycle distribution, reactive oxygen species (ROS) assay and transcriptome sequencing analysis of xenografts were performed selected to clarify the mechanism.

Results

CS2164 combined with radiation effectively decreased growth of the above tumor cells, compared with CS2164 or radiation alone. The sensitivity enhancement ratio (SER, SER=D0(control group) / D0(treated group)) of CS2164 was 1.406 in HeLa cells and 1.123 in Huh-7 cells, respectively. Similar antitumor effects were observed in xenograft radiation models. The results of GO, KEGG pathway and PPI analysis screened out a series of genes that related to DNA damage repair process, oxidation-reduction reaction, and multiple cell death types. Therefore, γ-H2AX expression, cell cycle distribution and ROS assay were detected. There was no significant difference in the number of γ-H2AX foci between control and CS2164-treated cells. While the numbers of γ-H2AX foci increased in the CS2164-treated (3µM) cells after radiation (4 Gy) at 3 and 6 h, compared to cells treated with radiation alone (p < 0.05). Meanwhile, more cells were observed to be blocked in G2/M phase after treatment of CS2164 combined with radiation, compared with CS2164 or radiation alone. Moreover, the ROS level increased after treatment of CS2164 combined with radiation, which could be partially rescued by oxidation scavenger, NAC.

Conclusion

Our study demonstrates anti-tumor efficacy of CS2164 combined with radiation in treatment of malignant tumors.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-026-15865-y.

Keywords: CS2164, Radiation, DNA damage response, Redox imbalance

Background

Radiotherapy (RT) is an important treatment for more than 50% of cancer patients [1]. In the management of cervical cancer, radical radiotherapy possesses considerable significance, particularly those locally advanced patients. However, intrinsic or acquired radio-resistance remains a major problem that impedes the efficacy of RT and combination therapy [2, 3], therefore leading to local recurrence and distant metastasis. The intricate mechanism of radiation resistance involves a multifaceted interplay, including DNA damage repair, the tumor micro-environment, hypoxia, cell cycle arrest, tumor stem cells, and epigenetic mechanisms [4]. The similar treatment challenge encounter in hepatocellular carcinoma (HCC). A variety of therapeutic options are available for HCC, encompassing surgical management with liver transplant or resection, other locoregional therapies (LRTs), and systemic therapies [5]. The systemic therapies are based on tyrosine kinase inhibitors (TKIs) and immunotherapy-based regimens. RT were found to be a promising therapeutic, based on the pooled hazard ratios (HRs) from six studies that comparing RT-containing treatment with other therapies [6]. Moreover, radiotherapy combined with targeted and immunotherapy could improved overall survival (OS) and progression-free survival (PFS) [7, 8]. How and when to use one intervention or combination therapies requires robust patient-centered and multidisciplinary discussion. Therefore, a comprehensive understanding among tumor could improve the efficacy of combination therapies.

Previous studies showed that RT inhibits tumor growth through inducing endogenous and exogenous DNA damage [9]. Ionizing radiation can trigger exogenous DNA damage and promote a prompt set of signaling events known as the DNA damage response (DDR) [10, 11]. These coordinate the DNA repair process, and ultimately induce cell death or senescence [1, 12–14]. After irradiation, endogenous DNA damage is induced by cellular factors such as hydrolysis, oxidation, alkylation, or reactive chemical species (e.g., reactive oxygen species (ROS), reactive nitrogen species). However, tumor cells that undergo repair of single- or double-strand breakages (DSBs) could ultimately survive, inducing radio-resistance. Therefore, common strategies to improve the efficacy of radiotherapy include interfering with the DNA damage repair process [15, 16], inducing cell cycle arrest [17], and promoting oxidation-reduction imbalance [18–20]. Small molecule compounds that target the above signaling pathways could enhance the efficacy of radiotherapy, leading to a synergistic anti-tumor effect.

