XN (purity >98.6%) was provided by Yumen Tuopu Science and Technology Development LLC (Yumen, China), in accordance with the chemical structure of XN as shown in Fig. 1B. Anti-mouse CD3 (FITC), anti-mouse CD4 (PE), anti-mouse CD8 (PE), anti-mouseCD25 (APC), anti-mouse IL-4 (APC), anti-mouse IFN-γ (DAPI), anti-mouse perforin (FITC), anti-mouse granzyme B (FITC), flow cytometry staining buffer, fixation/permeabilization concentration, fixation/permeabilization diluent and permeabilization buffer were purchased from eBioscience, Inc. (San Diego, CA, USA). Mouse IL-2, mouse IL-4, mouse IL-10 and mouse IFN-γ ELISA kits were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). Anti-T-bet and anti-GATA-3 antibodies were purchased from Abcam (Cambridge, UK). Ki-67 (D3B5) was purchased from Cell Signaling Technology (CST; Boston, MA, USA). Anti-phospho-STAT4 (Tyr693), anti-STAT4, anti-STAT6, and anti-phospho-STAT6 (Tyr641) were purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Total protein extraction kit was purchased from BestBio Co., Ltd. (Shanghai, China). Mouse 1X lymphocyte separation medium was purchased from Dakew (Shenzhen, China).

The 4T1 cell line was purchased from the China General Microbiological Culture Collection Center (Shanghai, China). The 6- to 8-week-old BALB/c female mice, weighing 18±2 g, were purchased from Lanzhou Veterinary Research Institute (Chinese Academy of Agricultural Sciences, Lanzhou, China). All the animals were acclimatized for one week. Living environment was maintained under controlled conditions (24±2°C, 50±10% relative humidity, with a rhythm of 12 h light/dark cycles).

All animal methods in the experiments were in line with ‘Guidelines for the Humane Treatment of Laboratory Animals’, which was issued by the Ministry of Science and Technology of the People's Republic of China in 2006.

Mice were randomly assigned into five groups and named the healthy, control, 25 mg/kg XN and 50 mg/kg XN groups, and 10 mg/kg cyclophosphamide (Cy) group. Cy has been reported to shift Th1/Th2 immune response from Th2 to Th1 (26,27); we used it as a positive treatment. A mouse model of breast cancer was constructed by subcutaneous injection of 4T1 cells (1×10⁵-10⁶ cells/ml) into the right posterior leg. Drugs and solvent were administered by gavage after three days of injection, and treatment was administered for two weeks. The healthy and control groups received only solvent. The treatment schedule is shown in Fig. 1C. Two weeks later, mice were anesthetized with ether and peripheral blood was collected by removal of the eyeballs. Finally, tumors and spleens were removed from the mice.

Calculation of the tumor volume (V) was as follows: V_tumor = (L × W²)/2 (L, is the longest diameter; W, is the shortest diameter) (28), and the diameters were measured by electronic calipers. Tumor inhibitory rate = (tumor weight of treatment group - tumor weight of control group)/tumor weight of control group × 100%. Rate of body weight change = (body weight at the end of experiment - body weight at the beginning of experiment)/body weight at the beginning of experiment × 100%. The above experiments were performed at least three times.

We homogenized the tumor tissues and obtained total protein using a total protein extraction kit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) was performed on 30 µg of each sample protein, and then the samples were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Blocking of non-specific protein occurred at room temperature in Tris-buffered saline-Tween-20 (TBST; TBS containing 0.01% Tween-20)-5% non-fat milk. Following incubation with the primary antibody at 4°C overnight, incubation with the secondary antibody was carried out at room temperature for 4 h. Membranes were washed with TBST for 5×5 min and peroxidase activity was visualized with an enhanced chemiluminescence western detection system (Perkin-Elmer Life Sciences, Boston, MA, USA).

Tumor tissues (3 µm) were cut and fixed in formalin buffer after being paraffin-embedded. Paraffin-embedded cancer tissue sections were deparaffinized and dewatered first. Then, slides were incubated with 0.3% H₂O₂ for 10 min and washed with ddH₂O for 3×3 min. Antigen retrieval was undertaken in a pressure cooker for 10 min with 0.01 M citrate buffer and washed with phosphate-buffered saline (PBS) for 3×5 min. Bovine serum albumin (BSA) (5%) was applied to the specimen sections to block non-specific protein for 20 min at room temperature. The primary antibody was incubated for 2 h at 37°C and washed with PBS for 3×3 min. After washing, specimen slides were incubated with the secondary antibody for 20 min at 37°C and washed with PBS for 3×3 min. Finally, the specimen slides subsequently underwent 3,3-diaminobenzidine (DBA) staining for 10 min. Slides were assessed using microscopy; positive cells are shown in brown or yellow and cell nuclei are shown in blue.

