HT-29 cells (cat. no. HTB-38) and CCD-18Co cells (cat. no. CRL-1459) were purchased from the American Type Culture Collection. IK was isolated from Perilla frutescens Britt by the supercritical carbon dioxide (SC-CO₂) method (17). DMEM (cat. no. SH30285.02), fetal bovine serum (FBS; cat. no. SH30088.03IR25-40), trypsin (cat. no. SH30042.02) and penicillin-streptomycin (cat. no. SV30010) were purchased from HyClone (Cytiva). Cell Counting Kit-8 (CCK-8) assay kit (cat. no. PA137267) was purchased from Pierce (Thermo Fisher Scientific, Inc.). Specific antibodies against HIF-1α (1:1,000; cat. no. 36169), PI3K (1:1,000; cat. no. 4292), phosphorylated (p)-PI3K (1:1,000; cat. no. 4228), AKT (cat. no. 9272), p-AKT (1:500; cat. no. 9271), mTOR (1:500; cat. no. 2972), p-mTOR (1:1,000; cat. no. 2971), BCL-2 (1:500; cat. no. 15071), BAX (1:1,000; cat. no. 2772), LC3 I/II (1:1,000; cat. no. 4108S), Beclin-1 (1:1,000; cat. no. 3738) and β-actin (1:1,000; cat. no. 4967) were purchased from Cell Signaling Technology, Inc. Specific antibodies against γH2AX (1:1,000; cat. no. ab81299) were purchased from Abcam. A total of 40 male BALB/c nude mice (age, 4 weeks-old; weight, 18~22 g) were purchased from Beijing HFK Bioscience Co. Ltd. X-ray irradiator (X-ray RAD 320; Precision X-ray Inc.). All mice were housed in an SPF environment with free access to food or water at a temperature of 21–25°C and humidity of 60–70%, ensuring 12 h of alternating light.

HT-29 and CCD-18Co cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin, and placed in an incubator at 37°C with 5% CO₂ and saturated humidity. After 48 h of incubation, cells were rinsed twice with PBS, and 1 ml trypsin was added for digestion at 37°C for 5–6 min. After complete digestion, 2 ml fresh DMEM was added to terminate the reaction, and a cell suspension was prepared, which was pipetted into a 10-ml Eppendorf tube and centrifuged at 1,000 × g for 3 min at room temperature. Next, fresh DMEM was added in a 1:3 ratio, and the mixture was transferred to a flask and incubated in a 37°C in a 5% CO₂ incubator. HT-29 and CCD-18Co cells were incubated with different concentrations of IK (0, 50, 100 or 200 µg/ml) for 48 h at ≤65% cell confluence. At the same time, all grouped cells were irradiated with different doses (0, 1, 2, 4, 6, 8, 12 or 16 Gy) of X-rays (X-ray RAD 320 irradiator; Precision X-ray, Inc.). Cells from each group were seeded in 96-well plates at a concentration of 6×10³ cells/well. A total of 10 µl CCK-8 solution was added to each well and incubated at 37°C for 4 h. Finally, the optical density (OD) 450 nm values were determined by using a plate reader. Cell viability was calculated by average cell viability of control wells. DNA damage was detected by single cell gel electrophoresis at 0, 15, 30, 60 and 120 min after X-ray irradiation. Olive tail moment (OTM) was analyzed with Comet assay software project (http://casplab.com/).

BALB/c nude mice were fed normally for 7 days after acquisition. A mouse tumor model was established by injecting cancer cells into mice. Briefly, log-proliferating human colon cancer HT-29 cells were washed with PBS and digested with trypsin for 4–6 min, and the digestion was terminated by adding fresh DMEM. After centrifugation, cell suspensions were obtained after two resuspensions in serum-free DMEM. The cell concentration in the mixture was adjusted to 2.5×10⁷ cells/ml. A total of 40 healthy BALB/c-nu nude mice were inoculated subcutaneously with 150 µl (3.75×10⁶ cells) cell suspension on the dorsal side of the right axilla to obtain a HT-29 tumor-bearing nude mouse model. All HT-29 tumor-bearing nude mice were randomly divided into i) control group, ii) RT, iii) IK and iv) RT + IK groups (n=10). Mice in both the RT and combination groups were treated with 8 Gy doses of X-ray radiation once a day for 14 consecutive days, while mice in the IK and combination groups were intraperitoneally injected with 100 mg/kg IK daily for 14 consecutive days. Placebo was administered during RT to the control group. Tumor size was measured every other day and the survival of mice was counted. Mice were euthanized when the tumor size exceeded 3,000 mm³. All surviving mice were anesthetized 24 h after the last administration. The anesthetic agent used was sodium pentobarbital, prepared as a 3% sterile saline solution at a dose of 30 mg/kg body weight. Sodium pentobarbital was administered via intraperitoneal injection, and the animals' responses were continuously observed. Once the animals were fully anesthetized, they were euthanized by cervical dislocation.