CS2164 [21–25] is a novel oral multi-target inhibitor that targets the angiogenesis-related kinases (VEGFR1, VEGFR2, VEGFR3, and c-Kit), the mitosis-related kinase Aurora B, and the chronic inflammation-related kinase CSF-1R. Previous studies found that CS2164 inhibited the proliferation of acute myeloid leukemia [22], Non-Hodgkin’s lymphomas [23], and hepatocellular carcinoma (HCC) [26] by altering angiogenesis and mitosis, modulating ROS, as well as regulating the immune microenvironment. In early-phase clinical trials, CS2164 shows anti-tumor effect with escalated dose of 65 mg/d [27]. The combination of CS2164 (50 mg/d) with chemotherapy have also been explored [24]. While CS2164 has shown anti-tumor activity in preclinical models and early clinical trials, its potential role in combination with radiation has not been explored. Here, for the first time, we investigated the combination of CS2164 with radiotherapy and its underlying mechanisms in preclinical models of HCC and cervical cancer. We found that low concentration of CS2164 (3µM in vitro, 10 mg/d in vivo) could improve the radiosensitivity of tumor cells by interfering with DNA damage repair process. These findings may provide a theoretical basis for the potentially application of CS2164 combined with radiation in malignant tumors.

Methods

Cells and reagents

The Huh-7 human HCC cells were purchased from the American Type Culture Collection (ATCC), Manassas, Virginia, USA. The HeLa human cervical cancer (CC) cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Huh-7 cells were cultured in standard Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin solution (15070063, GBICO, Grand Island, NY, USA). HeLa cells were maintained in Minimum Essential Medium (MEM) (11095080, GBICO, Grand Island, NY, USA) with 1% MEM Non-Essential Amino Acid Solution (11140050, GBICO, Grand Island, NY, USA), 10% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin solution. All cell lines were cultured in a humidified CO2 incubator at 37 °C, and authenticated by short-tandem repeat (STR) analysis before the start of this study. All of the cells were passaged for less than 3 months before renewal from frozen [28, 29].

Cell growth and colony formation assays

Cell growth was measured via cell counting kit-8 (CCK-8) assay (HY-K0301, MedChemExpress, NJ, USA). Tumor cells (2,000/well) were plated into each well of 96-well plates and incubated for 24 h. Then, CS2164 solutions of various concentrations were added into each well. The concentrations used in our in vitro studies (1–16 µM) were chosen based on the previously published articles of CS2164 [22, 30]. After 48 h of incubation, the medium was carefully removed, and CCK-8 working solutions were added for another 2 h. Finally, absorbance was measured with a microplate reader at test and reference wavelengths of 450 nm. The percentage of growth is shown relative to the untreated controls. Each experiment was done in triplicate.

Colony formation assay was used to evaluate the radio-sensitivity of tumor cells. Tumor cells were plated into each well of 6-well plates and incubated for 24 h. Then, CS2164 or equivalent DMSO solution was added. After a further 12 h incubation, all cells were exposed to radiation using a linear accelerator (Elekta Infinity; Stockholm, Sweden) at a dose of 0, 2, 4, 6, and 8 Gy. Then, the cells were further cultured under standard conditions for 10–14 days and fixed with 4% paraformaldehyde (PFA) for 30 min, followed by staining with 0.5% crystal violet at room temperature for 20 min. The colonies containing ≥ 50 cells were counted, and the cell survival curves were fitted with the single-hit multi-target model (y = (1 − e − D/D0) n). The sensitivity enhancement ratio (SER, SER=D0(control group)/D0(treated group)) was determined as the ratio of the mean inactivation dose under radiation-only conditions to the mean inactivation dose after CS2164 plus radiation treatment [31, 32].

Immunofluorescence staining, cell cycle detection, and reactive oxygen species detection

Tumor cells (10,000/well) were plated into 24-well plates containing adhesive glass slide and incubated for 24 h. Then, CS2164 (3µM) or equivalent DMSO solution was added. After a further 12 h incubation, all cells were exposed to radiation at a dose of 0–4 Gy, and continued to incubate under standard conditions. Finally, tumor cells were sequentially collected for further 0, 1, 3, 6 h after radiation. Finally, the above cells were collected and performed by immunofluorescence staining, as previously described [28, 33]. The human anti-phospho-Histone H2A.X (Ser139, 1:400 dilution, #9718), and anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate, #4412) were purchased from Cell Signaling Technology. Antifade solution with DAPI (#S2110) was purchased from Solarbio life sciences, Beijing, China. The γ-H2AX foci numbers were counted using the image and analysis processing by ImageJ software (National Institutes of Health, USA).

Tumor cells (100,000/well) were plated into 6-well plates, and treated with CS2164 (3µM) and/or radiation (4 Gy), according to the above method. Then, all the treated cells were incubated for further 24 h after radiation, and detected by cell cycle staining kit (lot #70-CCS012, MultiSciences, China) or ROS assay kit (S0033S, Beyotime Biotechnology, Shanghai, China), according to the manufacturer’s instructions. NAC (C8460, Solarbio, Beijing, China) was purchased to rescue the ROS level at a concentration of 5µM. The mean fluorescence intensity (MFI) and mean specific fluorescence intensity (MSFI) were measured by flow cytometer to evaluate the ROS level. The FlowJo software was used for cell cycle analysis. Each experiment was performed in triplicate.

