Alcohol Extract Of Platycarya Strobilacea Sieb.et Zucc Induces Nasopharyngeal Carcinoma Cell Methuosis By Regulating RAS/MAPK/FOS Pathway

BACKGROUNDPlatycarya Strobilacea Sieb.et Zucc belongs to Platycarya and Juglandaceae.It is widely distributed in provinces of Shanxi,Gansu,Henan and the Yangtze River to the south.Its infructescence can clear away the heat-evil,expel superfidal evils,activate blood circulation to dissipate blood stasis,and dispel wind-evil.It has been used as a useful drug for treating nasal sinusitis and nasopharyngeal carcinoma in Chinese people.Nasopharyngeal carcinoma(NPC)is a tumor arising from the epithelial cells that cover the surface and line the nasopharynx.It is one of the most common malignant tumors in Southern China,especially in Guangdong,Guangxi,Hunan,Fujian and Jiangxi province.NPC is seen primarily in middle-aged persons ranged from 30 to 50 years and few in young persons.Radiotherapy and chemotherapy are currently main treatments,whereas the long-term efficacy is still unsatisfactory and the 5-year-survival rate is only 50%?70%after radiotherapy and chemotherapy,radioresistance,chemoresistance and severe complications lower the effect of combining treatment for Nasopharyngeal Carcinoma.Therefore,the search for more effective anti-NPC drugs with less toxic side effects and the improvement of clinical treatment efficacy are urgently needed.Cell death is a major physiological or pathological phenomenon in the life activities.The classic forms of cell death were apoptosis,necrosis,autophagy,and so on.Recently,a novel type of cell death has been observed and termed methuosis,in which the excessive stimulation can induce the cytoplasmic uptake and accumulation of small bubbles that gradually merge into giant vacuoles,eventually leading to the decreased cellular metabolic activity,cell membrane rupture and cell death.According to the morphological classification proposed by the Nomenclature Committee on Cell Death(NCCD)(2009),it suggestivs as methuosis.Traditional Chinese medicine(TCM)is derived from Chinese people's life and treatment of diseases,proven to be effective.Its mechanism of anti-tumor mainly lies on its functions of inhibiting tumor cell proliferation,inducing tumor cell apoptosis,repressing tumor angiogenesis,and reversing tumor multidrug resistance,etc.In thegiven references,PSZ has cytotoxic,inhibition of harmful bacteria in the intestine,antioxidant,cancer prevention and anti-aging effects.In addition,its compound herb has been reported to treat acute and chronic rhinitis and sinusitis,however.The induction of autophagy by Platycarya strobilacea sieb.et Zucc in nasopharyngeal carcinoma cells has still been not reported.In the present project,we based on the folk herbal medicine,determined the anti-NPC effects of Platycarya strobilacea extract using in vitro and in vivo experiments,and for the first time preliminarily explored its underlying mechanism.OBJECTIVESTo study the antitumor activity of alcohol extract of Platycarya Strobilacea Sieb.et Zucc,a NPC inhibitor,and investigate the relative mechanisms.In addition,we further evaluate the antitumor activity of alcohol extract of Platycarya Strobilacea Sieb.et Zucc in nude mice model of cervical cancer xenogRAFts.METHODPart 1 Extraction of the effective components from Platycarya strobilaceaAccording to dissolve the nature of incense Platycarya Strobilacea Sieb.et Zucc drawing Northwestern University Dr.Gao Rong of incenset Platycarya Strobilacea Sieb.et Zucc active ingredient extraction,separation,purification and other research programs,the use of ethanol was extracted,dried rotary evaporator.HPLC and GC-MS analysis of the main monomer components.Part 2 Alcohol extract of Platycarya strobilacea sieb.et Zucc induces nasopharyngeal carcinoma cell Methuosis2.1 Cell morphology observationCNE1/CNE2 cells were seeded in 6-well plates(1.2? 105 cells/ml,2ml/hole),37 ? 5%CO2 incubator for PSZ treatment groups after 24h,we observe the changing in morphology of the cells in each group after training 4h,8h,12h,24h,48h.CNE1/CNE2 cells were seeded in 6-well plates(1.2 ? 