br FOXP is an important transcription factor controlling di
FOXP3 is an important transcription factor controlling diﬀerentia-tion and function of Tregs and is found in tumors like PC and melanoma (Karanikas et al., 2008). The role of FOXP3 in cancer has been reported especially in PC: this tumor can accumulate Tregs in/near the tumor and may ruin the immune system (Wang et al., 2017). Additionally, the
same research group discovered that FOXP3 in cancer interacts with CCL5 to turn on the CCL5–CCR5 signaling, which is strongly involved in Treg recruitment, and this eﬀect is attenuated by the CCL5 antibody (Wang et al., 2017).
Treg recruitment to tumors from peripheral regions is regulated by chemokine receptors, e.g., CCR, and their ligands, such as CCL (Adams
& Eksteen, 2006). CCL22 in human ovarian cancer is transactivated with CCR4 on Tregs to drive them into the tumor, and CCL17 or CCL22 in gastric cancer have been reported to participate in Treg cell re-cruitment to the tumor (Curiel et al., 2004; Mizukami et al., 2008). In another murine model of PC, CCR5+ Tregs infiltrated the tumor de-pending on tumor CCL5 levels to create a favorable tumor
Fig. 8. HPLC analysis of β-carotene (A), NL (B), and TL (C). Chromatographic separation was carried out on a C30 column (250 × 4.6 mm, 5 μm particle size) using an isocratic solvent system composed of water and acetone at a 1:9 ratio. The flow rate was adjusted to 1.0 mL/min, the detection wavelength was 450 nm, and 10 μL of each sample was injected. The analytical process was performed at 30 C. β-carotene was detected at RT 40.5 min, and it was observed in TL extract. According to reference previously reported, 1st peak (RT 7.4 min), 2nd peak (RT 7.7 min), 3rd peak (RT 9.2 min) and 4th (RT 10.5 min) were assumed as lutein, zeaxanthin, β-apo-8′-carotenal and β-cryptoxanthin though the resolution between 1st and 2nd peaks were poor (Howe & Tanumihardjo, 2006).
microenvironment; this eﬀect was attenuated by treatment with short hairpin RNA (Tan et al., 2009). Recently, CCL5–CCR5 axis interactions were reported to also play a key role in migratory and invasive prop-erties of PC Gastrin I in three human PC cell lines (Singh et al., 2018). Likewise, many tumor CCL and Treg CCR types are involved in the pathogenesis, and CCL5–CCR5 signaling dominates in case of PC (Wang et al., 2017). Accordingly, we measured the CCR5 and CCL5 expression in tumors and in the spleen and found that the TL extract blocked this expression successfully.
Moreover, the two treatments inhibited mRNA expression levels of IL-10 and TGF-β in tumor tissue and in the spleen. IL-10 and TGF-β are Treg-associated cytokines having an anti-inflammatory activity to de-crease an immune response (Bettini & Vignali, 2009). Therefore, both treatments contributed to an unfavorable microenvironment for PC, thereby resulting in a smaller size than that in the no-treatment control group, but the eﬀect of TL treatment was more stable and powerful.
As seen above, general immune responses can be aﬀected by Treg recruitment inducing locally immunosuppressive conditions. Consequently, we checked the levels of several cytokines. IFN-γ pro-duced and released by T helper 1 (Th1) cells triggers the protective defenses of the immune system eradicating pathogens such as viruses, bacteria, parasites, and tumor cells (Schoenborn & Wilson, 2007). TNF-
α is produced mainly by macrophages, but it can be also produced by a wide variety of cell types including CD4+ T cells, lymphocytes, and mast cells. It is involved in the induction of inflammation as well as inhibition of tumorigenesis (Feng et al., 2015). Both treatments here increased TNF expression, and the NL extract increased the level of IFN-
γ, but the TL extract’s eﬀect did not reach significance. IL-4 and IL-13 produced and secreted by Th2 cells into the supernatant of cell culture after anti-CD3 stimulation were quantified next. Th2 cells producing these cytokines perform a key function by managing immunity eﬀectors against extracellular antigens like parasites. IL-4 particularly induces diﬀerentiation of Th2 cells and stimulates B cells to produce IgE anti-bodies activating mast cells. Both treatments increased IL-4 expression. This finding suggested that in terms of cytokine levels after anti-CD3 stimulation, both treatments strongly enhanced immune responses at a peripheral site, the spleen; this phenomenon is closely associated with the survival of patients with PC (Yamamoto et al., 2012).
Overall, not only TL but also NL extract had a prophylactic or therapeutic eﬀect on PC; however, the TL extract exerted more stable and robust activity in terms of tumor size in the in vivo assay, Treg recruitment, and expression of the relevant biomolecules. There is, of course, a limitation in the eﬀects of both natural substances that we tested here: we observed relatively mild eﬀects as compared to that of a synthetic drug. Nonetheless, TL gave many clues about its prophylactic or therapeutic eﬀect on PC.