The Bioactive Agent From Avenanthramides And Molecular Mechanism Of Anti-colorectal Cancer Effects

Cancer is one of the major public health challenges globally that seriously threaten human life and health.In light of the global cancer report from World Health Organization(WHO),the number of cancer patients worldwide are expected to amount to 24 million by 2035.Colorectal cancer(CRC)is a common malignant gastrointestinal tumor with a mortality rate of 11.6 % in 2018,which ranks as the second leading cause of cancer-related death.In recent years,China has been a high-incidence area of colorectal cancer as the lifestyle changes.Although the present chemotherapies for colorectal cancer improve the patients’ overall survival,their effectiveness remains not ideal because of the toxicity to normal tissues and drug resistance phenotype of cancer cells,which obviously limits the success in war on cancer.Therefore,screening novel bioactive compounds from natural sources with anticancer property and low toxicity and bid a tremendous opportunity for developing as safe and effective chemopreventive and anticancer drugs.Oat(Avena sativa L.)is a wholegrain cereal known as an important edible crop,which contains several bioactive ingredients including β-glucan,oats saponins,flavonoids,vitamins and avenanthramides(AVNs).Epidemiological studies have revealed that oats consumption in daily life can improve the digestive health and reduce the risk of CRC.At present,among the most widely used plant-derived antitumor agents in clinical applications,several alkaloids,such as camptothecin and vincristine have become generally accepted as the first choice for tumor treatment with the advantages of high therapeutic effect and low toxicity.AVNs belong to the phenolic alkaloids found exclusively in oats and possess anti-inflammatory,antioxidant,anti-atherogenic and anti-carcinogenic properties.However,the specific compounds responsible for anti-carcinogenic characteristics,the direct intracellular potential targets and the underlying molecular mechanisms remain unclear.In this study,renewable oats bran was utilized as raw material to extract AVNs by ethanol abstraction method.The antitumoral bioactivity of AVNs was evaluated both in vitro and in vivo for the first time.In addition,AVN A was identified as the main bioactive compound in AVNs extracts responsible for the antitumor effect via GE Healthcare Biacore T200 biomolecular interaction analysis system.Furthermore,the therapeutic potential of AVN A against CRC was assessed by the combination of commercial cell lines,nude mice transplanted tumor model and the murine AOM/DSS colitis-associated carcinoma model.In addition,the underlying mechanisms of AVN A-mediated antitumor effects were systematic illuminated from the perspective of cellular apoptosis,cellular senescence and chemosensitization.The main research contents and results are listed as follows:(1)The preparation and biological evaluation of anti-carcinogenic properties of AVNs in vitro and in vivo.Avenanthramides were extracted using ethanol abstraction and subsequently separated and purified by AB-8 macroporous resin.The results of HPLC/MS analysis identified that the phytochemical compounds in oats bran extracts were AVNs.MTT assay,clonogenic assay,apoptosis analysis and Ed U assay were applied to determine the effects of AVNs on CRC cells proliferation.The results indicated that AVNs significantly attenuated cell growth and viability in CRC cells.Furthermore,our study demonstrated that AVNs exert more significant effects on tumor cells under stress milieus such as glucose starvation,hypoxia and acidic extracellular p H.The xenograft tumor model was constructed using BALB/c nude mice to assess the antitumor effects of AVNs in vivo.The results suggested that AVNs administration dramatically suppressed the tumor growth.(2)The underlying molecular mechanism for AVNs induces CRC cells apoptosis.Morphological observation by using immunofluorescent analysis and transmission electron microscopy(TEM)demonstrated that AVNs treatment can trigger pharmacological changes such as mitochondrial swelling and increased fragmentation type.AVN treatment significantly reduced the ATP production by retarding the cellular oxygen consumption rate(OCR)of mitochondrial respiration.Furthermore,AVNs administration impaired ETC complex,which further resulted in the oxidative stress and excessive ROS generation to surpass a fatal threshold,with ensuing permeability transition pore(PTP)activation,mitochondrial membrane potential(MMP)loss,cytochrome c release,culminating in apoptosis of CRC cells.Further study found that AVNs exhibited antitumor activities by targeting DDX3,an ATPase-dependent RNA helicase,which is implicated in mitochondrial translation.AVNs induced-DDX3 degradation via ubiquitylation-dependent proteolysis led to impaired translation of ETC complexes components and ROS-mediated bioenergetics catastrophe and apoptosis.