The Preparation Of Foxtail Millet Bran-derived Bound Polyphenol And Molecular Mechanism Of Anti-colorectal Cancer Effects

With the improvement of living standards,people's diet structure has changed.Moreover,pesticide residues and irrational use of food additives have accelerated the incidence of digestive system tumors.Colorectal cancer?CRC?is the most common digestive system malignancy and the main treatment method easy to produce drug resistance and strong toxic and side effects.Many clinical anti-colon cancer drugs have been obtained from natural products,such as taxol,camptothecin derivatives?irinotecan?and Changchun alkaloids.From the above three kinds of drugs,it can be concluded that natural products are a huge treasury for drug development.Obtaining efficient and non-toxic anticolon cancer drugs from natural products is a hot field in the development of cancer drugs.China has a rich natural resources and a long medicinal history.The better utilization of the natural resources is the correct direction for the development of new drugs in China.Foxtail millet?Setaria italica?is annual gramineous plant,which originated from China,and is now planted all over the world.Millet bran is a by-product during the millet production.Recent studies indicate that millet bran contains an abundance of phytochemicals,particularly polyphenols.In nature,polyphenols generally occur in the free and bound forms,but most of them are bound.These studies showed that bound polyphenols possess a higher antioxidant capacity compared to free polyphenols.It has been reported that bound polyphenols exhibit a wide range of pharmacological and medicinalproperties,includinganti-inflammatory,anti-carcinogenic,anti-bacterial and immunomodulatory effects,which have been mostly attributed to their free radical scavenging,metal chelating and antioxidant activities.Therefore,bound polyphenols have attracted more attention as potential agents for precaution and therapy of oxidative stress-related diseases.In this study,we extracted a series of polyphenol compounds from foxtail millet bran and determined the content of each polyphenol.Screening of antitumor activity of polyphenols in the cell level and its components were identified.Furthermore,we affirmed that the effective ingredient of the anti-tumor activity polyphenol.The anti-cancer effects of foxtail millet bran-dirived polyphenol were studied in vitro and in vivo.The molecular mechanism of its-induced anti-colon cancer effects was investigated from several aspects:oxidative stress,anti-inflammation and remodeling tumor metabolism.The main research contents and results are as follows:?1?Extraction of polyphenols from foxtail millet bran and analysis of antioxidant capacity.Foxtail millet bran was mixed with acetone for extraction of free polyphenols.The residue obtained after extraction of acetone was hydrolysed with NaOH for extraction of bound polyphenols.Samples polyphenols content was measured by Folin-phenol reagent method.Bound polyphenol content of outside the shell was the highest followed by the bound polyphenols from inside the shell.MTT and cell cloning experiments determined the antitumor activity of polyphenol.The results showed that bound polyphenols from inside the shell can not only inhibit the proliferation of HCT-116 cells but also other tumor cell proliferation.In addition,the results found that the antioxidant capacity of BPIS in vitro was better than that of vitamin C.?2?The ingredient of BPIS was analyzed by UPLC-Triple-TOF.Results showed that it contained vanillic acid 4-O-?-D-glucopyranoside,glucosyringic acid,ferulic acid 4-O-?-D-glucopyranoside,4-Hydroxybenzoic acid,vanillic acid,syringic acid,p-coumaric acid,vitexin,ferulic acid,isoferulic acid,biferulic acid,4,4'-dihydroxy-3,5'-dimethoxy,3'-bicinnamic acid.The results of MTT assay showed that the antitumor effect of BPIS was the best.The anti tumor effect of BPIS is better than its component of molecular weight less than 200.?3?The molecular mechanism of BPIS induced apoptosis in colon cancer cells.The effects of ROS levels of BPIS on HCT-116 cells were detected using DCFH-DA fluorescent probe labeling method.BPIS could significantly increase the levels of ROS in the HCT-116 cells.The effects of BPIS on HCT-116 cell apoptosis were determined by Hochest staining.HCT-116 cells exhibited the apoptotic characteristics of chromatin condensation with typical fragmented nuclei after BPIS treatment.The expression of caspases family protein detection of mitochondrial membrane potential and western blotting flow cytometry.The results showed that BPIS significantly decreased the mitochondrial membrane potential of HCT-116 cells.The caspase-9,3 protein were sheared.Accompanied by up regulation of proapoptotic protein Bax expression and decline expression of antiapoptotic protein Bcl-2.We determined that BPIS induced mitochondria-mediated apoptosis.After adding ROS inhibitor?NAC?,the effect of BPIS induced apoptosis was significantly reversed,indicating that BPIS induced the