The procedures for sampling tumor and intestinal tissue were as follows: Using surgical instruments, the skin over the tumor site of the mouse was carefully cut and exposed. Next, the tumor was carefully separated from the surrounding tissues and the intact tumor tissue was placed in a sterile container for subsequent experimental analysis or preservation. For intestinal tissues, upon disinfecting the skin with alcohol, the abdominal skin and muscle layers were cut open to expose the abdominal cavity. After locating the intestine, the desired intestinal segment was excised. All operations were performed under sterile conditions to ensure that the samples were not contaminated. The obtained tumor and intestinal tissues were washed with normal saline to remove residual blood, dried with absorbent paper and stored at −80°C for later use. Intestinal tissues were fixed in a 4% paraformaldehyde solution, then embedded in paraffin and cut into 5-µm thick sections. The sections were sequentially stained with hematoxylin for 5 min and eosin for 2 min at room temperature. Intestinal images were obtained using a light microscope. Peripheral blood was collected from mice and analyzed for peripheral blood leukocyte, neutrophil and monocyte numbers using a LH-750 hematology analyzer (Beckman Coulter, Inc.).

The present animal studies complied with all relevant national regulations and institutional policies, and were approved [approval no. 2023 (30)] by the Animal Experimental Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China).

The levels of malondialdehyde (MDA) (cat. no. ab238537; Abcam), TNF-α (cat. no. ab181421; Abcam), NF-κB (cat. no. ab279874; Abcam) and IL-1β (cat. no. BMS6002-2TEN; Invitrogen; Thermo Fisher Scientific, Inc.), and the activities of glutathione (GSH) (cat. no. EEL155; Invitrogen; Thermo Fisher Scientific, Inc.) and catalase (CAT) (cat. no. KTB9040; Abbkine Scientific Co., Ltd.) were measured by ELISA. Briefly, intestinal tissue frozen at −80°C was mixed with 1 ml tissue lysis buffer, ground to homogenate in a glass mill in an ice bath, lysed at 4°C for 30 min and centrifuged at 3,000 × g for 20 min at 4°C. The levels of MDA, TNF-α, NF-κB and IL-1β in tissue homogenate, and the activities of GSH and CAT were measured using commercial ELISA kits.

Western blotting was used to analyze protein expression levels in different cells and tissues. Briefly, tumor and intestinal tissues frozen at −80°C were mixed with 1 ml tissue NP-40 lysis buffer (cat. no. P0013F; Beyotime Institute of Biotechnology), ground to homogenate in a glass mill in an ice bath, lysed at 4°C for 30 min and centrifuged at 3,000 × g for 20 min at 4°C. A total of 10 µl HT-29 cell homogenates, tumor tissue homogenates or intestinal tissue homogenates were isolated by 10% SDS-PAGE and transferred to nitrocellulose membranes, which were then blocked with 5% bovine serum albumin (cat. no. ST2249-5g; Beyotime Institute of Biotechnology) for 1 h at room temperature and washed with TBS-Tween 20 (TBST; 20 mM Tris buffer, 0.1% Tween 20, pH 7.4) for 5 min. Subsequently, the membranes were incubated at 4°C with a primary antibody (1:500) overnight and then with a horseradish peroxidase-labeled secondary antibody (1:1,000; cat. no. 58802; Cell Signaling Technology, Inc.) for 1 h at room temperature. After washing with TBST, the membrane was immersed in an enhanced chemiluminescence solution (SuperSignal™ West Atto; cat. no. A38556; Thermo Fisher Scientific, Inc.). The gray value of the bands was analyzed by ImageJ software (version 1.8.0; National Institutes of Health). The target protein expression level was calculated as the target band gray value divided by the reference material strip gray value.

