RPMI-1640 medium, heat-inactivated fetal bovine serum (FBS), penicillin and streptomycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). ELISA kits were from Shanghai Enzme-linked Biotechnology Co., Ltd. (Shanghai, China). BALB/c nude mice were from Vital River Laboratory Animal Technology Co. Ltd., Beijing, China. Antibodies to cyclin B1, Cdc25B, PI3K, Akt and p-Akt were purchased from Abcam (Cambridge, MA, USA). The PrimeScript™ RT-PCR kit and SYBR Premix Ex Taq™ kit were obtained from Takara (Takara, Kusatsu, Japan).

All procedures performed in studies involving animals were in accordance with the ethical standards of the Authors' institution.

SPS (AR, 90%) was purchased from Xi'an Reain Biotechnology Co. Ltd, Xi'an, China. The SPS was dissolved with DMSO to different concentrations: 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, and 2.56 mg/ml.

Human lung cancer cell lines, A549 and YTMLC-90 were purchased from the Cell Library Committee on Type Culture Collection of the Chinese Academy of Sciences, Beijing. Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified, 5% CO₂ atmosphere at 37°C. The cells were plated at a density of 10⁵ cells per well and grown for 24 h.

The MTT assay was used to evaluate cell viability. Cells were seeded in 96-well plates at a density of 5×10³ cells per well and grown for 24 h. Cells were then treated with various concentrations of SPS ranging from 0.04 to 2.56 mg/ml for 24, 48, and 72 h. Cells treated with an equivalent volume of DMSO were regarded as the control groups. Each group was treated in triplicate. Following treatment, 10 µl MTT was added to each well and incubated for 4 h. Finally, blue formazan crystals of viable cells were solubilized in 100 µl DMSO. The absorbance was measured at 450 nm using a microplate reader.

Apoptosis was assessed in A549 and YTMLC-90 cells using an Annexin V-FITC/propidium iodide (PI) staining assay. Cells were cultured in 6-well plates at a density of 2×10⁵/ml per well overnight. Cells were then treated with various concentrations of SPS ranging from 0.04 to 2.56 mg/ml for 48 h. Control cells were treated with culture medium containing DMSO. Each group was treated in triplicate. After treatment, cells were washed with cold PBS and resuspended in binding buffer (100 mM HEPES, pH 7.4, 100 mM NaCl, 25 mM CaCl₂). Cells were stained with Annexin V-FITC/PI at 4°C for 30 min. Apoptotic cells were analyzed using a fluorescence-activated cell-sorting (FACS) flow cytometer.

The expression levels of bax, caspase-3 and cdc25B were measured using real-time PCR. Cells were treated with various concentrations of SPS ranging from 0.04 to 2.56 mg/ml for 48 h. Following treatment, cells were collected and their total RNA extracted using TRIzol reagent. cDNA was synthesized using the PrimeScript RT-PCR kit (Takara). Real-time PCR was performed using SYBR Premix Ex Taq kit (Takara). Primer sequences were as follows: cdc25B: 5′-TTC ATC AGG GAA CGA GA CCG TG-3′, 5′-TTC ACA GAA GTT CGG GTG CTG AG-3′; bax: 5′-GGA GCT GCA GAG GAT GAT TG-3′, 5′-CCT CCC AGA AAA ATG CCA TA-3′; caspase-3: 5′-ATG GAG AAC ACT GAA AAC TCAG-3′, 5′-GAC CGA GAT GTC ATT CCA GTG-3′; GAPDH: 5′-GAA GGT GAA GGT CGG AGT C-3′, 5′-GAA GAT GGT GAT GGG ATT TC-3′. The reaction was repeated 3 times and carried out in an ABI7500 Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA). Templates were initially denatured at 95°C for 5 min followed by 40 cycles at 95°C for 5 sec and 60°C for 34 sec. The relative expression levels of tested genes were calculated using the 2^−ΔΔCT method.

PI staining followed by flow cytometry was used to assess the effect of SPS on the cell cycle of A549 and YTMLC-90 cells. Cells were treated with different concentrations of SPS for 48 h. Cells were then washed with cold PBS with 75% ethanol for 1 h at 4°C. The protocol was then followed as previously described (18). Cells were suspended in 1 ml of PBS that contained 1 mg/ml RNase and 50 µg/ml PI, followed by 30 min of shaking at 37°C in the dark. DNA content was detected using a flow cytometer (BD FACSCalibur System, San Jose, CA, USA).

Protein expression levels of cyclin B1, Cdc25B, PI3K, Akt and p-Akt were measured by western blot according to a previous study (19). After treatment, cells were lysed in RIPA buffer. Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). Twenty micrograms of protein from each sample were separated by 12% SDS-PAGE and transferred to PVDF membranes. Primary antibodies (rabbit monoclonal anti-human cyclin B1, 1:3000 dilution; rabbit polyclonal anti-human Cdc25B, 1:1000 dilution; rabbit monoclonal anti-human Akt, 1:1000 dilution; rabbit monoclonal anti-human p-Akt, 1:1000 dilution) were then applied and incubated at 4°C overnight, after which the appropriate HRP-conjugated secondary antibody was added and incubated for 1 h at room temperature. Proteins were detected using the ChemiDoc XRS imaging system and analysis software Quantity One (Bio-Rad).

To further analyze the effect of SPS injection in vivo, BALB/c nude mice (7 weeks old) were purchased from Vital River Laboratory Animal Technology Co. Ltd. Murine tumor models were induced by subcutaneous injection of A549 cells (5×10⁶ cells in 0.2 ml of PBS) at one site in the right flank, and tumors allowed to develop for 20 days. When tumors reached ~100 mm³ in volume, 40 animals were divided randomly into 4 groups (n=10 for each group): low dose (15 mg/kg), middle dose (45 mg/kg) and high dose (135 mg/kg) injection groups, and a control group that was treated with an equal volume of normal saline (NS). All injections were administered intraperitoneally every day. At 15, 20, 25, and 30 days post-injection, tumor size was measured using calipers and tumor volume was estimated according to a previous study (18). Blood samples were taken from a tail vein to measure the contents of TNF-α and IL-6 using ELISA kits.

