Oxymatrine and Cisplatin Synergistically Enhance Anti-tumor Immunity of CD8+ T Cells in Non-small Cell Lung Cancer

Abstract

Oxymatrine (OMT) has shown broad antitumor activities for the treatment of several types of cancers. However, little is known about its effect on anti-tumor immunity. Combination therapy is a potentially promising strategy of cancer to enhance anticancer activity, overcome drug resistance, and lower treatment failure rate. In the present study, we demonstrated that the combination of OMT with cisplatin (DDP) synergistically inhibited non-small cell lung cancer (NSCLC) cells growth when co-cultured with peripheral blood mononuclear cells in vitro. Furthermore, the combination of OMT with DDP significantly inhibited the growth of Lewis lung cancer (LLC) mouse xenograft tumors. Flow cytometry analysis revealed that OMT and DDP synergistically increase the CD8⁺/ regulatory T cells ratio and enhanced more CD8⁺ T cells secreted cytokines of IFN-γ, TNF-α, and IL-2 in vivo. Mechanistically, upregulation of miR-155 and downregulation of suppressor of cytokine signaling-1 (SOCS1) were confirmed as a target signaling pathway to positively regulate the anti-tumor response of CD8⁺ T cells. Overall, OMT in combination with DDP showed outstanding synergistic anti-tumor immunity, suggesting that this beneficial combination may offer a potential immunotherapy for NSCLC patients.

Keywords: CD8+ T cells; NSCLC; anti-tumor immunity; cisplatin; oxymatrine.

Figure 1

OMT and DDP synergistically inhibit the growth of NSCLC cells co-cultured with PBMCs in vitro. Cells were treated with the indicated concentrations of OMT or DDP for 72 h, and cell survival was determined by MTT or CCK-8 assay and summary survival curves (A) and IC₅₀ values in the indicated cells (B) were shown. Co-cultured NSCLC cells (target cells) with PBMCs (effect cells) at ratios of 1:0, 1:2, 1:4, and 1:6 were treated with OMT (3 mM) and DDP (2 μM) alone or combination for 24 h. Quantified results were shown in (C). The values presented are the means ± SD for each group. *P < 0.05 and **P < 0.01 vs. corresponding control.

Figure 2

OMT and DDP synergistically inhibit NSCLC xenografts growth in vivo. Each mouse was injected subcutaneously with LLC cells (2 × 10⁶ in 100 μl of PBS) in right scapular region. When the subcutaneous tumors were approximately 0.3 × 0.3 cm² (two perpendicular diameters) in size, mice were randomized into four groups, and were injected intraperitoneally with vehicle alone (0.9% saline), OMT alone (100 mg/kg body weight per day), DDP alone (2 mg/kg body weight every 2 day), or a combination of OMT and DDP (administration method is as same as the relevant single drug group). The body weights and tumor volumes of mice were recorded. The mice were anesthetized after experiment, and tumors were excised from the mice and weighted. The original tumors (A), tumor volumes (B), tumor weights (C), body weights (D), and summary data (E) were shown. The values presented are the means ± SD for each group. *P < 0.05 and **P < 0.01 vs. corresponding control.

Figure 3

OMT and DDP synergistically increase the CD8⁺/T_reg ratio in vivo. Isolated PBMCs, spleen lymphocytes and tumor infiltrating lymphocytes were stained with indicated antibodies and analyzed by flow cytometry. Representative flow plots and quantified results of CD8⁺ T cells and T_reg cells in PBMCs (A), spleen lymphocytes (B), and tumor infiltrating lymphocytes (C) were shown. The CD8⁺/T_reg ratios were quantified (D). *P < 0.05 and **P < 0.01 vs. corresponding control.

Figure 4

OMT and DDP synergistically enhance CD8⁺ T cells anti-tumor immune response. Spleen lymphocytes and tumor infiltrating lymphocytes were isolated, and intracellular IFN-γ, TNF-α, and IL-2 were determined by flow cytometry. Representative flow plots and quantified results of intracellular IFN-γ, TNF-α, and IL-2 expression in CD8⁺ T cells of spleen lymphocytes (A) and tumor infiltrating lymphocytes (B) were shown. *P < 0.05 and **P < 0.01 vs. corresponding control.

Figure 5

OMT and DDP synergistically upregulate miR-155 and downregulate SOCS1 expressions in splenic CD8⁺ T cells. Splenocytes CD8⁺ T cells were separated by magnetic bead from MACS and the total RNAs were extracted immediately. Expression of miR-155 and SOCS1 in splenic CD8⁺ T cells were determined by RT-qPCR. U6 and β-actin were used as the normal controls. Data shown are representative of three independent experiments. *P < 0.05 and **P < 0.01 vs. corresponding control.