(3)We cloned the full length DDX3 to identify bioactive ingredients in AVNs extracts that target DDX3 to exert antitumor properties.Through the combination of surface plasmon resonance(SPR),1H NMR,HPLC/MS and molecular docking,we identified AVN A as a novel and effective DDX3 ligand.Autodock 6 software suite was used to construct a model of AVN A-DDX3 complex,and the analysis demonstrated that AVN A formed hydrogen bonds with Lys255,Arg287,Ser290 and Arg294 residues in ATP-binding cleft of DDX3.We then constructed the site-directed mutations of DDX3 based on 4 potential binding sites,and uncovered that AVN A bound to the Arg287 and Arg294 residues to induce DDX3 degradation.Subsequently,we evaluated the suppressive effects of AVN A on an azoxymethane/dextran sodium sulfate(AOM/DSS)induced colorectal carcinogenesis mouse model.The results demonstrated that AVN A treatment significantly suppressed the tumor incidence and growth,supporting the chemopreventive effects of AVN A on colon carcinogenesis and development.In addition,1H NMR spectra of mice serum samples demonstrated that AVN A exerted remarkable antitumor properties was closely related to the mitochondria metabolism dysfunction.(4)Immunochemistry staining was carried out to check the expression of Ki67 and γ-H2 AX in colon tissues from AOM/DSS mice.Our data have revealed that AVN A treatment decreased the expression of Ki67 and increased the level of γ-H2 AX.Noteworthily,H&E staining of the organs including the heart,liver,spleen,and kidney in mice receiving oral administration of AVN A did not show any detectable morphological changes.Further investigations disclosed that AVN A treatment triggered the cellular senescence in human colon cancer cells as shown by enlarging cellular size,upregulated β-galactosidase activity,γ-H2 AX positive staining,and G1 phase arrest.Interestingly,AVN A did not cause senescence of normal colonic epithelial cells(FHC).Moreover,AVN A treatment significantly increased the expression of mi R-129-3p,which markedly repressed the E3 ubiquitin ligase Pirh2 and two other targets,IGF2BP3 and CDK6.The Pirh2 silencing by mi R-129-3p led to a significant increase in protein levels of p53 and its downstream target p21,which subsequently induced cell senescence.(5)The mechanism study for the combination of AVN A with 5-Fluouracil(5-FU)chemosensitize CRC cells to chemotherapy.The results indicated that AVN A combined with 5-FU treatment dramatically induced pro-apoptotic protein Bim expression and sensitized CRC cells to cytotoxicity of 5-FU via attenuating oncogenic mi R-17-92 cluster expression.Using bioinformatic approach and functional analysis further confirmed that histone lysine-specific demethylase 4C(KDM4C)is a direct transcription regulatory factor of mi R-17-92 cluster host gene MIR17 HG.Ch IP assays indicated that both AVN A treatment and KDM4 C silencing resulted in an increase of H3K9me3 levels on the promoter region of MIR17 HG.The result showed that AVN A caused transcriptional inhibition of mi R-17-92 cluster was reversed by KDM4 C overexpression,manifesting the role of AVN A on MIR17 HG transcriptional repression.Collectively,AVN A enhanced therapeutic efficacy of 5-FU on colon cancer through inhibition of KDM4C/MIR17HG/Bim signaling axis.In addition,a xenograft model was employed to evaluate the potential effect of AVN A and 5-FU combination in vivo.The volume and weight of the tumor tissues in the combined treatment group were significantly smaller as compared to the treatment with either AVN A or 5-FU alone.The results showed that AVN A treatment effectively mitigates KDM4 C and mi R-17-92 expression level of tumor tissues.Strikingly,5-FU-caused systemic adverse reactions such as weight loss,elevated activity of aspartate aminotransferase(AST)and alanine transaminase(ALT),intestinal injury and decreased granulocytes,were partially alleviated by AVN A treatment.In summary,our findings imply that AVNs display antitumor activities against CRC both in vitro and in vivo.Through the mechanism by which AVNs induced CRC cell apoptosis,DDX3 was recognized as the critical intracellular target.AVN A was identified as the bioactive ingredient in AVNs extracts-mediated the anti-cancer effects via direct molecular fishing strategy.Evidenced by oxidative stress and high expression of γ-H2 AX after AVN A treatment in CRC cells,which promoted us to speculate the likelihood of cellular senescence.Subsequent studies support this hypothesis that AVN A induces cell senescence via mi R-129/Pirh2/p53 signaling pathway.Considering the potential application of phytochemicals in clinical combined therapies,the effects of combination with AVN A and 5-FU were explored.AVN A remodels H3K9me3 occupancy on MIR17 HG promoter by suppressing the demethylase activity of KDM4 C,which prevents biogenesis of oncogenic mi R-17-92 cluster,leads to promotion of pro-apoptotic protein Bim expression and subsequently triggers CRC cells apoptosis.Additionally,it is also documented that p53 and p21 are targets of mi R-17-92,which indicates the network regulatory roles of AVN A in CRC treatment.These results suggest that dietary AVN A has advantages of good biosafety and high bioavailability and may be developed as a safe and adjuvant chemotherapeutic agent for CRC treatment.Taken together,the present study aims to provide scientific data and theoretical basis for further utilization and in-depth exploration of AVN A,and it provides an appealing alternative therapy based on nutrition intervention for CRC patients.