Data were expressed as mean ± SD and considered significant at P<0.05. Statistical analysis was performed using a Student's unpaired t-test (SPSS release 19.0; SPSS Inc.).

To evaluate the effect of SPS on A549 and YTMLC-90 cell proliferation activity in vitro, an MTT assay was conducted. Results of the MTT assay are shown in Fig. 1A and B. These results demonstrated that the proliferation of A549 and YTMLC-90 cells was inhibited by different concentrations of SPS. Compared with the CK group, concentrations of 0.04 to 2.56 mg/ml of SPS suppressed the viability of A549 and YTMLC-90 cells, with the exception of 0.04 mg/ml at 24 h (P<0.05). According to Fig. 1C and D, a time-dependent inhibitory effect of SPS on cell survival was found in A549 and YTMLC-90 cells. At 0.64 mg/ml of SPS, the inhibition rate peaked after 72 h of treatment, being 76.66 and 75.47% in A549 and YTMLC-90 cells, respectively. These results suggested that SPS inhibited A549 and YTMLC-90 cell proliferation.

Since a significant decrease in cell viability was found after treatment with SPS, we further tested the effect of SPS treatment on apoptosis. The results of Annexin V-FITC/PI staining showed a dose-dependent effect of SPS on apoptosis in A549 and YTMLC-90 cells (Fig. 2A, B). Compared with CK, apoptosis in A549 and YTMLC-90 cells was significantly increased (P<0.05). Even at 0.64 mg/ml, SPS treatment induced the highest apoptosis rate in both cell lines (P<0.05). When the treatment concentration was >0.64 mg/ml, the level of increase in the apoptosis rate was decreased. To further analyze the mechanism of apoptosis, we also investigated expression levels of bax and capase-3. For the two cell lines, expression levels of bax and capase-3 were both notably induced after treatment with various concentrations of SPS. The expression level of bax peaked at 0.64 mg/ml of SPS treatment, increasing 2.4- and 3.01-fold in A549 and YTMLC-90 cells, respectively, and the transcription level of capase-3 was increased 3.89- and 3.72-fold, respectively (Fig. 2C and D). This result was consistent with the apoptosis rate. In brief, SPS induced apoptosis in A549 and YTMLC-90 cells.

To further analyze the effect of SPS treatment on the cell cycle, we examined both mRNA and protein expression levels of cdc25B and cyclin B1 (Fig. 3). The results of real-time PCR showed that after treatment with SPS, the expression levels of cdc25B and cyclin B1 were both decreased with increasing concentration of SPS when compared with the CK group in A549 and YTMLC-90 cells, reaching their lowest levels at 0.64 mg/ml SPS (Fig. 3A and C). Western blot results also confirmed that the activity of cdc25B and cyclin B1 were both decreased, especially after treatment with 0.64 mg/ml of SPS in A549 or TYMLC-90 cells (Fig. 3B and D). As shown in Table I and Table II, when cells were treated with SPS for 48 h, their DNA contents were significantly increased in the G2/M phase. In the 0.64 mg/ml SPS treatment group, DNA content was markedly increased from 11.1 to 60.3% in A549 cells, and from 4.2 to 61.8% in YTMLC-90 cells. In contrast, the G0/G1 phase population in the SPS treatment group decreased from 65.7 to 29.3% in A549 cells, and from 68.2 to 28.9% in YTMLC-90 cells. These results suggested that SPS induced cell cycle arrest in the G2/M phase.

To investigate whether the inhibitory effect of SPS treatment on NSCLC cell proliferation was mediated by the PI3K/Akt pathway, we analyzed protein expression levels of the PI3K/Akt pathway. In these experiments, cells were treated with SPS (0.04, 0.64, and 2.56 mg/ml) for 48 h. As seen in Fig. 4, we found that compared with the CK group, 0.04 mg/ml of SPS treatment had no effect on the expression of Akt, p-Akt, and PI3K, whereas 0.64 mg/ml of SPS treatment markedly decreased the expression of Akt, p-Akt and PI3K; 2.56 mg/ml of SPS treatment also decreased the expression of these four proteins, but the decreased level was lower than in the 0.64 mg/ml group. This result suggested that SPS inhibited NSCLC cell proliferation by decreasing protein expression levels of the PI3K/Akt pathway.

BALB/c nude mice were used to determine the anti-lung cancer effect of SPS in vivo. Mice were injected with different concentrations of SPS (15, 45, and 135 mg/kg). As shown in Fig. 5A, compared with the control group, SPS injection significantly decreased tumor volume (P<0.05). SPS injection of 45 mg/kg and 135 mg/kg dramatically suppressed tumor growth, with the inhibition rate reaching 75 and 65% 25 days after injection (Fig. 5B). This result indicated that SPS inhibited tumor growth in mice. TNF-α and IL-6 levels were measured to evaluate the immunomodulatory activities of SPS injection in mice. According to Fig. 5C and D, SPS significantly increased TNF-α and IL-6 levels when compared with the control group (P<0.05). For instance, 30 days after injection of SPS (45 mg/kg) in mice, TNF-α was increased by 33.55%, relative to the control group. The level of IL-6 in the 45 mg/kg SPS injection group was increased by 39.50% relative to the control group. These results indicated that SPS injection increased immunomodulatory activities in mice in vivo, suggesting a potential antitumor application in tumor-bearing mice.