Data were analyzed using SPSS 25.0 statistical software (IBM Corp.), and the data were expressed as the mean ± SD. Inter-group comparisons were performed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether IK can synergistically promote the inhibitory effect of X-rays on the proliferation of human colorectal cancer cells, the viability of HT-29 cells treated with RT combined with IK was examined by CCK-8 assay. As shown in Fig. 1A, RT at different irradiation doses inhibited the viability of HT-29 cells. In addition, RT in combination with different concentrations of IK further reduced HT-29 cell viability. Notably, for normal human colon fibroblasts (CCD-18Co cells), IK effectively reversed the RT-induced decrease in cell viability (Fig. 1B). The optimal dose ratio of RT in combination with IK was further analyzed (Fig. 1C and D). At different doses of RT and IK, the proliferation inhibition rate of HT-29 cells in the combination group was significantly greater than that in the RT alone or IK alone groups (both P<0.05). In addition, the proliferation inhibition of CCD-18Co cells in the combination group was significantly lower than that in the RT alone group (all P<0.01). CCD-18Co cell viability was significantly decreased (P<0.05) in the 12 Gy/200 µg/ml group compared with the RT/IK dose of 8 Gy/100 µg/ml, but had no significant effect on HT-29 cell viability (P>0.05). Therefore, 8 Gy/100 µg/ml was selected as the optimal RT/IK dose ratio for subsequent studies. The combination index (CI) values were further calculated (data not shown), and they were observed to be <1 at each concentration, suggesting that RT combined with IK had a synergistic inhibitory effect on the proliferation of colon cancer cells.

After X-ray irradiation, the repair time of DNA damage is usually 4–6 h. Therefore, single cell gel electrophoresis was used in the present study to analyze the initial DNA damage in HT-29 cells at 0, 15, 30, 60 and 120 min after irradiation. As demonstrated in Fig. 2A, OTM in control HT-29 cells remained low from 0 to 120 min. RT alone significantly increased the OTM levels of HT-29 cells at 0, 15, 30, 60 and 120 min compared with the controls (P<0.01). Notably, IK alone also slightly increased OTM levels in HT-29 cells at 0 and 15 min (both P<0.05). In addition, RT in combination with IK increased the OTM levels of HT-29 cells to a greater extent and significantly higher than either treatment alone (all P<0.001).

The expression levels of HIF-1α and the P13K/AKT signaling pathways in HT-29 cells were analyzed by western blotting. As revealed in Fig. 2B-E, RT or IK alone significantly decreased the expression levels of HIF-1α, p-PI3K and p-AKT compared with controls (all P<0.01). In addition, RT in combination with IK further reduced the expression level of HIF-1α and the phosphorylation level of the PI3K/AKT signaling pathway.

The expression of apoptosis and autophagy-related proteins in HT-29 cells was further analyzed by western blotting. As demonstrated in Fig. 3A-C, both RT or IK alone significantly increased the expression of the proapoptotic factor BAX and decreased the expression of the anti-apoptotic factor BCL-2 compared with the control group (all P<0.05). As expected, RT in combination with IK increased BAX but decreased BCL-2 expression, compared with controls (both P<0.05), and the extent of the change was significantly higher than either treatment alone. In addition, RT or IK alone promoted the conversion of LC3 I to LC3 II and increased Beclin-1 expression levels compared with controls (both P<0.05). Notably, the combination treatment group further increased the changes in these levels. These results suggest that RT combined with IK can significantly promote apoptosis and autophagy in HT-29 cells.

Based on the efficacy of RT in combination with IK in inhibiting HT-29 cell proliferation, the in vivo antitumor potential of this combination therapy was further evaluated in HT-29-bearing mice. As shown in Fig. 4A and B, the tumor volume of control mice continued to increase over the 14-day treatment period, but there was no significant change in body weight. The maximum tumor diameter and volume was 16.12 mm and 1,425 mm³, respectively. Both RT and IK treatments significantly reduced tumor size in mice compared with controls (both P<0.001). In addition, the combination of RT and IK significantly reduced tumor size in HT-29-bearing mice compared with controls (P<0.001), and to a significantly greater extent than RT or IK alone. Notably, after 14 consecutive days of RT in combination with IK, the transplanted tumors in HT-29-bearing mice were almost completely eliminated, followed by no tumor recurrence. The survival rates of different groups of mice during 14 days of continuous treatment were statistically analyzed (Fig. 4C). Xenograft mice in the control group commenced to succumb to disease 6 days after modeling and all succumbed within 14 days. Treatment with RT or IK alone significantly improved mouse survival compared with controls, but survival was only 40–60%. Of note, xenograft mice in the combination treatment group did not die during 14 consecutive days of treatment (survival rate of 100%), indicating that RT combined with IK treatment is effective in prolonging the survival of HT-29-bearing mice.

