Study On The Active Ingredients And Mechanism Of The Zhuang Drug Cycad In Inhibiting The Growth Of NSCLC

Objective:Lung cancer is the most common malignancies in clinical practice,it is one of the leading causes of cancer-related deaths worldwide and non-small cell lung cancer(NSCLC)was especially considered as the most common form,accounting for approximately 85%of all lung cancer cases.The 5-year survival rate of NSCLC remains as low as about 15%because of fast growth,early metastases and the resistance to chemotherapy and radiotherapy.NSCLC is a malignant disease with activation of multiple major signaling pathways,which results in cancer cell survival and chemoresistance.At present,the treatment of lung cancer mainly based on surgery,radiotherapy and chemotherapy,so,development of novel therapeutic agents is urgently needed to treat this lethal disease.Apoptosis and autophagy are two important physiological activities that control cell survival and cell death.The crosstalk between autophagy and apoptosis influences cell homeostasis,cargo clearance of dying cells and clinical therapeutics.In recent years,traditional Chinese medicine and its effective parts or components of anti-cancer research work have made a series of achievements,and become the hotspots of oncology research.Cycas reivolutla is an evergreen palm woody plant of Cycas revoluta Thunb.It has ornamental,medicinal and edible value.Its main components are double flavonoid compounds,amino acids and sugars,etc.,Ancient records of its sweet,flat,astringent,small poison,has the effect of clearing away heat,stopping bleeding and dispersing stasis.in this study,the pharmacognosy identification of Cycas revoluta was first carried out,then the project carried out an in vivo animal experiment on the total flavonoids of Cycas revoluta and discussed the preliminary mechanism of anti-lung cancer.In order to further clarify the specific effective components and mechanism of Cycas revoluta against lung cancer,the flavonoids in Cycas revoluta were isolated and identified.The sotetsuflavone were identified as the next experimental object by literature review and experimental screening.Further,in vitro experiments were conducted to investigate the molecular mechanism of sotetsuflavone against lung cancer growth and metastasis,and to identify its main pathways and targets from the perspective of apoptosis and autophagy.Methods:(1)The original plant morphology,character,microscopic and thin-layer chromatography method of Cycas revoluta were identified.(2)84 successful tumor-bearing mice were selected and then randomly divided into seven groups:model group,control group,high-dose group,medium-dose group,low-dose group,cis-platinum group,and combination group,12 mice in each group.Drug intervention for 14 days respectively,On the fifteenth day,pick the eyeball method for blood,use conventional methods to strip tumor tissues,lung,spleen and thymus and weight them,then calculate the inhibition rate,lung index,spleen index and thymus index.Using the enzyme-linked immunosorbent assay(ELISA)to detect the interleukin-2(IL-2)and interleukin-10(IL-10)leves in serum.The expressions of VEGF,bFGF,HIF-1? and NF-?B in tumor tissues were detected by immunohistochemistry and Western blot.The expression of VEGF,bFGF,HIF-1? and NF-?B mRNA in tumor tissues were detected by RT-PCR.(3)15 kg of Cycas revoluta leaves were taken,pulverized into coarse powder,and then immersed in 75 ethanol for 1 h,then refluxed(3×100 L×1.5 h),at last,we concentrated under reduced pressure at 45? and obtained 1.3 Kg of total extract thick paste.After the total extract was dissolved in an appropriate amount of water,it was extracted with dichloromethane and n-butanol respectively.Finally,we obtained 200 g of dichloromethane extract and 375 g of n-butanol extract.Then,we used silica gel column chromatography,Sephadex LH-20,HPLC and other separation methods to separate and purify the dichloromethane extract,and identified the structure by means of nuclear magnetic spectroscopy and literature data.The isolated flavonoid monomer compounds were screened for antitumor activity.