Antitumor Mechanism Of Chelidonium Majus L. Targeting Adenosine Signaling Pathway Within The Tumor Microenvironment

Despite the success of Immune checkpoint blockade therapies(ICBs)in advanced cancer,many cancers do not benefit which suggests the non-redundant immunosuppressive mechanisms co-exist within the tumor microenvironment(TME).Extracellular adenosine(e ADO)signaling is a common immunosuppressive pathway,blockade of adenosine generation and signaling has been used in clinical research on cancer.Nevertheless,fairly short half-life,notoriously difficult preparation and unpredictable side effects of the inhibitors greatly limited clinical therapeutic effectiveness.Here,we found easily achieved alternative molecules with good pharmacological activity from Chelidonium majus L.(C.majus L.)identified by systems pharmacology strategy exhibit anti-tumor effect largely through targeting TME and reversing immunosuppression induced by e ADO.The main findings of this work are summarized as follow:1.Combined the systems pharmacology approach with TCGA database analysis,we uncovered the anti-tumor effect of C.majus L.The main contents include:(1)Through ADME screening,22 active ingredients with good pharmacokinetic properties were obtained from C.majus L.;(2)The C-T network analysis showed that the 22 candidates acted on 97targets,and all compounds or targets displayed poly-pharmacology properties,indicating that C.majus L.has multi-directional pharmacological properties and synergistic effects in the treatment of cancer.(3)The TCGA analysis confirmed that the targets related to cancer and immunity of C.majus L.were highly positively correlated with the overall survival of patients with 8 cancer types;(4)The antitumor effect of C.majus L.was verified by inhibition of cancer cell viability in vitro and tumor-bearing mouse model experiment in vivo.2.(1)The synergism of Chelerythrine and Chelidonine(CC)was then confirmed by PEA combination prediction and Combination Index;(2)pharmacodynamic evaluation showed that 2.5 mg/kg CC had good antitumor effect on tumor volume growth rate and survival time of tumor-bearing mice.3.Through GOBP enrichment,TCGA database analysis together with detection of the key factors in TME,we found CC downregulates the gene transcription and protein expression of HIF-1α,CD39 and CD73 by targeting the TME in the LLC-bearing mice.These data demonstrated that CC favors anti-tumor responses by overcoming e ADO-induced suppress barrier through CD39/CD73 axis driven by HIF-1αenhancing the immune response of CD8~+T cells within TME.The main contents include:(1)HIF1A was identified as the potential anti-tumor target gene,and it was found that HIF1A was highly positively correlated with ENTPD1 and NT5E in the e ADO pathway in clinical lung cancer;(2)CC significantly down-regulated the gene transcription and protein expression of HIF1A,CD39 and CD73 in TME,which suggesting that the potential mechanism of CC is the regulation of HIF-1α-driven CD39/CD73 axis by targeting TME and relieving immunosuppression of e ADO;(3)CC reversed the activity of ADA and promoted the activation of adenosine consumption pathway;(4)Data of the flow cytometry that impeding the generation of e ADO facilities anti-tumor immunity of CD8~+T cells based on A2AR-mechanism by modulating the proliferation and efficacy of effectors;(5)CC improved the abnormal activation of the downstream signaling pathway of A2AR,c AMP/PKA/CREB/NF-κB.4.Animal pharmacodynamics combined with RNA-Seq technology illustrated that CC might be sensitized to anti-PD-L1-mediated immunotherapy of the established tumors by delaying tumor progression.Taken together,the approach for seeking potential drugs will help develop adenosine-based strategy to sensitized ICBs in oncology.