Xenograft tumor experiments

BALB/c nude mice (6 weeks old) were purchased from Beijing SiPeiFu Biotechnology and maintained in a specific pathogen-free (SPF) room at the experimental animal center of the fifth affiliated hospital of Sun-Yat Sen University, Zhuhai, China. Tumor cells (5 × 106 per mouse) were injected subcutaneously into the nude mice. A priori power analysis (α = 0.05, power = 0.8) indicated that a minimum of n = 5 mice per group was required to detect difference in tumor volume between the radiation alone and combination groups. When the average volume of the xenografts grew to 150–200 mm3 (volume = (width2 × length) × 0.5), nude mice were randomized into four groups (n = 5 per group): control group, CS2164 treatment group, radiation treatment group, and CS2164 + radiation treatment group, using a random number generator. Then, CS2164 (10 mg/kg/day) or its control co-solvent was administered daily by oral gavage. Before radiation, all mice were anesthetized intraperitoneally with 100-150ul of 0.3% sodium pentobarbital, and shielded by a lead shield, so that only the xenograft tumor exposed, as previously described [34]. Radiation treatments were given from Monday to Friday at a dose of 2 Gy with a dose rate of 1 Gy/min. The total therapeutic radiation dose was 2 Gy × 10 F on weekdays. The mice of non-irradiation group only received anesthesia. Then the anesthetized mice were placed in a warm environment for resuscitation until complete recovery. After the last radiation treatment, all the mice were sacrificed by cervical dislocation after deeply anesthetized intraperitoneally with 500ul of 0.3% sodium pentobarbitalto, and the xenograft tumors were collected after 6 hours of irradiation. Investigators measuring tumor volume and performing histological analysis were blinded to the treatment groups. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Fifth Affiliated Hospital of Sun Yat-sen University (No. 00234, Zhuhai, China), and were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines.

Immunohistochemistry (IHC)

IHC staining was performed on 3 μm paraffin sections, as previously described [35]. The anti-CD31 monoclonal antibody (1:800 dilution, #3528) and Ki-67 monoclonal antibody (1:400 dilution, #9027) were purchased from Cell Signaling Technology. The horseradish peroxidase-conjugated secondary antibodies (SP-9000, PV-9000) and DAB staining solution (SP-9000-D) were purchased from Beijing Zhongshan Gold Bridge Biotechnology Co., Ltd. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using TdT-mediated TUNEL method, performed according to manufacturer’s instructions of Apoptosis Detection System (Promega). The IHC staining was evaluated by two pathologists.

Transcriptome sequencing and bioinformatics analysis

Xenografts were collected from nude mice and further analyzed by next-generation sequencing technology to obtain gene expression profiling at the level of the transcriptome. The RNA-seq methods have been described in the supplementary materials. Then, differentially expressed genes (DEGs) between the CS2164 plus radiation treatment group and the control group were identified using |log2FoldChange| ≥ 1 and p < 0.05. In total, 16,707 genes were detected, and 233 DEGs were screened, with 211 genes up-regulated and 22 genes down-regulated. Then Gene ontology (GO), which included Biological Process, Molecular Function, and Cellular Component, was analyzed and visualized [28, 36] with Cytoscape ClueGO (two-sided hypergeometric test, adjusted p-value < 0.05 corrected with the Benjamini-Hochberg procedure) and the R package Circlize. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs was performed with the cluster Profiler R package. To further explore changes in metabolism, significant pathways were divided according to nucleotide, amino acid, lipid, and glucose metabolism. In addition, to further show changes in cancer signaling pathways, the top eight pathways were mapped.

A protein-protein interaction (PPI) network was constructed with STRING database analysis to evaluate interactive associations among all DEGs. The combined node scores ranged from 0.900 to 0.999, and the co-expression coefficient was more than 0.700. Among the regulatory networks, a series of DEGs with high interaction scores (> 0.9), high combined scores (> 0.9), and high co-expression coefficients (> 0.9) were screened out. Then, the network was clustered by MCL inflation parameter.