Non-specific immunity plays an important role in the treatment of solid tumors. Therefore, the levels of leukocytes, neutrophils and monocytes in the peripheral blood of different groups of HT-29-bearing mice were further analyzed. As revealed in Fig. 4D-F, RT alone significantly increased the levels of white blood cells, neutrophils and monocytes in the peripheral blood of mice compared with controls (all P<0.05). In addition, the levels of neutrophils in mice treated with IK alone were significantly higher than those in the control group (P<0.05), whereas the levels of white blood cells and monocytes were not significantly different (both P>0.05). As expected, the levels of white blood cells, neutrophils and monocytes in peripheral blood were significantly enhanced in the combination group, and were significantly higher than in the monotherapy or control groups.

Western blotting was used to study the expression of apoptosis and autophagy-related proteins in tumor tissues of HT-29-bearing mice. As shown in Fig. 5A-C, RT or IK alone significantly upregulated the expression of BAX and downregulated the expression of BCL-2 compared with the control group (all P<0.01). Furthermore, RT in combination with IK further significantly upregulated BAX expression and downregulated BCL-2 expression compared with both treatment groups alone (both P<0.01). On the other hand, both RT and IK alone significantly upregulated LC3 II expression and downregulated LC3 I expression compared with controls, indicating an increased conversion of LC3 I to LC3 II (both P<0.01) (Fig. 5D). In addition, treatment with RT or IK alone also significantly upregulated Beclin-1 expression (both P<0.01) (Fig. 5E). The combination of RT and IK further significantly enhanced the conversion of LC3 I to LC3 II (both P<0.001), and significantly upregulated Beclin-1 expression (both P<0.001) compared with the two treatment groups alone. These results suggest that RT combined with IK can promote tumor cell apoptosis by regulating the expression of apoptosis-related proteins, and ultimately inhibit the xenograft tumor growth.

Radiation therapy damages normal tissue around the lesion. Oxidative stress and inflammation are the main manifestations of radiation side effects. Therefore, the present study further investigated the protective effect of IK on normal intestinal tissue after RT in mice. As demonstrated in Fig. 6A-C, after RT, MDA levels in normal intestinal tissues of xenograft mice were significantly increased (P<0.001), whereas the activities of GSH and CAT were significantly decreased (both P<0.001), suggesting development of oxidative stress. Notably, IK treatment significantly decreased MDA levels in the intestinal tissue of irradiated mice (P<0.01), and significantly increased the activities of GSH and CAT (both P<0.01). On the other hand, RT treatment significantly increased the levels of inflammatory factors, including TNF-α, NF-κB and IL-1β, compared with controls (all P<0.001) (Fig. 6D-F). In addition, these changes in inflammatory cytokine levels were significantly reversed by IK treatment (all P<0.01). These results suggest that IK significantly ameliorates radiation-induced oxidative stress and inflammation.

The results of H&E staining in normal intestinal tissues of xenograft tumor models are shown in Fig. 7A. Intestinal microvilli were structurally intact in the control and IK groups. By contrast, in the RT group, the tip of some villi in intestinal mucosa was necrotic, the height and width of villi were disorganized, and some villi exhibited shortening and atrophy. In the IK group, only partial villus shortening and structural integrity were observed in intestinal microvilli. In the RT+IK group, histopathological changes were relieved, and the submucosal, muscular and serosal tissue structures were significantly improved.

The PI3K/AKT/mTOR signaling pathway plays an important role in inflammation and oxidative stress. Activation of the PI3K/AKT/mTOR signaling pathway promotes the expression of numerous proinflammatory cytokines as well as the development of oxidative stress. Thus, the expression of proteins of the PI3K/AKT/mTOR signaling pathway in normal intestinal tissues of tumor-bearing mice was further analyzed by western blotting. As revealed in Fig. 7, RT treatment significantly upregulated the expression level of γH2AX and the phosphorylation levels of PI3K, AKT and mTOR compared with controls (all P<0.001). In addition, the expression level of γH2AX and the phosphorylation levels of PI3K, AKT and mTOR in the intestinal tissue of mice treated with IK alone did not change significantly compared with the control group (all P>0.05). As expected, IK treatment significantly reversed the RT-induced phosphorylation of PI3K/AKT/mTOR (all P<0.01). These results suggest that IK can ameliorate radiation injury in the intestinal tissue of xenografted mice by inhibiting the phosphorylation of the PI3K/AKT/mTOR pathway.