Rhus Verniciflua Stokes Inhibits PD-1 Expression and Induces Anticancer Effects by Enhancing T Cell Function

Abstract

Background: Over the last decade, the anticancer effects of Rhus verniciflua Stokes (RVS) have been reported in various preclinical or clinical studies. However, the effects of RVS on immuno-oncology, especially on the functional properties of T cells and their phenotypes, remain unclear. Here, we planned to investigate the impact of RVS on immuno-oncology, specifically focusing on its effects on T cells.

Methods: Peripheral blood mononuclear cells (PBMCs) from breast cancer patients were isolated to obtain cytokine-induced killer cell populations with >85% CD3+ T cells. The anticancer activity of these T cells was evaluated by introducing red fluorescent protein (RFP) into HLA-A02:01 type-matched breast cancer cell lines (MCF7 and MDA-MB-231) and analyzing the results using flow cytometry. The effect of RVS extracts on T cell phenotype was assessed using markers such as CTLA-4 and PD-1, as well as mRNA expression levels of key genes (IFN-γ, TNF-α, and IL-2).

Results: RVS treatment significantly enhanced the anticancer activity of T cells against breast cancer cells. Specifically, T cells treated with 100 µg/mL of RVS showed a 20.6% increase in cytotoxicity against MCF-7 cells and a 36.2% increase against MDA-MB231 cells compared to the control. Additionally, RVS treatment led to a significant reduction in PD-1 expression on T cells.

Conclusion: Our findings demonstrate that RVS treatment enhances T cell function against breast cancer cells by reducing PD-1 expression. These results suggest that components of RVS may serve as potential candidates for restoring exhausted T cells in cancer therapy.

Keywords: PD-1; Rhus verniciflua Stokes; T cell co-culture; anticancer; breast cancer; immuno-oncology.

Figure 1.

Quantitative analysis of marker compounds in RVS extract. The chemical structure of four reference marker compounds such as butein, fisetin, fustin, and sulfuretin in the extract of RVS (A). The Chromatogram of the four reference marker compounds at 1 ppm concentration (B). The Chromatograms (C) and composition pie chart (D) comparing the peak areas of butein, fisetin, fustin, and sulfuretin in RVS extract.

Figure 2.

The viability of breast cancer cells treated with RVS extract. MCF-7 and MDA-MB-231 cells were treated with varying concentrations of RVS (0.5, 1, 5, 10, 25, 50, 100, 250, and 500 µg/mL) for 48 hours ((A and B), respectively). The cells were then subjected to the MTT assay to determine cell viability. Data are presented as the mean ± standard deviation of 3 independent experiments performed in triplicate. Immune cell toxicity analysis of RVS treatment using 7AAD staining and flow cytometry (C). Representative flow cytometry plots of 7AAD staining for untreated and RVS-treated immune cells. Statistical significance is indicated as follows: P < .05*.

Figure 3.

Effect of RVS treatment on cancer cell killing by immune cells in co-culture at different E:T ratios. RFP killing assay was performed by co-culturing immune cells with breast cancer cells and treating them with RVS at 10 and 100 µg/mL for 48 hours, followed by RFP measurement using flow cytometry in MCF-7 (A) and MDA-MB-231 (C). The experiment was repeated 3 times and presented as mean ± standard deviation. Statistical significance was determined by t-test. Trypan blue staining was performed to observe the cell morphology under co-culture conditions with Effector:Target (E:T) ratios of 1:1 and 1:5 in MCF7 (B) and MDA-MB-231 (D). Magnification was 200x and statistical significance is indicated as follows: P < .05* and P < .01**.

Figure 4.

Phenotypic analysis of immune cells following RVS treatment. Flow cytometry analysis (lower) and table (upper) showing the phenotype of immune cells following marker staining (A). Immune cells from 7 cases were treated with RVS at 10 µg/mL for 48 hours, and the phenotype was compared using marker staining (B). Data represents mean ± standard deviation of n = 3 independent experiments, analyzed using t-test. Statistical significance is indicated as follows: P < .05 *.

Figure 5.

Increased Secretion of IFN-γ and TNF-α in RVS Treated CIK Cells. IFN-γ and TNF-α detected by ELISA assay. The serum cytokine concentrations of (A) IFN-γ, (B) TNF-α and (C) IL-2 were detected by ELISA assay. The levels of IFN-γ and TNF-α were significantly increased in the RVS treated group. Statistical significance is indicated as follows: P < .05* and P < .01**.