Statistical analysis

All the data are presented as the mean ± standard deviation (SD) of three independent experiments. Two-tailed Student’s t test and one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons were used for group comparisons using SPSS software (SPSS, version 26.0, IBM Corp., Armonk, USA). variance homogeneity was checked using Levene’s test before applying ANOVA. A P value < 0.05 was considered statistically significant.

Results

CS2164 combined with radiation significantly decreased the proliferation of malignant tumor cells

CS2164 is a promising multi-target inhibitor of mitosis, angiogenesis, and chronic inflammation-related kinases [21]. However, the added clinical benefit of CS2164 combined with radiation and its underlying mechanisms remain unknown. Radiotherapy plays a crucial role in the comprehensive treatment regimen for patients with hepatocellular carcinoma (HCC) and cervical cancer (CC) [6, 37], and the radiosensitivity of tumor significantly impacts the clinical efficacy and patient survival. To assess the anticancer effects of CS2164, HCC and cervical carcinoma (CC) cells were selected due to their moderate sensitivity to radiotherapy. As shown in Fig. 1a-b, CCK-8 assays showed that CS2164 decreased the growth of Huh-7 (HCC HBV−) and HeLa (CC HPV+) cells in a dose-dependent manner. The IC50s were 11.31 (95% CI, 10.40–12.40), and 10.99 (95% CI, 9.44–13.17) µM in Huh-7 and HeLa, respectively.

Fig. 1.

Fig. 1

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CS2164 combined with radiation significantly decreased the proliferation of malignant tumor cells. a, b The growth of Huh-7and HeLa cells at different concentration of CS2164. c-f Colony formation of Huh-7 and HeLa cells at 0, 2, 4, 6 Gy dose of radiation with or without the treatment of CS2164. Data are presented as the means ± standard deviation (SD), n ≥ 3; ns, not significant; *p < 0.05; **p < 0.01, ***p < 0.001

Colony formation assays were performed to further evaluate the effect of CS2164 combined with radiation. Results showed that the SER was 1.123 and 1.406 in Huh-7 and HeLa cells, respectively (Fig. 1c–f). The above results indicated that CS2164 in low concentration combined with radiation may effectively inhibit the growth and enhance the radiosensitivity of malignant tumors in vitro.

CS2164 combined with radiation decreased the growth of xenografts in vivo

Because in vitro experiments cannot evaluate angiogenesis and changes in the tumor microenvironment, we further evaluated the effect and safety of CS2164 combined with radiation in vivo. Nude mouse xenograft models were constructed as previously described [38]; the flow diagram of the therapeutic design is shown in Fig. 2a. In the Huh-7 xenografts, the tumor volume was decreased in the CS2164 and radiation groups with statistically significance, compared with the control and the radiation groups (Fig. 2b-c, p < 0.001, p = 0.006, respectively). CS2164 combined with radiation effectively suppressed tumor growth compared with the control group. Similar results were found in HeLa xenografts (Fig. 2d-e, Fig. S1a-b, p < 0.001, p = 0.018, respectively). Additionally, there was no significant difference in body weight among the four groups of mice, suggesting that the combination treatment was well tolerated in nude mice (Fig. S1c-d).

Fig. 2.

Fig. 2

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CS2164 combined with radiation decreased the growth of xenografts in vivo. a The flow diagram of in vivo experiment. Tumor cells were injected into the right thigh of each mouse, then drug and radiation were delivered when the tumor reached 150–200 mm3. Drug: CS2164 (10 mg/kg) or placebo was delivered daily by oral gavage for 14 days. Radiation was delivered at a dose of 2 Gy for 10 times, and fractionated for 5 times a week. Mice were sacrificed after all treatment finished. b, c Xenograft images (b) and tumor volumes at 0, 3, 6, 9, 12 days (c) in four groups of Huh-7. d-e. Xenograft images (d) and tumor volumes at 0, 3, 6, 9, 12 days (e) in four groups of HeLa. Data are presented as the means ± standard deviation (SD), n ≥ 3; ns, not significant; *p < 0.05; **p < 0.01, ***p < 0.001

The H&E staining results indicated no significant morphological abnormalities among the above-mentioned groups (Fig. 3a-b). Additionally, the percentage of Ki-67 positive cells and the area of CD31-positive endothelial cells significantly decreased in the combination treatment group compared to control group (Fig. 3a-b, S2a-b). Meanwhile, higher proportion of TUNEL positive cells were detected after treatment of CS2164 combined with radiation, compared with CS2164 or radiation alone. These results indicated that CS2164 combined with radiation could effectively inhibit the growth of malignant tumors in vivo.

