Kaempferol blocks neutrophil extracellular traps formation and reduces tumour metastasis by inhibiting ROS-PAD4 pathway

Kaempferol (kaem) is a dietary flavonoid found in a variety of fruits and vegetables. The inhibitory effects of kaem on primary tumour growth have been extensively investigated; however, its effects on tumour metastasis are largely unknown. In the present study, we found that kaem significantly suppresses both primary tumour growth and lung metastasis in mouse breast tumour model. Furthermore, decreased expression of citrullinated histone H3 (H3-cit), a biomarker of neutrophil extracellular traps (NETs), had been founded in metastatic lung upon treated with kaem. The reduction of H3-cit is not, however, due to the cytotoxicity of kaem on neutrophils since the frequency of CD11b(+)Ly6G(+) neutrophils did not change in lung, tumour or blood in the presence of kaem. We then confirm the anti-NETs effects of kaem in vitro by co-culturing mouse neutrophils and kaem. Supplementing the neutrophils with GSK484, a potent NET inhibitor, totally abrogated the inhibitory effects of kaem on tumour metastasis while having little or no impact on primary tumour growth, indicating the specificity of kaem acting on NET formation and tumour metastasis. We also found that kaem suppressed ROS production in mouse bone-marrow derived neutrophils. Supplementing with the ROS scavenger DPI abrogated kaem's effects on NET formation, suggesting the involvement of kaempferol in NADPH/ROS-NETs signalling. Finally, we applied the kaem on NET-deficient PAD4(-/-) mice and found decreased primary tumour volume and weight but similar lung metastatic tumour with kaempferol treatment. Therefore, our findings reveal a novel mechanism of kaem in breast cancer development by targeting NETs induced tumour metastasis.