10scells/ml,2ml/hole),37 ?,5%CO2 incubator for PSZ treatment groups after 24h,cells to be recovered by centrifugation of about 5 ×106 were washed twice with PBS,discard supernatant,add 1ml 2.5%glutaralde hyde suspension cells.They were transferred to 1.5ml Ep tube and stained.Hitachi H-7650 transmission electron microscope observated and photographed.2.2 Cell lysosomes and nucleus observation with fluorescence microscopy CNE1/CNE2 cells were seeded in 6-well plates(1.2?105cells/ml,2ml/hole),37? 5%CO2 incubator for PSZ(1.0mg/ml)and quercetin(50?g/ml)treatment groups after 24h.Adding 37 ? preheated probe lysosomal dye concentration of l?M,PI concentration of 5?g/ml,incubated in the dark 0.5h.Cells morphology were observed and photographed changes under an microscope in each group.2.3 Effect of Platycarya strobilacea sieb.et Zuce extract on the proliferation of CNE1 and CNE2 cellsCNE1/CNE2 cells were treated with PSZ(0.25mg/ml,0.50mg/ml,1.0mg/ml?1.5mg/ml,2.0mg/ml for 24 h.After treatment,MTT solution dissolved in the culture medium was added to each well and the plates were incubated for another 4 h.DMSO was then added and vibrated.The colored formazan product was determined photometrically at 490 nm in a multiwall plate reader.2.4 Flow cytometry was used to determine the extent of apoptosisCNE1 and CNE2 cells were incubated for 24 h with PSZ at 1.0mg/ml.Then harvested cells with trypsin(no EDTA)and washed cells twice with cold PBS after centrifugation,and then resuspended cells in 100 ?LBinding Buffer,according to the FITC Annexin V Apoptosis Detection Kit,added 5 ?L of FITC Annexin V and PI,gently vortexed the cells and incubate for 15 mill at RT(25 ?)in the dark.400?L of lx Binding Buffer was added to each tube.Stained cells were analyzed within 1 h and the ratio of apoptosis were analyzed by Flow cytometer.2.5 Flow cytometry was used to determine the cell-cycle distributionCNE1 and CNE2 cells were incubated for 24 h with PSZ at 1.0mg/ml,cells were collected and washed twice with PBS,then fixed in 75%ice-cold ethanol ovemight.Cells were harvested and washed once with PBS.Fixed cells were resuspended in 500 ?L lxPBS containing 50 ?g/mL propidiun iodide(PI),10 ?g/mL RNase-A,and then incubate for 30 min at 37 ? in the dark.The distribution of cell cycle was analyzed by flow cytometer within 1 h.Part 3 Alcohol extract of Platycarya strobilacea sieb.et Zucc induces nasopharyngeal carcinoma cell Methuosis by regulating RAS/MAPK/FOS pathway3.1 High-throughput RNA sequencing and transcriptome analysis of differential gene expressionCNE1 and CNE2 cells were incubated for 24 h with PSZ at 1.0mg/ml,cells were collected and washed twice with PBS,extracting total RNA,using Illumina HiSeq2500 were PE 100 dual terminal sequencing.Sequencing results GO,KEGG analysis to determine the target genes Methuosis and investigate signaling pathways.3.2 Quantitative real-time PCR(Real-time qPCR)to detect the expression of related genesRNA was extracted from each group of cells,reverse transcription were amplified DNA amplification,Real-time qPCR relative quantification method to compare the differences in gene expression RAS/DUSP5/MAPK1/MAPK3/SRF/FOS/FOSB.The blank group was as control,GAPDH as internal control,calculate Act values.3.3 Western Blot detection RAS/DUSP5/MAPK/SRF/FOS related protein expression differencesCNE1 and CNE2 cells were incubated for 24 h with PSZ(1.0mg/ml)and quercetin(50?g/ml),then cells were dissolved and proteins were quantified.The results of western blotting suggested that inactivate.After treatment,AKT?p-AKT?ERK,P-ERK,EGFR,RAS,RAF,FOS etc proteins were detected by western blotting.Part 4 Depressive effect of Platycarya strobilacea sieb.et Zucc extract on the growth of transplanted tumor in nude miceLogarithmic growth phase of human nasopharyngeal carcinoma cells CNE2,to cells(3 × 106/0.2ml)were inoculated in weighing 15.3?21.3g of male nude mice subcutaneously right.Mice were randomly divided into a control group,the experimental group of low concentration and high concentration in the experimental group(n = 10)with the experimental group alcohol extract PSZ gavage doses of 4.0g/kg/d,8.0g/kg/d,orally volume of 0.4ml/only twice a day;control group 0.4ml/only saline intragastrically 10-20 days,observed and recorded growth