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  • br Fig ART treatment decreased the frequencies


    Fig. 3. ART treatment decreased the frequencies MDSCs and Tregs in the spleens and tumors of 4T1 TB mice. Flow cytometric analysis of MDSCs (CD11b+ Gr-1+) and Tregs (CD4+ CD25+ Foxp3+) in both the spleen and tumor (A and B). P < 0.05 compared to the control group.
    2.8. RNA isolation and real-time RT-PCR
    Trizol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from excised tumors (~100 mg) per the manufacturer's instruc-tions. The RNA was then quantified at 260 nm using a UV–VIS spec-trophotometer (PYE-UNICAM, USA). To remove any contaminant genomic DNA, the RNA was treated with DNaseI and cDNA was syn-thesized using the PrimeScript™ RT reagent kit with gDNA Eraser kit (Takara, China). PCR was then performed using the cDNA as a tem-plate. Table 1 shows the specific oligonucleotide primers used for the PCR reaction. Quantitative PCR was performed on an ABI 7500 real-time PCR system (ABI, USA) using a SYBR® Premix Ex Taq™ reagent kit (Takara) according to the manufacturers' instructions. β-actin amplifi-cation served as an internal control. Gene Verteporfin was quantified using the 2− CT method.
    2.9. Statistical analysis
    Results are presented as the mean ± standard deviation of three experiments. One-way ANOVA and two-tailed Student's t-tests were used to test for statistical significance. The Kaplan–Meier method was 
    used to calculate survival analysis. P < 0.05 was considered statisti-cally significant.
    3. Results
    3.1. ART inhibits proliferation of 4T1 cells
    ART has been shown to considerably inhibit the proliferation of certain breast cancer cells, but this effect has not been demonstrated in 4T1 cells. Therefore, we performed an MTT assay to examine 4T1 cell proliferation after ART treatment. High doses (10 μM and 100 μM) of ART significantly inhibited the proliferation of 4T1 cells, beginning 24 h post treatment. Lower doses (0.01 μM, 0.1 μM, and 1 μM) of ART also inhibited proliferation, but not until 48 h post treatment. Furthermore, ART treatment also induced apoptosis of 4T1 cells, as 100 μM ART significantly increased the rate of tumor cell apoptosis (P < 0.01). Additionally, transforming growth factor β (TGF-β) levels in the supernatant significantly decreased following ART treatment (concentrations ranging from 1 to 100 μM, P < 0.001) (Fig. 1).
    Fig. 4. ART promoted T cell-mediated anti-tumor immune responses. Flow cytometric analysis of CD4+ IFN-γ+ T cells and CTLs in the spleen and tumor from 4T1 mice treated with ART or PBS (control). P < 0.05 compared to the control group.
    3.2. ART inhibits the growth of 4T1 breast cancer cells and prolongs the survival of 4T1 TB mice
    To determine whether ART could suppress tumor development and enhance the survival of 4T1 TB mice, mice were given ART daily for 20 days. We found that the tumor volume in the ART group was re-duced compared to that of the control group (Fig. 2B). Similarly, the weight and size of the tumors were significantly decreased in the ART group (P < 0.001) compared to the control group (Fig. 2A). No dif-ferences in body weights were seen between the two groups (Fig. 2C). All the control mice died 28–45 days post 4 T1 cell inoculation. How-ever, ART supplementation significantly extended the survival of 4T1 mice, with no deaths seen before 38 days post inoculation (P < 0.05; Fig. 2D).
    3.3. ART inhibits Treg and MDSC expansion in the spleen and tumor
    CD11b+ Gr-1+ MDSCs suppress immune responses in the tumor microenvironment and directly stimulate tumorigenesis, thereby pro-moting tumor development [24]. To assess whether ART treatment could inhibit the expansion of MDSCs, the frequencies of MDSCs in the spleens and tumors of 4T1 TB mice were analyzed by flow cytometry. We found that MDSC frequencies were significantly lower in both the splenic (P < 0.05) and tumor samples (P < 0.05) from ART-treated 4T1 TB mice compared to the control mice (Fig. 3A).