Fig. 3.

Fig. 3

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CS2164 combined with radiation effectively inhibited the proliferation in vivo. a The H&E, Ki-67 and TUNEL staining images (left), positive percent rates of Ki-67 (medium) and positive percent rates of TUNEL staining (right) in Huh-7 xenografts. b The H&E, Ki-67 and TUNEL staining images (left), positive percent rates of Ki-67 (medium) and positive percent rates of TUNEL staining (right) in HeLa xenografts. Data are presented as the means ± standard deviation (SD), n ≥ 3; ns, not significant; *p < 0.05; **p < 0.01, ***p < 0.001

A comprehensive landscape changes after treatment of CS2164 combined with radiation based on transcriptome sequencing results

Transcriptome sequencing using Huh-7 xenografts was performed to clarify the anti-cancer mechanism of CS2164 combined with radiation. Differentially expressed genes between the combination treatment group and the control group were identified for GO and KEGG pathway analysis (Fig. S3-5, Fig. 4a-c). As shown in Fig. S3-5, Biological Process enrichment analysis indicated that signatures were related to response to ROS metabolic processes, cell migration, kinase activity, response to lipids and immune response. Furthermore, the results of the Cellular Component analysis showed that most DEGs were distributed in the plasma membrane, microtubule bundles and collagen, synapses, etc. Moreover, enrichment related to oxidoreductase activity, signaling receptor binding, transporter activity, phospholipid binding, cell adhesion molecule binding, were identified, based on Molecular Function analysis. Among the KEGG results, most DEGs were closely associated with metabolic pathways such as purine metabolism, valine, leucine, and isoleucine degradation, pyruvate metabolism, glycerophospholipid metabolism, fatty acid metabolism, and glycolysis (Fig. 4a, b). Moreover, a series of DEGs were enriched in cancer-related signaling pathways (PI3K-Akt, MAPK, phosphatidylinositol, tumor necrosis factor (TNF), HIF-1, ErbB, and Notch signaling) (Fig. 4a, c). Meanwhile, a series of signatures related to cell death types were identified including lysosome, cellular senescence, apoptosis, oxidative phosphorylation, and peroxisomes (Fig. 4a).

Fig. 4.

Fig. 4

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Significantly changes among the CS2164 combined with radiation group compared with the control group. a The KEGG analysis results based on Differentially expressed genes (DEGs) between the CS2164 plus radiation treatment group and the control group. The top changed pathways were displayed using a bubble chart. The significantly changed pathways were arranged according to GeneRatio values. The size of the circle represented the number of DEGs in each pathways, and the color of the circle from blue to red represented an increase in p-value. b The detail pathways enriched in the Metabolic pathways were displayed according to GeneRatio values. The size of the circle represented the number of DEGs in each pathways, and the color of the circle from blue to dark represented an increase in p-value. c The detail pathways enriched in the Pathways in cancer were displayed according to GeneRatio values. The size of the circle represented the number of DEGs in each pathways, and the color of the circle from white to blue represented an increase in p-value

Regarding changes in the tumor environment, the actin cytoskeleton and various immune-related pathways were screened and mapped, as shown in Fig. 4a. These included the T cell receptor signaling pathway, PD-1/PD-L1 signaling pathway, and inflammatory mediator regulation of TRO channels. Based on the above transcriptomics changes, we hypothesized that CS2164 combined with radiotherapy might decrease the growth of malignant tumors by multiple cell death types through multiple signaling pathways.

Protein-protein interaction (PPI) regulatory network analysis identified the molecular changes of CS2164 combined with radiation treatment

After mapping the comprehensive landscape (Fig. S3-5, 4a-c), we then identified the core regulatory molecules. STRING database was utilized to construct a PPI regulatory network, based on DEGs with a threshold of log2FC ≥ 1 and p < 0.05 (Fig. S6). In total, 19 clusters were mapped and marked with distinct color, respectively. The dark golden cluster comprised H2AC11, H2BC5, H2BU1, and H4C3, which are the core components of nucleosome that regulate the DNA damage repair process. And calcium channels cluster, which contains CABP4, CACNA1D, CACNA2D2, CACNB3, CATSPERE, GNG3, were colored in red. Regarding the tumor microenvironment, the cluster that related to microtubule and collagen formation were mapped, including COL1A2, COL1A2, COL11A2, FMOD, TIMP1, COL12A1, ANXA1, COL7A1, LAMC2. Additionally, MUC1, MUC13, MUC3A, MUC6 and GALNT5 were grouped in a brown cluster, which are involved in cell adhesive and anti-adhesion process. Those findings may help shed light on key molecules for overcoming the radiation resistance in malignant tumors.