(4)The sotetsuflavone and A549 cells were determined by literature review and experimental screening,the trypan blue staining method was used to detect the toxic effect of sotetsuflavone on normal lung epithelial cells BEAS-2B.then,with the concentration gradient of Sotetsuflavone in A549 cells by MTT assay on cell growth inhibition.Lactate dehydrogenase(LDH),5-ethynyl-20 treatment-deoxyuridine(EDU)and colony formation experiments were used to further investigate whether cycadine can inhibit the proliferation of A549 cells.Observed the effects of Sotetsuflavone on migration and invasion of A549 cells by cell scratch text and transwell assay.The periodic distribution of A549 cells,the ROS level and the change of mitochondrial membrane potential were detected by flow cytometry.The level of apoptosis was detected by double staining with Annexin V-FITC/PI and Hoechst 33258 staining.The formation of autophagosomes in cells was observed by transmission electron microscopy.The effect of sulphonic flavonoids on the formation of autophagosomes was detected by acridine orange staining.(5)The expression changes of VEGF,TNF-?,NF-?B,Vimentin,Snail,N-cadherin,PI3K,AKT,Beclinl,Atg5,Atg7 and P62 mRNA in A549 cells were detected through real-time quantitative PCR.VEGF,TNF-?,NF-?B,Snail,MMP-9,MMP-13,E-cadherin,HIF-1?,PI3K,AKT,Bax,p-PI3K,p-Akt,p-mTOR,p-Raptor,p-P70S6K,LC3 and p62 protein expression were detected by Western blot.The expression levels of Angiostatin,E-cadherin,HIF-1?,MMP-9,MMP-13 and LC-3 were analyzed by immunofluorescence after treatment with different concentration of Sotetsuflavone.(6)The progect further used the activator IGF-1 and the inhibitor LY294002 to determine whether cycadine inhibits the PI3K/Akt pathway,and uses molecular docking to verify the docking between cycadine and the target.Results:(1)The main distinguishing features of morphology,characters,microscopic and thin-layer chromatography were determined(2)Compared with the model group,the tumor weight of the mice from the high-dose group,medium-dose group,low-dose group,cis-platinum group,and combination group were decreased.the difference was highly significant(p<0.01).the spleen index and thymus index of the high-dose group,medium-dose group and low-dose group were significantly increased,and far higher than the cis-platinum group and combination group.The ELISA results showed that compared with the model group,The IL-2 levels of the high-dose group,medium-dose group,low-dose group,cis-platinum group and combination group were significantly increased,and the IL-10 levels were significantly higher than the other groups in addition to the medium-dose group.The results of immunohistochemistry,Western blot and real-time PCR showed that the results were basically the same,compared with model group,the expression of VEGF,bFGF,HIF-1?,NF-?B mRNA and the expression of VEGF,bFGF,HIF-1? and NF-?B were decreased,the difference was highly significant(n<0.05).(3)Eighteen compounds were isolated from the dichloromethane extra ction site of Cycas irevoluta,and ten of them were identified:4-hydroxybe nzaldehyde(1),vanillin(4-hydroxy-3-methoxybenzaldehyde,2),2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one(3),Amentoflavone(4),4-(4-(ben zo[d][1,3]dioxol-5-yl)tetrahydro-1H,3H-faro[3,4-c]furan-l-yl)-2-methoxyphenol(5),4-[(2R,3R,4S,5S)-5-(4-hydroxy-3-methoxyphenyl)-3,4-dimethyloxolan-2-y 1]-2-methoxyphenol(6),(4aS,10aR)-6-hydroxy-1,1,4a-trimethyl-7-propan-2-yl-3,4,10,10a-tetrahydrophenanthrene-2,9-dione(7),sultane flavone(8),6-hydro xy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one(9),naringenin(5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one,10)(4)It was found that 0-160?mol/L sotetsuflavone had no significant toxic effect on BEAS-2B cells,while 200?mol/L