The spleen from each sacrificed mouse was aseptically removed and put into a 35-mm culture dish containing cold PBS. Total lymphocytes were separated using the Mouse 1X Lymphocyte Separation Medium. Then, the lymphocytes were resuspended at a concentration of 1×10⁷ cells/ml and transferred (100 µl) to a 1.5 ml tube. The antibody was then labeled. For the Th1 and Th2 subset analysis, Th1 subsets were labeled as CD3⁺CD4⁺IFN-γ⁺, Th2 subsets were labeled as CD3⁺CD4⁺IL4⁺ (29,30). The ratio of Th1/Th2 = percentage of CD4⁺IFN-γ⁺/percentage of CD4⁺IL4⁺. Cell surface antigens CD3 and CD4 were labeled in the dark for 30 min. Then, cells were fixed and permeabilized in fixation/permeabilization mixture in dark for 1 h. Lastly, the anti-mouse IFN-γ and IL4, which are intracellular antigens, were incubated in the dark for 30 min. For the perforin and granzyme B subset analysis, splenic lymphocytes were fixed and permeabilized first, and then the anti-mouse perforin and granzyme B antibodies, which are also intracellular antigens, were incubated in the dark for 30 min. For the CD8 and CD25 subset analysis, the CD8 subset was labeled as CD3⁺CD8⁺, and the CD25 subset was labeled as CD3⁺CD25⁺. After antibodies were labeled, lymphocytes were fixed in 1% paraformaldehyde, and detected by flow cytometer. The above procedures were performed on ice and repeated at least three times.

Serum was separated from peripheral blood via centrifugation (12,000 rpm, 10 min, 4°C) and stored at −20°C. Four cytokines (IL-2, IL-4, IL-10 and IFN-γ), and the key tumor-specific markers (CA15-3) were detected using mouse cytokines ELISA kit. All steps were followed in accordance with the manufacture's protocol. The cytokines and marker were detected within seven days after serum separation, and above procedures were performed on ice and repeated at least three times.

Values are presented as mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism 5. The results of flow cytometry were analyzed using FlowJo 7.6.1. Grayscale quantitative analysis was performed using Image Pro-Plus 6.0. Probability values <0.05 were considered to indicate statistical significance.

Body weight, tumor volume and tumor weight were assessed to evaluate the effects of XN on breast cancer. As shown in Fig. 2A and B, the tumor growth of the XN-treated groups was evidently slower than that noted in the control group. Fig. 2D shows that tumor weight was significantly decreased in the 50 mg/kg XN-treated group compared with the control group (p=0.036). Meanwhile, Table I shows that inhibitory rate of tumor growth in the 25 and 50 mg XN groups and Cy-treated group was 25.68, 37.72 and 29.85%, respectively. Body weight did not obviously change in the four groups (Fig. 2C), while 10 mg/kg Cy caused negative growth rate (−0.93%) (Table I).

CA15-3 and antigen Ki-67 are the most common markers for breast cancer (31,32). IHC analysis (Fig. 3A) showed that expression of Ki-67 in the tumor tissues was significantly inhibited after treatment. Concomitantly, we detected the concentration of CA15-3 in serum using mouse CA15-3 ELISA kit. Results demonstrated that the CA15-3 level in the control group was evidently higher than levels in the other groups (p<0.05) (Fig. 3B). These results demonstrated that XN restrained the growth of breast cancer cells in the mouse model.

CTL responses are very important for cellular immunity, and it is associated with Th1 polarization (19). The ratio of CD8⁺/CD25⁺ lymphocytes was detected by flow cytometry. As shown in Fig. 4A and D-a, XN increased the ratio of CD8⁺/CD25⁺ when compared with that noted in the control group. Fig. 4B, C and D-b and -c indicate that expression of perforin and granular enzyme B were also markedly upregulated by treatment with XN (compared with control group, all p<0.05).

Numerous studies have shown that cancer disrupts the Th1/Th2 balance, and this is known as ‘balance shifting’ (33–35). IFN-γ and IL-4 are pivotal cytokines which are produced by Th1 and Th2 cells, respectively. We investigated Th1 and Th2 cytokines in serum by ELISA kit. As shown in Fig. 5, expression of Th1 cytokines IL-2 and IFN-γ was markedly enhanced; in contrast, expression of Th2 cytokines, (including IL-4 and IL-10) was evidently weakened after XN therapy (p<0.05). Fig. 6A and B show the flow cytometric analysis of Th1 (CD4⁺IFN-γ⁺) and Th2 (CD4⁺IL4⁺) cells, respectively. Fig. 6C-a and -b indicates that the percentage of Th1 cells was significantly increased, while the percentage of Th2 cells was markedly decreased after treatment with 50 mg/kg XN (compared with control group, p<0.05). Fig. 6C-c indicates that the ratio of Th1/Th2 in the 50 mg/kg XN-treated group was significantly increased compared with the control group (p<0.05).

Based on the above results, XN regulated the Th1/Th2 balance drifting to Th1, indicating that there may be merit in exploring the mechanisms of the balance drifting. We examined expression levels of key proteins in Th1 and Th2 development with western blotting and IHC, respectively. As shown in Fig. 7 the expression of T-bet and p-STAT4 were markedly enhanced, while GATA-3 was significantly weakened (p<0.05). Meanwhile, p-STAT6 did not obviously change (p>0.05). Simultaneously, the results of IHC corroborated those of western blotting. The above results indicated that STAT4, mediating Th0 differentiate to Th1, may be involved in the influence of the Th1/Th2 balance by XN.