br Statistical analysis br All
2.9. Statistical analysis
All graphical results described in this study are presented as the mean ± SEM of at least three independent experiments analyzed with two-tailed Student’s unpaired t test. A diﬀerence was considered
significant when the p value was below 0.05.
3.1. Cytotoxicity to Panc02 cells in vitro and dose determination for the in vivo assay
We first tested whether these extracts had any cytotoxicity toward Panc02 cells in an in vitro system. TL showed stronger cytotoxicity as compared to NL (half-maximal inhibitory concentration [IC50] for NL was 1.7 mg/mL and for TL was 0.3 mg/mL; Fig. 2A). Additionally, to determine the treatment dose for an in vivo assay, 20, 40, or 80 mg/mL NL or TL was administered once a day intraperitoneally to mice for 3 days in a row. Neither body weight loss nor abnormal signs were observed (Fig. 2B–D). Thus, the 80 mg/mL dose was chosen for the animal experiments.
3.2. Evaluation of tumor size and the immune-cell population profile in a tumor
The size of tumor decreased significantly in TL-treated mice com-pared to that in the control group on days 19 and 25 after cancer cell inoculation, whereas no significant change was observed in NL-treated mice (Fig. 3).
Regarding the immune-cell population profile in the tumor, both NL- and TL-treated mice showed decreased levels of natural killer (NK) cells as compared to the control group (Fig. 4A). On the other hand, the numbers of 1 \'-hydroxy Midazolam significantly increased only in the TL-treated group compared to that in the control group (Fig. 4F). No significant change was detected in other immune cells like macrophages and CD4+ or CD8+ T cells. TL treatment recruited more B cells with less suppression of NK cells as compared to NL treatment (Fig. 4B–E). In addition, Tregs are known to help Panc02 cells to proliferate through local immune suppression (Hinz et al., 2007); this eﬀect was reduced by TL treatment (Fig. 4G).
3.3. Expression levels of anti-inflammatory cytokines and genes involved in Treg recruitment
Expression levels of IL-10 and TGF-β (generally regarded as anti-inflammatory cytokines) and FOXP3, CCR5, and CCL5 (known to be associated with Treg recruitment and their chemotaxis) were measured by PCR. Both treatments significantly reduced TGF-β and CCR5 mRNA
25. At the last two time points, TL treatment significantly suppressed tumor growth as compared to that in the animals inoculated only with tumor cells. The
data are presented as the means ± SD from three independent experiments. Statistical significance was evaluated by Student’s t test (*p < 0.05 Cancer vs TL or NL) (Cancer: group inoculated with cancer; NL: NL treated group in-oculated with cancer; TL: TL treated group inoculated with cancer). Journal of Functional Foods 58 (2019) 301–310
levels, but FOXP3 mRNA was downregulated only by TL treatment (Fig. 5A, C, and D). Even though the mRNA levels of IL-10 and CCL-5 were not significantly altered, both treatments showed a trend toward the decreased expression of these genes (Fig. 5B and E). Thus, both treatments interrupted the accumulation and polarization of Tregs in tumors by inhibiting the production of related proteins; TL had a stronger eﬀect than NL.
3.4. Immune alteration in the spleen by treatment with TL or NL
Regarding the immune-cell population profile in the spleen, de-creased numbers of dendritic cells were observed in tumor-inoculated mice, but this eﬀect was reversed by treatment with NL and TL. Moreover, TL treatment significantly increased the number of CD8+ T and B cells, whereas NL did not (Fig. 6A–F). As for T-cell activation (CD8+/CD4+), analysis of the tumor-injected mice revealed that as naïve-T-cell numbers increased, eﬀector T cells tended to decrease in number as compared to that in the group without tumor inoculation; however, this outcome was attenuated by NL and TL extracts (Fig. 6G–L). It should be stated that TL showed a stronger influence. NL and TL reduced the expression of genes involved in recruitment and polarization of Tregs upregulated by the tumor cell inoculation; TL exerted this eﬀect more clearly (Fig. 6M–Q).
3.5. Analysis of cytokines and Treg-related genes in splenocytes after anti-CD3 stimulation for 2 days
Splenocytes extracted from the mice were stimulated with the anti-CD3 antibody for 2 days to check concentrations of cytokines like IFN-γ, TNF-α, IL-4, IL-13, and IL-10 secreted into the culture medium and the expression of genes such as FOXP3, IL-10, TGFβ, CCR5, and CCL5 re-levant for Treg recruitment. The mRNA level of FOXP3 was decreased by the TL extract (Fig. 7A) and both treatments suppressed CCL5 mRNA expression (Fig. 7E), but no significant change was observed in the expression of other genes (Fig. 7B–D). A higher IFN-γ protein level was seen after NL treatment, and both treatments increased TNF-α and IL-4 protein secretion as compared to cancer cell–inoculated as well as naïve groups (Fig. 7F–H).