Jing Du1, Xiangfeng Chen1, Chenying Wang1, Hongxiang Sun2. 1. College of Animal Sciences, Zhejiang University, Yuhangtang Road 866, Hangzhou, Zhejiang 310058, China. 2. College of Animal Sciences, Zhejiang University, Yuhangtang Road 866, Hangzhou, Zhejiang 310058, China. Electronic address: sunhx@zju.edu.cn.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Actinidia eriantha Benth have been used clinically to treat a variety of cancers in traditional Chinese medicine. The polysaccharide from this drug (AEPS) was previously reported to be a potential antitumor agent with immunomodulatory activity. However, the mechanisms of its antitumor action in immunomodulation have not yet been well-defined. AIM OF THE STUDY: To investigate the effects of AEPS on the phenotypic and functional maturation of dendritic cells and to explore the intracellular signaling mechanisms of its antitumor action in the immunomodulation. MATERIALS AND METHODS: The effects of AEPS on the phagocytic activity, expression of surface molecules, mRNA and protein expression levels of cytokines and chemokines in mouse bone-marrow derived dendritic cells (BMDCs) were determined by flow cytometry, qRT-PCR and ELISA, respectively. The transcriptional profile induced by AEPS was established using oligonucleotide microarray, and Ingenuity Pathway Analysis (IPA) was used to identify potential signaling pathways. Western blotting, neutralization experiments and inhibition assay were performed to confirm signaling pathway involved in maturation of DCs induced by AEPS. Furthermore, we discussed the downstream effects of the action of AEPS using clustering, network and pathway mapping approaches. RESULTS: AEPS could significantly reduced phagocytic activity, promoted expression of accessory and co-stimulatory molecules, and up-regulated the mRNA and protein expression levels of cytokines and chemokines in BMDCs. Microarray assay revealed that AEPS induced significantly differential expression of 452 genes including up-regulated cytokines (IL-6, IL-1β, TNF-α, IL-10, IL-12p40, IFN-β and IFN-γ), chemokines (MIP-1α, MIP-1β, CCL5, MDC and MCP-1), transcription factors (STAT1, STAT2, STAT5b, IRF1 and IRF7) and pattern recognition receptors (TLR3, DDX58, DHX58 and IFIH1) in the BMDCs. AEPS-induced production of TNF-α and IL-12p40 from BMDCs was inhibited by antibodies against TLR2 and TLR4. Furthermore, AEPS induced the phosphorylation of NF-κB p65 in a time-dependent manner, and BAY 11-7082, an inhibitor of NF-κB, remarkably suppressed the production of cytokines induced by AEPS. CONCLUSION: AEPS triggers the phenotypic and functional maturation of DCs via TLR2/4 and NF-κB signaling pathway, resulting in augmented antitumor immune responses. Our results suggested that AEPS might be helpful in potentiating the efficiency of DC-based cancer immunotherapy. This study further expanded current knowledge on the mechanisms of antitumor action of AEPS.
ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Actinidia eriantha Benth have been used clinically to treat a variety of cancers in traditional Chinese medicine. The polysaccharide from this drug (AEPS) was previously reported to be a potential antitumor agent with immunomodulatory activity. However, the mechanisms of its antitumor action in immunomodulation have not yet been well-defined. AIM OF THE STUDY: To investigate the effects of AEPS on the phenotypic and functional maturation of dendritic cells and to explore the intracellular signaling mechanisms of its antitumor action in the immunomodulation. MATERIALS AND METHODS: The effects of AEPS on the phagocytic activity, expression of surface molecules, mRNA and protein expression levels of cytokines and chemokines in mouse bone-marrow derived dendritic cells (BMDCs) were determined by flow cytometry, qRT-PCR and ELISA, respectively. The transcriptional profile induced by AEPS was established using oligonucleotide microarray, and Ingenuity Pathway Analysis (IPA) was used to identify potential signaling pathways. Western blotting, neutralization experiments and inhibition assay were performed to confirm signaling pathway involved in maturation of DCs induced by AEPS. Furthermore, we discussed the downstream effects of the action of AEPS using clustering, network and pathway mapping approaches. RESULTS: AEPS could significantly reduced phagocytic activity, promoted expression of accessory and co-stimulatory molecules, and up-regulated the mRNA and protein expression levels of cytokines and chemokines in BMDCs. Microarray assay revealed that AEPS induced significantly differential expression of 452 genes including up-regulated cytokines (IL-6, IL-1β, TNF-α, IL-10, IL-12p40, IFN-β and IFN-γ), chemokines (MIP-1α, MIP-1β, CCL5, MDC and MCP-1), transcription factors (STAT1, STAT2, STAT5b, IRF1 and IRF7) and pattern recognition receptors (TLR3, DDX58, DHX58 and IFIH1) in the BMDCs. AEPS-induced production of TNF-α and IL-12p40 from BMDCs was inhibited by antibodies against TLR2 and TLR4. Furthermore, AEPS induced the phosphorylation of NF-κB p65 in a time-dependent manner, and BAY 11-7082, an inhibitor of NF-κB, remarkably suppressed the production of cytokines induced by AEPS. CONCLUSION: AEPS triggers the phenotypic and functional maturation of DCs via TLR2/4 and NF-κB signaling pathway, resulting in augmented antitumor immune responses. Our results suggested that AEPS might be helpful in potentiating the efficiency of DC-based cancer immunotherapy. This study further expanded current knowledge on the mechanisms of antitumor action of AEPS.