sotetsuflavone had significant cytotoxicity to BEAS-2B cells.Sotetsuflavone have significant antiproliferative activity against A549 cells,At the same time,the reactive oxygen species(ROS)content increased while the mitochondrial membrane potential and the ratio of Bcl-2/Bax decreased.Cleaved caspase-3,cleaved caspase-9,cytochr-ome C and Bax expression increased,and Cyclin D1,CDK4,cleaved caspase-8 and Bcl-2 expression decreased.Interestingly,we demonstrated that sotetsuflavone could effectively inhibit the G0/G1 cycle progression,and then induce the endogenous apoptosis pathway.Our results show that sotetsuflavone could inhibit the growth of A549 cells by up-regulating intracellular ROS levels and causing the mitochondrial membrane potential to collapse,inducing G0/G1 phase arrest and endogenous apoptosis.(5)Sotetsuflavone could inhibit the metastasis of A549 cells,moreover,the epithelial mesenchymal transition(EMT)of A549 cells was inhibited,which reflected in the upregulation of E-cadherin,and downregulation of N-cadherin,Vimentin and Snail.mechanistically,our study showed that HIF-1? played an important role in the anti-metastatic effect of Sotetsuflavone in non-small cell lung cancer A549 cells,which could not only mediated VEGF expression,but also downregulated VEGF and upregulated Angiostatin,simultaneously,affected the expression of MMPs and decreased MMP-9 and MMP-13 expression.More importantly,HIF-1? expression may be regulated by the inhibition of PI3K/AKT and TNF-?/NF-?B pathways.(6)Blockade of the PI3K/Akt/mTOR pathway in sotetsuflavone mediated non-small cell lung cancer cells A549 cells was confirmed by decreasing the phosphorylation levels of PI3K,Akt,mTOR and p70S6K.Inhibition of PI3K/Akt signal transduction pathway was reversed by treatment with 20ng/mL of IGF-1 On the contrary,the LC3-II conversion was significantly increased in the non-small cell lung cancer A549 cell group treated with sotetsuflavone combined with 20?M LY294002.(7)sotetsuflavone interacts with Glu342,Ser429 and Lys346 of PI3K p85a through hydrogen bond and ?-hydrogen bond interactions.sotetsuflavone interacts with Asn53,Thr82,Gln203,Gln79 and Trp80 of Aktl through hydrogen bond,?-hydrogen bond and ?-? bond interactions.sotetsuflavone interacts with Trp2023,Glu2014 and Met2010 of mTORc1 through hydrogen bond interactions.Conclusion:(1)These characteristics can provide a basis for the pharmacognosy identification,quality standard of Cycas revoluta.(2)The total flavonoids from Cycas revoluta may modulate the expression level of the interleukin-2(IL-2)and interleukin-10(IL-10),inhibit the growth and metastasis of the model mice with Lewis lung cancer and improve the immune function.The mechanism of total flavonoids from Cycas revoluta in the treatment of lung cancer may be through inhibition of the expression of VEGF,bFGF,HIF-1?,NF-?B mRNA,and further inhibit the expression of VEGF,bFGF,HIF-1? and NF-?B in invasion and metastasis-related proteins,thus play a role of anti-lung cancer invasion and metastasis.(3)Compounds 4,8 and 10 were identified as flavonoids.Compounds(1?3),(5-7)and(9)were isolated from this genus for the first time.Compounds(4)and(8)were double flavonoids.(4)Sotetsuflavone could inhibit the growth of A549 cells by up-regulating intracellular ROS levels and causing the mitochondrial membrane potential to collapse and induce GO/G1 phase arrest and endogenous apoptosis.(5)Sotetsuflavone can reverse EMT,thereby inhibiting the migration and invasion of A549 cells.This process may be associated with PI3K/Akt and TNF-?/NF-?pathway.(6)Sotetsuflavone induced autophagy was associated with the inactivation of the PI3K/Akt/mTOR pathway in non-small cell lung cancer cells.(7)The different binding models between sotetsuflavoine and proteins result in different binding ability.The computational result indicates that sotetsuflavone can interact with PI3K p85a,Aktl and mTORcI and the binding ability is PI3K p85a>AKTl>mTORc1.