CS2164 induced γ-H2AX persistence and ROS accumulation in malignant tumors

The DDR process is an important repair way triggered by ionizing radiation. The DDR process disfunction after radiation may leads to redox imbalance and cell cycle arrest [39], therefore induce radiation resistance. Among the results of comprehensive landscapes and PPI regulatory network (Fig. 4a, Fig. S3-6), the pathways related to DDR process, the oxidation-reduction reaction, the microtubule formation were frequently screened out. Therefore, we detected the foci number of phosphorylated histone H2AX (γ-H2AX), ROS level and cell cycle distribution in tumor cells after treatment of CS2164 combined with radiation. γ-H2AX expression is a sensor of DNA double-strand breakages to evaluate the DDR process in tumor cells. As we previously found that CS2164 (3µM) plus radiation (4 Gy) reduced the survival rate in all four malignant tumor cell lines, compared to cells treated with radiation alone (Fig. 1e–h), Therefore, X-ray at a dose of 4 Gy was selected for the following experiments. As shown in Fig. 2a-b, there was no significant difference in foci number between control and CS2164-treated (3µM) cells at 0 h after radiation, indicating that CS2164 had no effect on the initial level of double-strand breakages. When CS2164 and radiation were combined, more extensive γ-H2AX foci were detected at 1 h compared to cells treated with radiation (4 Gy) alone in Huh-7 cells (34.86 ± 10.69 vs. 16.28 ± 5.05) and HeLa cells (32.50 ± 5.74 vs. 15.8 ± 4.06). Similar results were found at time of 3 houres and 6 h after radiation in Huh-7 cells (12.27 ± 2.52 vs. 9.73 ± 2.78; 7.35 ± 2.93 vs. 1.39 ± 1.12), as well as in HeLa cells (8.37 ± 2.13 vs. 3.79 ± 1.85; 6.35 ± 5.64 vs. 1.31 ± 1.12). These results indicated that CS2164 might enhance radiation-induced DNA DSBs and probably suppress the DNA damage repair process in malignant tumors.

To detect the cell cycle distribution and bulk cellular ROS level in tumor cells after treatment of CS2164 combined with radiation, flow cytometry was performed. As shown in Fig. 6a-b, more cells were blocked in the G2/M phase after treatment of CS2164 combined with radiation, compared with CS2164 or radiation alone (Fig. 6a-b, p < 0.05). Meanwhile, the ROS levels in tumor cells moderately increased after radiation (Fig. 6c-d). However, pretreatment with CS2164 significantly increased ROS levels in tumor cells, compared to the radiation group (10.14% ± 0.67%, p < 0.01). Besides, the increased ROS level after treatment of CS2164 combined with radiation, could be partially rescued by oxidation scavenger, NAC (5µM, Fig. S7a-b, p < 0.05). CS2164 (3µM) slightly decreased the p-Histone H3 level of Huh-7 and HeLa cells. After treatment of CS2164 (3µM) combined with radiation (4 Gy), the p-Histone H3 (Ser10) level further decreased, compared with radiation (4 Gy) alone (Fig. S7c-d). Taken together, these findings demonstrated that CS2164 combined with radiation could induce the γ-H2AX persistence and redox imbalance, leading to G2/M cell cycle arrest, therefore impaired the growth of malignant tumor.

Fig. 6.

Fig. 6

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CS2164 combined with radiation influenced the tumor microenvironment of tumor cells. a, b The cell cycle distribution condition of Huh-7 and HeLa cells after treatment of CS2164 (3µM)±Radiation (4 Gy). c, d The level of reactive oxygen species (ROS) in Huh-7 and HeLa cells after treatment of CS2164 (3µM)±Radiation (4 Gy). Data are presented as the means ± standard deviation (SD), n ≥ 3; ns, not significant; *p < 0.05; **p < 0.01, ***p < 0.001