apoptosis of HCT-116 cells by increasing the level of ROS.?4?The molecular mechanism of BPIS exerted anti-inflammatory effect.Lipopolysaccharide?LPS?was used to construct the inflammatory model of colon cancer HT-29 cells.ELISA assay showed that BPIS could significantly inhibit the secretion of proinflammatory cytokines IL-1?and IL-6,and enhance the secretion of anti-inflammatory factor IL-10.The results of western blotting showed that BPIS could inhibit the expression of NF-?B protein in HT-29 cells and block its nuclear transposition,and then inhibit the expression of total Akt protein and p-Akt^ser473er473 protein.IGF-1?Insulin-like Growth Factors?,a Akt protein activator,was co-culture with BPIS.Western blotting results showed that IGF-1 reversed the inhibitory effect of BPIS on the p-Akt^ser473er473 protein and NF-?B protein.It is suggested that p-Akt^ser473protein regulates the expression of NF-?B protein and its nuclear transposition.Akt was the target gene of miR-149 by the analysis of double luciferase reporter vector.The results of RT-PCR showed that BPIS could significantly increase the expression of miR-149 in HT-29 cells.The DCFH-DA fluorescence probe was used to detect the level of ROS in HT-29cells,and the results showed that BPIS could significantly increase the level of ROS in HT-29 cells.HT-29 cells were co-treated with NAC and BPIS.The results of RT-PCR showed that NAC could significantly reverse the up regulation effect of BPIS on miR-149.Therefore,we concluded that BPIS can enhance the expression of miR-149 by increasing the ROS level of in HT-29 cells which inhibit the expression of its target gene Akt.Akt inhibited the expression of NF-?B,and then inhibits the expression of a variety of inflammatory factors.Therefrom,BPIS plays an anti-inflammatory effect.?5?The molecular mechanism of BPIS reversed the energy metabolism of colon cancer.The results showed that BPIS significantly reduced glucose consumption and lactate production by inhibiting PKM2 protein in colon cancer HT-29 cells.The extracellular acidification rate?ECAR?was detected by the hippocampal XF24 analyzer,the results showed that BPIS could significantly reduce the glycolysis.On the other hand,BPIS can inhibit the GLS enzyme activity and inhibit the expression of SLC protein in RNA level and protein level.Therefore BPIS inhibited the glutamine metabolism.BPIS could reverse c-myc-mediated metabolism by western blotting.As previous studies,it has been shown that BPIS can significantly increase the expression of miR-149 in HT-29 cells.We speculated that miR-149 can negatively regulate the expression of c-myc protein.A series of bioinformatics software was used to predict c-myc as a target gene for miR-149.The dual luciferase reporter vector experiments confirmed that c-myc is the target gene of miR-149.This proved that miR-149 can negatively regulated c-myc.MTT and cell clone experiment proved that BPIS can significantly inhibit colon cancer HT-29 and DLD1 cell proliferation.Using the same method to detect miR-149 inhibitors on the proliferation of HT-29 and DLD1 cells,the results showed that miR-149 inhibitors can significantly reverse the proliferation effect of BPIS on colon cancer HT-29 and DLD1 cells,indicating that BPIS exerted anti-tumor effect by reversing miR-149 mediated colon cancer cell metabolism.?6?We performed xenograft studies in nude mice implanted with colon cancer cell.Colon cancer cell-bearing nude mice received intraperitoneal injections for seven injections of BPIS.The results showed that BPIS had the ability to suppress the xenografted tumor growth in nude mice,but the growth of nude mice was not affected.The relevant indicators?poptosis,inflammation and metabolism?of tumor tissues were detected by western blotting and immunohistochemical methods,including miR-149,p-Akt^ser473,NF-?B,PKM2 and c-myc etc.These results were consistent with the data from the in vitro experiments.In summary,BPIS is a bound polyphenol from inside the shell of foxtail millet bran.BPIS consists of 12 components.The component of molecular weight less than 200 in BPIS has anti-colon cancer activity,but the activity of anti-colon cancer is less than that of total BPIS.Therefore,we explored the antitumor molecular mechanism of BPIS in cell level and tumor model of nude mice.BPIS overproduction of ROS could activate mitochondria mediated apoptosis in colon cancer cells.Overproduction of ROS upregulated miR-149 that inhibited expression of downstream target gene Akt.Inhibiton of Akt reduced a variety of inflammatory cytokines production by inhibiting the expression of NF-?B.Hence,BPIS exhibited the effect of antiinflammation.Moreover,the increase expression of miR-149 reversed its target gene c-myc-mediated energy metabolism in colon cancer cells.Therefore,we imply that the molecular mechanism of BPIS exertes antitumor effect.Furthermore,BPIS has the potential to develop health products or antitumor drugs.These results will provide reliable theoretical and experimental basis for BPIS application.