YLSPS was prepared as previously described (10). The root of Millettia pulchra Kurz var. laxior (Dunn) Z. Wei was dried, and extracted three times with boiling water. The polysaccharide in the filtrate was precipitated fractionally with alcohol. The protein in the product was removed and further purified using diethylaminoethyl (DEAE) ion exchange cellulose (DEAE-52). Gas chromatography and thin-layer chromatography analysis demonstrated that YLSPS was composed of D-glucose and D-arabinose in a molar ratio of 90.79 and 9.21%, respectively, with an average molecular weight of ~14,301 Da. CTX injection was purchased from Shanxi Pude Pharmaceutical Co., Ltd. (Datong, China).

A total of 80 male and 80 female BALB/c mice (aged 4–6 weeks and weighing 20±2 g) were purchased from the Institute of Animal Care Center of Guangxi Medical University. The mice were acclimatized for 1 week prior to being used in the study. All animals were cared for in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. The experimental protocols of the study were approved by the Animal Care Committee at Guangxi Medical University (certificate no. SYKG 2013–0005).

Murine sarcoma 180 (S180) cells were injected into the peritoneal cavity of the mice and proliferated to produce ascites. The cell colonies were maintained by weekly transplantation of the tumor cells from the ascitic fluid into the peritoneal cavity of another mouse. S180 cells were isolated from the ascitic fluid and suspended in saline at a density of 1×10¹⁰ cells/l. Subsequently, 2×10⁶ cells (in 200 µl saline) were injected into the axillary fossa of the right forelegs to prepare the tumor-bearing mice. Two days after S180 cell implantation, the mice were randomly divided into five groups (10 mice per group), as follows: i) CTX group [0.02 g/kg, intraperitoneal (i.p.) injection on days 1, 4, 7 and 10]; ii) high-dose YLSPS group [0.6 g/kg/day, intragastric (i.g.) administration; iii) intermediate-dose YLSPS group (0.3 g/kg/day, i.g.); iv) low-dose YLSPS group (0.15 g/kg/day, i.g.); and v) control group (saline, 0.2 ml/10 g/day, i.g.). The animals were treated for 10 days and then sacrificed by cervical dislocation under ethyl ether anesthesia on day 12.

The tumor inhibition rate was calculated as follows: Inhibition (%)=(C-T)/C × 100%, where C and T represent the tumor weights (in mg) of control and treated mice, respectively. Based on the correlation between immune activity and the weights of the spleen and thymus, the relative weights of the spleen and thymus (in mg) with regards to the mouse body weight (10 g) were used to obtain the spleen index (SI) and thymus index (TI), as previously described (11,12).

B-cell lymphoma 2 (Bcl-2), p53 and Bcl-2-associated X (BAX) proteins were detected by IHC, as previously described (13). Briefly, all specimens were fixed in formalin, embedded in paraffin and cut into 4-µm sections for IHC. Bcl-2, p53 and BAX were detected by rabbit anti-mouse polyclonal antibodies (N-20; dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IHC was performed using the immunoperoxidase method as follows: Antigen retrieval was performed using cell conditioning 1 antigen retrieval buffer (pH 8.5; Ventana Medical Systems, Tucson, AZ, USA). The sections were incubated with a primary antibody in phosphate-buffered saline (pH 7.4) with 1% bovine serum albumin and stained on a BenchMark XT automated slide stainer using a diaminobenzidine detection kit (ultraView Universal DAB detection kit; Ventana Medical Systems). The positive reaction, shown by brown color, was evaluated under a light microscope and scored by two pathologists who were blinded to the origins of the sections. The staining was scored on semi-quantitative scales as follows: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining.

Spleens from S180 sarcoma-bearing mice treated with CTX and/or YLSPS were aseptically removed for the preparation of spleen lymphocytes. The removed spleens were homogenized in sterile Hank's balanced salt solution (HBSS) and the homogenized spleen tissue was passed through a mesh (200 mesh sieve) and washed twice with HBSS. The erythrocytes were lysed with red blood cell lysis buffer, while the remaining cells were suspended in RPMI-1640 medium with 10% fetal calf serum. Cells were counted with a hemocytometer using the trypan blue exclusion technique (14). Cell viability exceeded 95% and cells were resuspended with complete medium at a density of 1×10¹⁰ cells/l. ConA-stimulated cell proliferation was detected with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, cell suspensions (100 µl/well) were added to 96-well culture plates followed by the addition of 100 µl ConA (5 mg/l). The cells were then incubated at 37°C in a humidified, 5% CO₂ atmosphere for 68 h. Subsequently, 10 µl MTT (5 mg/ml) was added into each well followed by a 4-h additional incubation. The cultures were centrifuged at 444 × g for 5 min and the supernatant was carefully removed. After adding 200 µl dimethyl sulfoxide, absorbance was assessed with a microplate reader at a wavelength of 570 nm (A570; Jupiter ASYS, Montreal Biotech, Kirkland, PQ, Canada).

