Aqueous extract of Fritillariae cirrhosae induces cellular apoptosis through activation of STATs-mediated immunomodulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae cirrhosae (FC), referred to'Chuan beimu'in China. As an important edible and medicinal plant, the bulbs of F.cirrhosae is used traditionally in the treatment of pulmonary diseases associated with lung heat, inflammation and tumors. In the study, we investigated the effect of aqueous extract of FC (FC-AE) and elucidated its mechanism in non-small cell lung cancer A549 cells and a xenograft model of nude mice. MATERIALS AND METHODS: CCK-8 and plate colony formation assay were used to evaluate the effect of FC-AE in A549 cells in vitro, and the gene expression profile of FC-AE on A549 cells was assessed by RNA sequencing system. Then, the effects of FC-AE on cell cycle and apoptosis of A549 cells were analyzed by flow cytometry. In combination with RNA-seq data, RT-PCR and western blot were used to evaluate the expression of proteins related to apoptosis and immune regulation. A xenograft model of nude mice was used to assess the effect of FC-AE in vivo. RESULTS: CCK-8 and plate cloning assays showed that FC-AE inhibited the proliferation and colony formation of A549 cells. A549 cells treated with FC-AE can triggered apoptosis. GO and KEGG pathway enrichment analysis of RNA-seq data showed that most of the differentially expressed genes (DEGs) were related to immune response, apoptosis and cell cycle process. Several immune and apoptotic DEGs were identified by qRT-PCR which were consistented with RNA-seq data. In nude mice, FC-AE reduced the tumor size and promoted the secretion of cytokines IL12 and IFNγ. FC-AE up-regulated the two members (STAT1 and STAT4) of STATs and their target genes (IFNγ and IL-12, respectively) protein expressions, and actively regulates Bcl-2/Bax family proteins which resulted in cellular apoptosis in A549 cells. CONCLUSION: Our finding suggests that FC-AE mediates apoptosis through a STAT1 and STAT4-mediated co-regulatory network, which may be the key novel mechanism for its antitumor activity. The F. cirrhosa may be a promising antitumor drug for modulating immune responses to improve cancer therapy.

Transcriptome analysis reveals in vitro-cultured regeneration bulbs as a promising source for targeted Fritillaria cirrhosa steroidal alkaloid biosynthesis.

The bulbs of Fritillaria cirrhosa is wildly used in traditional Chinese medicine to treat lung-related disease, which has recently been found to have antitussive, anti-inflammatory, antihypertensive and anti-tumor activity. Steroidal alkaloids are the major effective ingredients of F. cirrhosa. In the current study, we demonstrated an efficient strategy for F. cirrhosa bulb regeneration in vitro by cytokinin/auxin induction. Our data showed that the regenerated bulbs accumulated higher alkaloid content that the wild ones. We further performed RNA-seq and bioinformatics analysis to study the gene expression profile, especially those related to alkaloids biosynthesis. KEGG pathway annotation identified genes related to "Metabolic pathways" were the most abundant (2644, 26.0%), followed by those for "Biosynthesis of secondary metabolites" (1319, 13.0%) among the 113,865 unigenes identified. Further analysis suggested MEP pathway, other than MVA pathway, might be the major route for steroidal alkaloid biosynthesis of F. cirrhosa, as all the key genes in this pathway were found to be unregulated in our study. We also showed that accumulation of different phytochemicals was linked to plant hormone addition. Our current study demonstrated that in vitro cultivation is a promising strategy for mass production of F. cirrhosa steroidal alkaloids for pharmacological industry.