Discussion

Hepatocellular carcinoma [40], characterized by active angiogenesis and metastasis, is one of the most lethal human malignancies due to the difficulty of early detection, chemoresistance, and radio-resistance [41]. Antiangiogenic therapy [42, 43] can sensitize tumors to radiotherapy by normalizing tumor blood vessels, reducing vessel density, and improving oxygen perfusion. Sorafenib is the first-line treatment for unresectable HCC [44], inhibiting the activation of VEGFR-2, VEGFR-3, Raf, and c-KIT. Yoshiyuki Wada found that sorafenib combined with radiotherapy dramatically prolonged the overall survival of patients with advanced HCC compared to sorafenib alone (31.2 months vs. 12.2 months) [45], suggesting a synergistic antitumor effect. However, a high incidence of severe toxic side effects limited its application. Apatinib, a highly selective inhibitor of VEGFR2, has been used in advanced HCC [46] and CC [47] patients in clinic. Previously studies reported that apatinib may also act as a radiosensitizer via suppressing PI3K/AKT signaling pathway [31], at the concentration of 10 µM in vitro experiments, as well as 150 mg/kg in vivo experiments. Furthermore, apatinib (200 mg/kg) significantly enhanced the anti-tumor efficacy of RT and prolonged the median survival of mouse models with cervical cancer [48]. In our study, CS2164 combined with radiation could effectively decreased the growth of HCC and CC, at a concentration of 3 µM in vitro and 10 mg/kg in vivo (Figs. 1 and 2). No intolerable toxic side effects were observed in nude mice. Those results suggested promising transitional value of CS2164. More phase I/II clinical trials could be conduct to assess the efficacy and safety of CS2164 combined with radiation in HCC and CC patients, particularly those with advanced and metastatic disease.

Previous studies have reported that CS2164 interfered with mitosis [21] and induced redox imbalance [25] in tumor cells. However, no studies have evaluated whether CS2164 can enhance the therapeutic effect of radiotherapy. In our study, anti-tumor effects were observed better in the CS2164 plus radiation group compared with radiation alone (Figs. 1 and 2), suggesting that CS2164 may act as a sensitizer. To further determine the mechanism, we observed γ-H2AX persistence in the CS2164 plus radiation group compared with other groups (Fig. 5a-d). Moreover, transcriptomics sequencing based on Huh-7 xenografts was performed, the results suggested that the mechanisms mainly involved the DNA damage repair process (Fig. S6). In addition, higher bulk cellular ROS levels and G2/M arrest were found in the CS2164 plus radiation group compared to the radiation only group (Fig. 6a-d). As a classical PARP inhibitor, Olaparib combined with radiation has been reported to effectively suppress tumor growth HCC by interfering with DNA damage repair at a dose of 10 mg/kg in vivo [49]. In this study, we found that CS2164, at a dose of 10 mg/kg in vivo, also demonstrated inhibition effect in HCC and CC tumor xenografts when combined with radiation. It is meaningful to conduct head-to-head clinical trial on CS2164 and other DDR inhibitors (e.g., PARP, ATM inhibitors), combined with radiation. Moreover, further investigation of the ROS is needed to identify the specific subcellular source. More sequencing results of HeLa xenografts may help to perform the cross validation to provide more comprehensive information. In summary, these findings indicated that CS2164 combined with radiation could interfered the DDR process, leading to redox imbalance and cell cycle re-population of tumor cells.

Fig. 5.

Fig. 5

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CS2164 combined with radiation suppressed the DNA damage repair process. a-d The γ-H2AX foci numbers and statistical chart of Huh-7 and HeLa cells after radiation at 1 h, 3 h, 6 h with or without the treatment of CS2164. scale bar: 10 μm. Data are presented as the means ± standard deviation (SD), n ≥ 3; ns, not significant; *p < 0.05; **p < 0.01, ***p < 0.001

To further clarify the core regulatory pathways regulated by the CS2164 plus radiation treatment, comprehensive landscape analysis was performed. KEGG pathway analysis showed that metabolic pathways were the top signaling pathways affected (Fig. 4a-c). Specifically, the main changes were in purine, pyruvate, phospholipid, and glycolysis metabolism. Our team previously reported the close relationship between phospholipid metabolism and radiosensitivity and successfully constructed a metabolic prognostic model for lung adenocarcinoma [36]. In this study, a series of DEGs involved in phosphorylation were enriched in cancer-related pathways including PI3K-Akt, MAPK, TNF, ErbB, and the phosphatidylinositol signaling system (Fig. 4c). Phospholipids are an important component of cell membranes, and the phosphorylation status affects the activity of kinases. Therefore, it is meaningful to clarify the effect of CS2164 on tumor phospholipid metabolism in the future. In addition, previously study showed a synergistic anti-tumor effect, which was observed following combined treatment with CS2164 and L-asparaginase through an AIF-dependent pathway in extra-nodal NK/T-cell lymphoma [50]. Purines are the basic substrate for synthesizing nucleic acids, and glycolysis and pyruvate metabolic processes are important to tumor metabolic reprogramming. Therefore, it would be interesting to carry out further metabolism analyses of the metabolic changes induced by CS2164 plus radiotherapy [51].