in nude mice in each group and a tumor cases.RESULTS1.Totally 180g of extract was obtained from 4.5kg of Platycarya strobilacea,so the extract rate was 4.0%.Extraction rate of Platycarya strobilacea sieb.et Zucc essential oil.The essential oil rate was 0.12%.The main compounds of Platycarya strobilacea volatile oil were sesquiterpenes.Platycarya strobilacea sieb.et Zucc alcohol extract were analyzed by high performance liquid chromatograph,the main compounds of Platycarya strobilacea extracts were phenolic acids and flavonoids.Compounds have been identified mainly gallic,quercetin,ellagic acid,3,3'-dimethyl-1.3 ellagic acid.2.As shown in MTT assay,PSZ could inhibit nasopharyngeal carcinoma tumor cells grown according to concentration with time,and it basically lmg/ml with IC50 values of cells.This suggests that PSZ had significant cytotoxicity to nasopharyngeal carcinoma cells.3.CNE1 and CNE2 cells were incubated for 24 h with PSZ(1.0mg/ml),then observed with prolonged duration of action of PSZ,eells merging,shows a large cytoplasmic vacuoles,vacuoles merging,growing,and finally membrane rupture,the nucleus does not change dramatically.Cells were treated with different drugs,PSZ(1.0mg/ml),quercetin(25?M),after treatment 24h,lysosomal staining fluorescent probe,the normal control group,the number of cells in lysosomes,PSZ group were significantly decreased lysosomal fluorescence;after PI staining nuclei of PSZ the nucleus group did not change significantly,quercetin group nuclear firagmentation,ehromatin condensation.4.The extent of apoptosis was measured by the flow cytometric.The results indicated that no significantly increase in the rate of apoptosis..Flow cytometry was performed to analy the cell-cycle distribution.The resultsuggested that,treatment of cells with PSZ resulted in concentration dependent accumulation of cells in the G2/M phase.PSZ could arrest tumor cells in G2/M phase of cell cycle,like the paclitaxel.5.As shown in Sequence analysis that the cells after PSZ intervention CNE1,RAS gene expression was reduced,and found that the gene expression DUSP5,MAPK1,MAPK3,SRF,FOS,FOSB such as a significant difference.MAPK signaling pathways and genes are on biological pathways and KEGG Pathway analysis results coincide.6.According to the results of the analysis of high-throughput RNA,sequencing screened DUSP5,MAPK1,MAPK3,SRF,FOS,FOSB etc.Differentially expressed genes were validated qPCR results showed that CNE1 cells,DUSP5,FOS,FOSB gene expression level rise,RAS,MAPK1,MAPK3,SRF gene expression decreased.7.The results show that by western Infructescences of PSZ extract-treated group BECLIN1,P-AKT,PTEN protein were no significant changes occurred but MAPK1/MAPK3 treatment significantly increased the amount of protein,which is pre-the sequencing results consistent.RAS,FOS protein expression decreased,quercetin-treated group compared with the control group,EGFR,RAF,RAS,FOS protein expression levels decreased.8.Throughout the experiment,the control group and the experimental group nude body weight were increased,the difference was not statistically significant(P>0.05).The average tumor 8.0g/kg concentrations of heavy drug group,tumor volume compared with the control group was significantly(P<0.05).Inhibition rate was 59.57%,the results show PSZ in vivo inhibit the growth of nasopharyngeal carcinoma cells and to 8.0g/kg inhibited tumor growth in the drug group the most obvious effect.CONCLUSION:1.The main compound of Platycarya strobilacea sieb.et Zucc was sesquiterpenes.The extraction rate was about 4.5%by using alcohol refluxing method.2.Platycarya strobilacea sieb.et Zucc extracts can induce Methuosis of nasopharyngeal carcinoma cells in vitro,and thereby inhibit the growth of tumor cells.The extent of Methuosis was positively related to drug action time and concentration.3.Platycarya strobilacea sieb.et Zucc alcohol extract has anti-tumor activity in vivo.The moderate drug concentration group(8.0g/kg group)has the strongest inhibition effect.4.The mechanism by which Methuosis was induced by Platycarya strobilacea sieb.et Zucc extracts in nasopharyngeal carcinoma may be related to blocking of RAS/MAPK/FOS signal transduction pathway.