Mouse sarcoma samples were fixed with 4% paraformaldehyde and 1% glutaraldehyde. The fixed tissues were washed with phosphate buffer (pH 7.4) and post-fixed with osmium tetroxide. These tissue samples were dehydrated through a series of graded ethanol solutions and propylene oxide, and then embedded in epoxy resin. Ultrathin sections (0.5 µm) of each sample were stained with uranyl acetate and lead citrate and examined by transmission electron microscopy (TEM; JEM-1200EX, JEOL, Tokyo, Japan) with an accelerating voltage of 80 kV.

Numerical data were expressed as mean ± standard error. Statistical differences between the means for the different groups were evaluated with SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) using Student's t-test, with the level of significance set at P<0.05.

The tumor growth was significantly inhibited by YLSPS administration at a low dose (39.7%), intermediate dose (41.3%) and high dose (52.6%), compared with control mice. The dose-dependent pattern of YLSPS-induced inhibition indicated the therapeutic effect of YLSPS on S180 sarcoma tumors. To provide an antitumor effect comparable with commonly used anticancer drugs, CTX was also used at a standard dose (0.02 g/kg) in separate positive-control mice, resulting in a tumor inhibition rate of 71.1%.

Immunosuppression is considered to be a major side effect of anticancer agents. As such, the changes in the SI and TI induced by YLSPS and CTX treatment were compared. YLSPS increased the SI as well as the TI at all three dose levels. With increasing YLSPS dose (0.15, 0.30 and 0.60 g/kg/day), the SI values were 90.89±16.13, 92.93±7.26 and 104.52±8.22 mg/10 g, respectively, with related TI values of 29.49±7.11, 33.81±6.02 and 38.39±5.50 mg/10 g, respectively. The SI and TI values were 81.90±6.95 and 26.90±4.94 mg/10 g in the control group. By contrast, CTX significantly decreased the SI and TI compared with the YLSPS and control groups (28.45±7.65 and 8.76±2.59 mg/10 g, respectively).

The changes in p53, Bcl-2 and BAX expression following administration of YLSPS or CTX are summarized in Fig. 1. The accumulated scores represent the relative expression of the studied proteins in the sarcoma tissues of S180-bearing mice.

The majority of the cells in the sarcoma tissue were cerioid-shaped, with increased internuclear distance and atypical nuclei (Fig. 2A). S180 cells exhibited nuclear membrane integrity, typical organelles and mitotic activity (Fig. 2B). Following treatment with a low dose of YLSPS, the cell size decreased, with formation of mitochondrial vacuoles (Fig. 2C). S180-bearing mice treated with an intermediate dose of YLSPS exhibited an increased number of apoptotic cells with overt karyopyknosis and disordered cell structure (Fig. 2D). After treatment with a high dose of YLSPS (Fig. 2E), there was an increase in cell debris in the intercellular space accompanied by nuclear fragmentation and the formation of apoptotic bodies. Furthermore, in addition to organelle fragmentation and apoptotic body formation, the cell membrane structure completely dissolved following treatment with CTX (Fig. 2F).

The additive effect of YLSPS and CTX is shown by the q-value: q=EAB/[EA+(1-EA) EB], where EA and EB are the tumor inhibition rates of drug A and drug B, respectively, and EAB is the tumor inhibition rate of drugs A and B used in combination. In this case, q=0.85–1.25 indicates an additive effect of drugs A and B, q>1.25 indicates enhancement (or a synergistic effect) of the two drugs, and q<0.85 indicates that the two drugs are antagonistic (15). The tumor inhibition rate was 64.8% for the CTX treatment group, whereas for the high, medium and low doses of YLPS together with CTX, the tumor inhibition rates were 74.8, 76.1 and 67.4%, respectively, with related q-values of 0.905, 0.984 and 0.887, respectively. All the q-values were in the range of 0.85 to 1.25, which indicated that different doses of YLSPS may enhance the CTX antitumor effect on mouse S180 tumors in an additive manner.

To investigate the mechanisms through which YLSPS potentiates the antitumor effect of CTX, the SI, TI and peripheral blood leukocyte count were assessed following administration of various drug combinations. As shown in Table I, the administration of CTX significantly inhibited tumor growth, but its antitumor activity was accompanied by a decrease in SI, TI and peripheral blood leukocytes. However, the combination of CTX with YLSPS at the three tested doses achieved further tumor growth inhibition, but improved the SI, TI and peripheral blood leukocyte count compared with CTX alone. The results of spleen lymphocyte proliferation and tumor necrosis factor α (TNF-α) production analyses in S180-bearing mice were in accordance with the YLSPS-induced SI and TI changes following CTX treatment (Table II).