Proteome Analysis of Camellia nitidissima Chi Revealed Its Role in Colon Cancer Through the Apoptosis and Ferroptosis Pathway

Colon cancer is the third most common cancer in the world with a high mortality rate. At present, surgery combined with radiotherapy and chemotherapy is the primary treatment, but patient prognosis remains poor. Traditional Chinese medicine (TCM) has become a complementary and alternative source of anti-cancer drugs. Camellia nitidissima Chi (CNC) is a TCM used to treat a variety of cancers. However, the role of CNC in cancer remains elusive, and its effect and mechanism on colon cancer have not been reported. Here, we show that CNC exerts an excellent inhibitory effect on colon cancer proliferation and apoptosis induction in vitro and in vivo. We performed label free-based quantitative proteomic analysis to evaluate the HCT116 cells treated with CNC. Our data revealed a total of 363 differentially expressed proteins, of which 157 were up-regulated and 206 down-regulated. Gene Ontology enrichment analysis showed that these proteins were involved in tumor occurrence and development through multiple biological processes such as cell proliferation, cell apoptosis, cell cycle, and cell death. Interestingly, we also found significant changes in ferroptosis pathways. The role of essential proteins glutathione peroxidase 4 (GPX4) and heme oxygenase-1 (HMOX1) were verified. CNC decreased the expression of GPX4 and increased the expression of HMOX1 at the mRNA and protein levels in vivo and in vitro. Collectively, these findings reveal that CNC regulates colon cancer progression via the ferroptosis pathway and could be an attractive treatment for colon cancer.

Identifying Active Compounds and Mechanism of Camellia nitidissima Chi on Anti-Colon Cancer by Network Pharmacology and Experimental Validation

Camellia nitidissima Chi (CNC) is a traditional Chinese medicine (TCM) with anticancer property. However, its underlying mechanisms of anti-colon cancer (CC) remain unknown. Therefore, a systematic approach is proposed in the present study to elucidate the anticancer mechanisms of CNC based on network pharmacology and experimental validation. Initially, the potential active ingredients of CNC were verified via the TCMSP database based on the oral bioavailability (OB) and drug-likeness (DL) terms. Hub targets of CNC were acquired from SwissTarget prediction and TCMSP databases, and target genes related to CC were gathered from GeneCards and OMIM databases. Cytoscape was used to establish the compound-target networks. Next, the hub target genes collected from the CNC and CC were parsed via GO and KEGG analysis. Results of GO and KEGG analysis reveal that quercetin and luteolin in CNC, VEGFA and AKT1 targets, and PI3K-Akt pathway were associated with the suppression of CC. Besides, the result of molecular docking unveils that VEGFA demonstrates the most powerful binding affinity among the binding outcomes. This finding was successfully validated using in vitro HCT116 cell model experiment. In conclusion, this study proved the usefulness of integrating network pharmacology with in vitro experiments in the elucidation of underlying molecular mechanisms of TCM.

Flavonoid 3'-hydroxylase of Camellia Nitidissima Chi. Can Promote the Synthesis of Polyphenols Better Than Flavonoids

Camellia nitidissima Chi is an ornamental plant of the genus Camellia L. Its flowers contain a lot of flavonoids and polyphenols. Flavonoid 3'-hydroxylase (F3'H) plays an important role in the synthesis of flavonoids, polyphenols and anthocyanins. We cloned CnF3'H from the petal of C. nitidissima (GenBank code: HQ290518.1). The full length of CnF3'H was 1859 bp, with an open reading frame of 1577 bp, and encoded 518 amino acid. A phylogenetic tree analysis showed that CnF3'H was closely related to Camellia sinensis L. and C. sinensis cultivar Zhonghuang. CnF3'H was expressed in flowers, leaves, fruits, sepals, petals and stamens of C. nitidissima, and during the flowering process the expression level in flower decreased initially and then increased. CnF3'H expression was significantly positive correlated with polyphenol contents in C. nitidissima. A CnF3'H-EGFP expression vector was constructed to do the subcellular localization, we found that CnF3'H was obviously localized in the nuclear envelope and cytomembrane. In transgenic tobacco flowers, the total polyphenol content and various polyphenol constituents were significantly increased with high CnF3'H expression level, while total flavonoid contents and some flavonol constituents were increased slightly. These findings suggest that CnF3'H can promote the synthesis of polyphenols better than flavonoids.

Exploring the adaptive mechanism of Camellia limonia in karst high calcium environment via an integrative analysis of metabolomics and metagenomics

Background: Yellow Camellia is a kind of rare plant with high economic and medicinal value. It is known as the "giant panda" of the botanical world. Camellia group of plants (Camellia Sect. Chrysantha Chang) is the only golden flower of Theaceae. Compared with Camellia nitidissima, Camellia limonia grows in karst areas, where the soil has the characteristic of high calcium content. However, there are few studies about the Camellia limonia in karst soil environment and the adaptation mechanism is no clear. Results: In this study, we found that under high calcium treatment, the chlorophyll content and leaf areas of Camellia limonia increased, while those of Camellia nitidissima decreased. The photosynthetic efficiency of Camellia limonia was more stable and higher than Camellia nitidissima. Compared with Camellia nitidissima, the conductance was larger and the degree of leaf shrinkage was smaller in Camellia limonia. The metabolomics analysis showed that the kaempferol-3-o-rutinoside, tyrosol, 6-o-methyldeacetylisoipecoside and (r)-mandelic acid are the main differently metabolic compounds . The results of karst high calcium soil metagenomics showed that microbacterium-testaceum, intrasporangium-calvum and rubrivivax-gelatinosus significantly changed. Through metabonomics and metagenomics integrative analysis, flavone and flavonol biosynthesis is suggested to be the main regulation pathway, which is regulated by apigenin, kaempferol, astragalin, isoquercitrin metabolites and TT7, UGT78D1, UGT78D2 genes. This metabolic pathway involves the synthesis of flavonoids. Flavonoids have the functions in drought and salt resistance, which play an important role in the adaptation of Camellia limonia in karst high calcium environment.Conclusion: This omics study identified key regulation metabolites and genes for Camellia and provided important basis for the adaptive mechanism of plants to adapt to the high-calcium environment and the protection of Camellia species.

Functional Diversification of the Dihydroflavonol 4-Reductase from Camellia nitidissima Chi. in the Control of Polyphenol Biosynthesis

Plant secondary metabolism is complex in its diverse chemical composition and dynamic regulation of biosynthesis. How the functional diversification of enzymes contributes to the diversity is largely unknown. In the flavonoids pathway, dihydroflavonol 4-reductase (DFR) is a key enzyme mediating dihydroflavanol into anthocyanins biosynthesis. Here, the DFR homolog was identified from Camellia nitidissima Chi. (CnDFR) which is a unique species of the genus Camellia with golden yellow petals. Sequence analysis showed that CnDFR possessed not only conserved catalytic domains, but also some amino acids peculiar to Camellia species. Gene expression analysis revealed that CnDFR was expressed in all tissues and the expression of CnDFR was positively correlated with polyphenols but negatively with yellow coloration. The subcellular localization of CnDFR by the tobacco infiltration assay showed a likely dual localization in the nucleus and cell membrane. Furthermore, overexpression transgenic lines were generated in tobacco to understand the molecular function of CnDFR. The analyses of metabolites suggested that ectopic expression of CnDFR enhanced the biosynthesis of polyphenols, while no accumulation of anthocyanins was detected. These results indicate a functional diversification of the reductase activities in Camellia plants and provide molecular insights into the regulation of floral color.