Sophoridine Inhibits the Tumour Growth of Non-Small Lung Cancer by Inducing Macrophages M1 Polarisation via MAPK-Mediated Inflammatory Pathway

Abstract

Lung cancer is one of the most common and lethal neoplasms for which very few efficacious treatments are currently available. M1-like polarised tumour-associated macrophages (TAMs) are key mediators to modulate the tumour microenvironment, which play a key role in inhibiting cancer cell growth. Sophoridine, a naturally occurring alkaloid, exerts multiple pharmacological activities including anti-tumour and anti-inflammatory activities, but it has not been characterised as a regulator of tumour microenvironment towards NSCLC. Herein, the regulatory effects of sophoridine on the polarisation of THP-1 cells into TAMs and the anti-tumour effects of sophoridine-stimulated M1 polarised macrophages towards lung cancer cells were carefully investigated both in vitro and in vivo. The results showed that sophoridine could significantly promote M1 polarisation of RAW264.7 and THP-1-derived macrophages, leading to increased expression of pro-inflammatory cytokines and the M1 surface markers CD86 via activating MAPKs signaling pathway. Further investigations showed that sophoridine-stimulated RAW264.7 and THP-1-derived M1 macrophages effectively induced cell apoptosis as well as inhibited the cell colony formation and cell proliferation in both H460 and Lewis lung cancer cells. In Lewis-bearing mice model, sophoridine (15 or 25 mg/kg) significantly inhibited the tumour growth and up-regulated the expression of CD86/F4/80 in tumour tissues. Collectively, the findings clearly demonstrate that sophoridine promoted M1-like polarisation in vitro and in vivo, suggesting that sophoridine held a great therapeutic potential for treating lung cancer.

Keywords: RAW264.7; THP-1 cells; macrophage differentiation; mitogen-activated protein kinase (MPKs) pathway; sophoridine.

Figure 1

Sophoridine promoted macrophages shifting to M1 phenotype. (A) Chemical structure of sophoridine (downloaded from PubChem). (B, C) The cell viability of sophoridine-stimulated RAW264.7 and THP-1-derived macrophages was detected by CCK-8 assay. (D, E) The percentage of CD86 of RAW264.7 and THP-1-derived macrophages. (F, G) The relative expression of IFN-γ, TNF-α, IL-6, iNOS, and IL-1β mRNA in RAW264.7 and THP-1-derived macrophages determined by RT-PCR. (H, I) The NO production of RAW264.7 and THP-1-derived macrophages assessed by Griess. (J, K) The protein expression (% of GAPDH) of TNF-α and IL-1β in RAW264.7 and THP-1-derived macrophages determine by Western blotting (n = 3), p < 0.05 (*).

Figure 2

Sophoridine induced macrophages M1 polarisation via MAPKs pathway.

Figure 3

The sophoridine induced macrophages differentiation and produced pro-inflammatory cytokines via the MAPKs signaling pathway (A, B) The relative protein expression of p-JNK, p-ERK, and p-P38 (relative to JNK, ERK, and P38, respectively) in RAW264.7 and THP-1-derived macrophages determine by Western Blotting. (C, D) The percentage of CD86 of sophoridine-stimulated RAW264.7 and THP-1-derived macrophages with or without SP600125 treatment. (E, F) The relative expression of IFN-γ, TNF-α, IL-6, iNOS, and IL-1β mRNA in sophoridine-stimulated RAW264.7 and THP-1-derived macrophages with or without SP600125 treatment determine by RT-PCR. (G, H) The NO production of sophoridine-stimulated RAW264.7 and THP-1-derived macrophages with or without SP600125 treatment (n = 3), p < 0.05 (*).

Figure 4

Sophoridine-stimulated macrophage-lung cancer cell crosstalk induced cell apoptosis, cell colony formation, and suppressed cell proliferation. (A) The cell apoptosis of H460 with or without infiltration of sophoridine-stimulated THP-1-derived macrophages, and Lewis lung cancer cells with or without sophoridine-stimulated RAW264.7 macrophages determine by flow cytometry analysis. (B) The cell colony formation of H460 with or without infiltration of sophoridine-stimulated THP-1-derived macrophages, and Lewis lung cancer cells with or without sophoridine-stimulated RAW264.7 macrophages. (C) The cell proliferation and apoptosis of H460 with or without infiltration of sophoridine-stimulated THP-1-derived macrophages, and Lewis lung cancer cells with or without sophoridine-stimulated RAW264.7 macrophages determine by EdU and Hoechst staining. (D) The percentage of cell number in the G1, S, and G2/M phase during the cell cycle of H460 with or without infiltration of sophoridine-stimulated THP-1-derived macrophages, and Lewis lung cancer cells with or without sophoridine-stimulated RAW264.7 macrophages detect by flow cytometry using PI staining. (E) ROS intensity with or without infiltration of sophoridine-stimulated macrophages (n = 3), p < 0.05 (*).

Figure 5

Sophoridine inhibited tumour growth of Lewis-bearing mice and promoted M1 polarisation of macrophages in vivo. (A) Administration of 15 and 25 mg/kg sophoridine decreased the tumour volume (mm³). (B) Administration of 15 and 25 mg/kg sophoridine decreased the tumour weigh. (C) Administration of 15 and 25 mg/kg sophoridine decreased the infiltration of inflammatory cells measured by H&E staining, and the expression of Ki67 measured by IHC staining, in tumour tissues from Lewis-bearing mice p < 0.05 (*).

Figure 6

(A) The expression of CD86 and F4/80, determined by IHC. Scale bar: 50 μm (B) Administration of 15 and 25 mg/kg sophoridine increased the expression of the ratio of CD86/F4/80 (n = 3), p < 0.05 (*).

Figure 7

The toxicity of sophoridine on the heart, liver, kidney, and spleen tissues in vivo. The sections were stained with H&E staining. The pictures are representative from control and 15 and 25 mg/kg sophoridine groups.