Calpain Inhibitor I ALLN br Results br ART inhibits prolifer
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 Calpain Inhibitor I ALLN 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 . 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).
Previous studies have shown that tumor-induced regulatory B cells (tBregs) can stimulate FoxP3+ Treg diﬀerentiation from resting non-regulatory CD4+ T cells, which suppresses host anti-tumor immunity and promotes metastasis . Therefore, we also measured the fre-quencies of Tregs in the spleens and tumors of ART-treated and non-treated 4T1 TB mice. ART treatment significantly decreased the per-centage of Tregs in TB mice (P < 0.05, Fig. 3B), indicating that ART may inhibit immunosuppression in the tumor microenvironment.
3.4. ART promotes the activation of T cell
T cells play an important role in the anti-tumor immune response and therefore in preventing tumor growth. To determine whether ART can induce the activation of T cells, we quantified the percentages of CD4+ IFN-γ+ T cells and granzyme B-expressing cytotoxic T lympho-cytes (CTLs) within the spleens and tumors of 4T1 mice. ART treatment resulted in significantly increased percentages of CD4+ IFN-γ+ T cells in both the spleen and tumor samples (Fig. 4A, P < 0.05). ART treat-ment also significantly increased the expansion of granzyme B+ CTLs (Fig. 4B, P < 0.05). These results indicate that ART can promote the activation of T cells.
Fig. 5. Transcription factor and cytokine expression induced by ART. T-bet, FOXP3, IFN-γ, TNF-α, TGF-β, and IL-10 mRNA levels within the tumor tissue were measured by qRT-PCR. P < 0.01 compared to the control group.
3.5. ART treatment enhances the expression of immune mediators in the tumor microenvironment
Various immune mediators in the tumor microenvironment, in-cluding cytokines and transcription factors, play crucial roles in in-hibiting the progression of breast cancer. We used qRT-PCR to measure the relative mRNA expression levels of the transcription factors T-bet and FOXP3, and the cytokines IFN-γ, TNF-α, TGF-β, and IL-10 in the tumor tissues from 4T1 mice. As shown in Fig. 5, expression of T-bet and the related pro-inflammatory cytokine IFN-γ, as well as TNF-α, increased after ART treatment (P < 0.01). TGF-β mRNA levels within the tumor significantly decreased after ART treatment (P < 0.01), while IL-10 levels did not change significantly. Expression of Foxp3, a transcription factor active in Tregs, also did not change significantly.