Since immune changes were enriched by KEGG pathway analysis, the role of CS2164 in regulating the tumor microenvironment aroused our interest. Zhou et al. identified the immunomodulatory effects of CS2164 in mouse HCC models [26]. This involved upregulation of CD4+ and CD8+ T cells in the spleen, as well as downregulation of immunosuppressive cells such as regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages in the spleen and tumor tissues. Furthermore, several proinflammatory cytokines, including TNF-α and IFN-γ, increased in tumor-related ascites after treatment with CS2164. In our study, a series of immune-related pathways such as the T cell receptor signaling pathway, PD-1/PD-L1 signaling pathway, and inflammatory mediator regulation of TRO channels were screened out based on tumor xenograft studies (Fig. 4a). The above results demonstrate the potential immune modification effect of CS2164. Humanized mice with patient-derived xenografts (PDX) might be an ideal preclinical model, but it may not be conducted due to the incomplete qualifications and ethic limitation yet. Future studies should be conduced by syngeneic mouse models to dissect the immunomodulatory effects of CS2164 in an immunocompetent condition. Moreover, survival and fractionation experiments can be carried out to assess long-term outcomes and dissect the specific radiobiological mechanisms involved in this combination therapy. Further phase I/II trials are necessary to evaluate the efficacy of CS2164 combined with immunotherapy, such as atezolizumab, pembrolizumab, tislelizumab. In addition, the effect of CS2164 combined with radiation could be assessed in more cancer types and cell lines, which could broad the application of CS2164 to a wider range.

Conclusion

In conclusion, our study demonstrated that CS2164 combined with radiation might inhibit tumor growth through ROS accumulation and γ-H2AX persistence. Our findings provide evidence for the possible application of CS2164 combined with radiation in malignant tumors.

Supplementary Information

Supplementary Material 1. (1.2MB, docx)

Acknowledgements

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Abbreviations

HCC

Hepatocellular carcinoma

CC

Cervical cancer

RT

Radiotherapy

DSBs

Double-strand breakages

DDR

DNA damage response

γ-H2AX

Phosphorylated histone H2AX

SER

The sensitivity enhancement ratio

ROS

Reactive oxygen species

DEGs

Differentially expressed genes

KEGG

Kyoto Encyclopedia of Genes and Genomes

GO

Gene ontology

PPI

Protein-protein interaction

HBV

The hepatitis B virus

HPV-16

The human papilloma virus type 16

STR

Short-tandem repeat

ATCC

The American Type Culture Collection

Authors’ contributions

X.P., Q.L.: Conceptualization, Methodology. S.P., Y.Z., X.L., R.J.: Formal analysis, Investigation. X.L.: Data Curation. S.P., Y.Z., Q.L.: Visualization. S.Y.: Resources. S.P. Y.Z.: Writing - Original Draft, Software. X.P., Q.L., S.P.: Writing - Review & Editing, Supervision, Funding acquisition.

Funding

This study was supported by the Health Commission of Guangdong Province (Grant No. 20201124205311824 awarded to Qiao-dan Liu), the National Natural Science Foundation of China Grant (82303868 awarded to Shun-li Peng), and the Guangdong Basic and Applied Basic Research Foundation (Grant No.2020A1515110599 awarded to Shun-li Peng).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The RNA sequencing datasets generated during the current study are available in the GEO repository, GSE312448.

Declarations

Ethics approval and consent to participate

All animal experiments in this study were performed strictly in accordance with the Declaration of Helsinki, and approved by the animal ethic committee in the fifth affiliated hospital of Sun-Yat Sen University (NO.00234, Zhuhai, China).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Shun-li Peng, Yu-ling Zhou and Xuexia Liang contributed equally to this work.

Contributor Information

Qiao-dan Liu, Email: lqiaodan@mail.sysu.edu.cn.

Xiao-mou Peng, Email: pengxmou@mail.sysu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (1.2MB, docx)

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The RNA sequencing datasets generated during the current study are available in the GEO repository, GSE312448.

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