Combination Therapy of Sini Decoction and Cyclophosphamide Inhibits Tumor Growth in an Orthotopic Lung Cancer Model

Background: Lung cancer represents a frequently seen respiratory system malignancy. Sini Decoction combined with cyclophosphamide is demonstrated to remarkably extend survival and improve quality of life of these patients; however, the associated anti-tumor mechanisms are largely unexplored. Objective: The present work focused on investigating the inhibition of tumor cells by Sini Decoction plus cyclophosphamide within the orthotopic lung cancer model and exploring the mechanisms in terms of exosome-based tumor hypoxic microenvironment modulation. Methods: A549-luc2-tdT-2 cells were implanted in left lung of nude mice for establishing the orthotopic lung cancer xenograft model. After 5 days, bioluminescence imaging was conducted for model validation. Mice were later randomized as 4 groups: model, Sini Decoction , cyclophosphamide, as well as Sini Decoction plus cyclophosphamide. Bioluminescence imaging was conducted to assess anti-tumor effects. Enzyme-linked immunosorbent assay (ELISA) was performed for measuring liver and kidney function indicators (ALT, AST, Cr) in serum. Additionally, RT-qPCR and immunohistochemistry were carried out for detecting hypoxia-related factor levels (HIF-1α, VEGF, PDGF-β) within lung tissue. Additionally, characterizations of the separated exosomes were completed with transmission electron microscopy, BCA protein assay, nanoparticle tracking analysis, and Western blotting. Exosomal miR-20a-5p was chosen based on TargetScan database for analysis, while RT-qPCR was completed for validation. Results: Sini Decoction plus cyclophosphamide dramatically suppressed tumor growth within the orthotopic lung cancer model while ameliorating hepatorenal toxicities, as evidenced by serum liver and kidney function indicators and bioluminescence imaging. Meanwhile, immunohistochemistry showed that the combination therapy markedly downregulated HIF-1α expression in lung tissue and suppressed the expression of its downstream target genes VEGF and PDGF-β, which was also confirmed by RT-qPCR. Additionally, bioinformatic analysis suggested that tumor-derived exosomal miR-20a-5p could target HIF-1α. Isolation and RT-qPCR analysis of lung tissue-derived exosomes demonstrated that the combination therapy significantly downregulated the expression of exosomal miR-20a-5p. Conclusion: This study indicates that the combination of Sini Decoction and cyclophosphamide can effectively inhibit lung cancer growth and alleviate hepatorenal toxicity. The mechanism may be associated with the amelioration of the tumor hypoxic microenvironment, inhibition of the HIF-1α-mediated tumor hypoxia signaling pathway, and regulation of exosomal miR-20a-5p expression.