CA was purchased from the National Institute for Food and Drug Control (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

The present study used MSC-2 cells (MDSCs immortalized using a retrovirus encoding the v-myc and v-raf oncogenes) were provided by Dr Francois Ghiringhelli, (Department of Medical Oncology, Center GF Leclerc, Dijon, France). CT26 and RAW 264.7 cells (macrophage cell line) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). MC38 tumor cells (murine colon adenocarcinoma cell line) were provided by Professor Yangxin Fu (Chinese Academy of Sciences, Beijing). Cell lines were maintained at 37°C in a 5% CO₂ humidified chamber in Dulbecco's modified Eagle's medium (DMEM) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Pan Biotech, Aidenbach, Bavaria, Germany) and 100 U/ml penicillin/streptomycin.

BALB/c mice (n=32; age, 6–8 weeks; female; weight, 18–22 g) and C57BL/6 mice (n=20; age, 6–8 weeks; female; weight, 18–22 g) were purchased from the Weitonglihua Company (Beijing, China). Toll-like receptor 4^−/− (TLR4^−/−) mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China). All mice were maintained in a specific pathogen-free environment at 21–25°C and 50% relative humidity, with a 12 h light/dark cycle at the Institute of Biophysics, Chinese Academy of Sciences (Beijing, China). Three mice were housed in each cage and provided with sterilized food and water. Mice were grouped randomly, with 8 mice in each group, and all protocols were approved by the appropriate authorities. To generate tumor models, 5×10⁵ MC38 tumor cells or CT26 tumor cells (purchased from ATCC) were injected subcutaneously in the flank of TLR4^−/− C57BL/6 mice and BALB/c wild-type mice, and splenocytes were isolated after 21 days. Animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Health, and were approved by the Biological Research Ethics Committee (Institute of Biophysics, Chinese Academy of Sciences). The maximum size that tumors were permitted to grow to was 1,000 mm³.

Spleens were dipped into 75% alcohol for 1 sec, then dipped into PBS for 5 sec. Next, spleens were ground using frosted glass slides in PBS, and filtered through a mesh filter (150 µm), centrifuged at 524 × g for 3 min at 4°C. The cell pellets were resuspended in red blood cell lysis buffer (Cowin Biosciences Co., Ltd., Jiangsu, China). After 1 min, 5 ml PBS was added, and cells were filtered and centrifuged at 524 × g for 3 min at 4°C. The cell pellets were resuspended with PBS with 2% FBS and prepared for detection.

An MTT assay was used to assess the cell viability of MSC-2 cells and the macrophage RAW264.7 cell line. MSC2 and RAW264.7 were seeded in 96-well culture plates, respectively, at 3×10³ cells/well. Following exposure to various concentrations (0, 1, 2 and 4 µg/ml) of CA for 72 h at 37°C, 10 µl MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) solution (5 mg/ml in PBS) was added to each well, and the plates were incubated for an additional 4 h at 37°C in a CO₂ incubator. A total of 100 µl formazan lysis solution (5% 2-methyl-1-propanol, 10% SDS, 0.012 mol/l HCl) was added to each well. Following a 6-h incubation at 37°C, the absorbance was read at 570 nm on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

In brief, splenocytes isolated from BALB/c mice bearing CT26 tumors were cultured in 6-cm plates. Following a 4-h incubation at 37°C, adherent cells (8×10⁴ cells/well) were seeded into 24-well plates. Following treatment with serial concentrations of CA for 48 h, the floating and trypsinized adherent cells were harvested via centrifugation at 1,500 × g for 2 min at 4°C. The cell pellets were washed with cold PBS and prepared for detection according to the manufacturer's protocol for an Annexin V-FITC/PI kit (GenStar, Beijing, China). Samples were analyzed with a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo 7.6 software (FlowJo LLC, Ashland, OR, USA).

MDSCs treated with CA (0, 1, 2 and 4 µg/ml) or dimethyl sulfoxide (DMSO) were labeled for immunofluorescence and analyzed by flow cytometry. Antibodies including APC-anti-Gr1 (cat. no. 553129; dilution, 1:200; BD Biosciences) and PE-anti-CD11b (cat. no. 553311; dilution, 1:1,200; BD Biosciences), as markers of MDSCs, from mice bearing CT26 and MC38 tumors, respectively, and APC-Rat IgG1 isotype antibody (cat. no. R20011-11A; dilution, 1:200; Sungene Biotech, Tianjin, China) and PE-Rat IgG1 isotype antibody (cat. no. R20011-09A; dilution, 1:200; Sungene Biotech) were diluted in PBS with 2% fetal calf serum (Pan Biotech), and incubated for 30 min on ice. Next, the cells were washed with PBS plus 2% fetal calf serum twice. The data were analyzed using FlowJo 7.6 (FlowJo LLC).

MSC-2 cells were lysed by RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.0% Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and 1 mM EDTA] supplemented with 100 mM phenylmethylsulfonyl fluoride, 25 µg/ml aprotinin, 1 mM sodium orthovanadate and 50 nM NaF. Total protein was quantified using a bicinchoninic acid assay, and samples (30 µg/sample) were separated via 10% SDS-PAGE under denaturing conditions and then transferred onto a nitrocellulose membrane (GE Healthcare, Milwaukee, WI, USA) for 1 h at 100 V. The membranes were blocked with 3% BSA in PBS-T (0.1% Tween-20) for 2 h at 4°C and then incubated overnight at 4°C with the following primary antibodies: p53 (cat. no. 2524; dilution, 1:1,000), caspase-3 (cat. no. 9662; dilution, 1:1,000), caspase-9 (cat. no. 9508; dilution, 1:1,000) (Cell Signaling Technology, Inc., Danvers, MA, USA). Following three washes with PBST, the membrane was incubated with HRP-conjugated goat anti-mouse (cat. no. A3683; dilution, 1:5,000) or goat anti-rabbit IgG (cat. no. A6154; dilution, 1:3,000) secondary antibodies (Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. The membrane was washed with PBST five times (5 min/time) and then incubated with a chemiluminescent substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 3 min at room temperature. Specific bands were visualized with a chemiluminescence imaging system (Clinx Science Instruments Co., Ltd., Shanghai, China) and analyzed by Clinx software (http://www.clinx.cn). β-actin (cat. no. 4970S; dilution, 1:3,000; Cell Signaling Technology, Inc.) was used as an internal standard.

Total RNA was extracted from stimulated MSC-2 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA was synthesized using the 5X All-in-One RT Master mix (Abcam, Cambridge, UK). qPCR was determined using SYBR Green II Mix (Thermo Fisher Scientific, Inc.) on an Applied Biosystems 7500 thermocycler. Primer sequences were as follows: Bax forward, 5′-CCAGGATGCGTCCACCAAG-3′ and reverse, 5′-AAGTAGAAGAGGGCAACCAC-3′; caspase-9 forward, 5′-TCCTGGTACATCGAGACCTTG-3′ and 5′-AAGTCCCTTTCGCAGAAACAG-3′; caspase-3 forward, 5′-ATGGAGAACAACAAAACCTCAGT-3′ and reverse, 5′-TTGCTCCCATGTATGGTCTTTAC-3′; caspase-8 forward, 5′-TGCTTGGACTACATCCCACAC-3′ and reverse, 5′-TGCAGTCTAGGAAGTTGACCA-3′; p53 forward, 5′-CTCTCCCCCGCAAAAGAAAAA-3′ and reverse, 5′-CGGAACATCTCGAAGCGTTTA-3′; and β-actin forward, 5′-GGAGATTACTGCCCTGGCTCCTA-3′ and reverse, 5′-GACTCATCGTACTCCTGCTTGCTG-3′. The housekeeping gene GAPDH was used as an internal control. The thermocycling conditions consisted of an initial denaturation step for 10 min at 95°C, then amplification for 40 cycles of 15 sec at 95°C and 1 min at 57°C. The qPCR procedure was repeated three times. qPCR data was analyzed using the 2^−∆∆Cq method (15).

All data are presented as the mean values ± standard deviation and were evaluated by one-way ANOVA with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. GraphPad 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for the analysis.

To examine the effect of CA on MDSCs, isolated splenocytes from mice bearing CT26 tumors and then treated the cells with CA at various concentrations (0, 1, 2 and 4 µg/ml) for 24 h. As illustrated in Fig. 1, CA reduced the MDSC (Gr1⁺, CD11b⁺) numbers in a dose-dependent manner and showed significant suppression at concentrations of 4 µg/ml (P<0.05).

To confirm the inhibition of MDSCs by CA and to examine the toxicity of CA, the viability and growth of the MSC-2 cells and RAW 264.7 cells was assessed using an MTT assay. The viability of MSC-2 cells was significantly reduced (P<0.05) by CA in a dose-dependent manner (Fig. 2A); however, the viability of RAW 264.7 cells was not reduced by CA (Fig. 2B).

As CA was demonstrated to markedly reduce the survival of MDSCs, the present study investigated the mechanism underlying this decrease in cell viability. Fig. 3A is a typical quadrant analysis of the adherent splenocytes isolated from the mice bearing CT26 tumors, treated with CA at different concentrations for 48 h, double stained with Annexin V-FITC/PI and subjected to flow cytometry. The percentage of cells in early apoptosis increased from 0.243 to 4.49% (4 µg/ml). This result demonstrates that the incubation with CA promoted apoptosis in MDSCs in a dose-dependent manner (Fig. 3B).

Apoptosis is classified into two categories depending on its mediation by the sequential activation of different caspase proteins (16). The present study investigated expression levels of various proteins involved in the intrinsic and extrinsic apoptosis pathways by western blotting. Caspase-9 and caspase-3 were activated by CA, as shown in Fig. 4A-C by a significant time-dependent increase (P<0.05), while the expression of p53 was also increased. A series of semi-quantitative and quantitative real-time RT-PCR assays were performed on MSC-2 cells treated with CA (4 µg/ml for different periods of time). The expression levels of apoptosis factors caspase-9, caspase-3 and Bax increased in a time-dependent manner. p53 also increased slightly (Fig. 4B). These results indicated that CA induced apoptosis in MDSCs via the intrinsic pathway.

To further clarify the mechanism by which extracellular CA promotes the intrinsic apoptosis pathway, splenocytes were isolated from TLR4^−/− mice with colon tumors. As shown in Fig. 5A and B, in TLR4^−/− mice, CA does not decrease the percentage of MDSCs unlike its effect in WT mice (Fig. 1B). The anti-MDSC activity of CA was abolished in these cells (Fig. 5A and B), suggesting that CA reduced MDSCs through a TLR4-dependent pathway.