The Mechanism Of Alcohol Extract Of Platycarya Strobilacea Sieb. Et Zucc Induced Methuosis In Nasopharyngeal Carcinoma Cells

Background:Nasopharyngeal carcinoma(NPC)is the most common malignant cancer originating in the epithelial cells of nasopharynx.It has a high rate of morbidity in South China.Radiotherapy and chemotherapy are definitely dominant clinical treatments for NPC.However,both therapies have a multitude of side effects resulting in a poor 5-year survival rate of NPC patients.Recently researches demonstrated that a variety of Chinese traditional herbal medicines are effective anti-tumor drugs.Additionally,clinical studies showed the incorporation of Chinese traditional herbal,radiation therapy and chemotherapy could improve cancer patients' quality of life.Chinese traditional herbal medicines have been demonstrated as an adjuvant therapy for malignant cancer.Platycarya Strobilacea Sieb.et Zucc is widely distributed in provinces of South China.Studies shown that Platycarya Strobilacea Sieb.et Zucc infructescence could inhibit EB virus antigen expression and have cytotoxicity on NPC cells.Methuosis is a type of cell death which mechanism is still unclear.Most studies considered it is formed by the unnormal progress of micropinocytosis.In pathological circumstances,macropinosomes will accumulated in plasm which results in a lower rate of cellular metabolic activity and rupture of cell membrane and cells eventually die.An array of studies have been shown that methuosis is a potential effectively therapy for refractory malignancies.Our group demonstrated that alcohol extract of Platycarya strobilacea sieb.et Zucc(PSZ)can inhibit the proliferate of CNE1/CNE2 and induces methuosis in CNE1/CNE2.However,the mechanism of methuosis is still unclearly.The goals of this study is to find out the relative signal pathways of methuosis and key targets.Objective:To demonstrate potent anti-tumor activity of PSZ in a wide-spectrum of NPC and investigate relative signal pathways and key targets of methuosis.Method:Part 1 Identifying the effective components of PSZUsing ethanol refluxing method to purify the PSZ.Condensing PSZ by rotary evaporator.Analyzing the main monomer components through High Performance Liquid Chromatography(HPLC).Part 2 Investigating the effect of PSZ on different NPC cellsSUNE1,CNE1,CNE2 and 5-8F cells were seeded in 6-well plates(1.2×105cells/ml,2ml/hole)and treated with PSZ.Observing the changes of morphology in NPC cells at 4h,8h,12h,24h and 48h.Part 3 The mechanism of PSZ induced methuosis3.1 Bioinformatics AnalysisAccording to the genome sequencing results which discovered by our group,we used KEGG database to screen signal pathways related to methuosis death.3.2 Detecting the expression of related genes by Quantitative real-time PCR(RT-qPCR)Extracting cells' RNA and using reverse transcription to amplify each group' s DNA.Comparing the differences in gene expression H-Ras,Arf6,Rac1,MAPK1,MAPK3 and FOS by RT-qPCR.The blank group was control group and GAPDH was internal control.Calculating ?Ct values.3.3 Detecting H-Ras and Racl protein expression differences through Western Blotting methodCNE1 and CNE2 cells were treated with PSZ(1.0mg/ml),then cells were lysised and proteins were quantified after 24h treatment.3.4 EHT 1864Seeding CNE1 and CNE2 cells in 6-well plates(1.2×105cells/ml,2ml/hole).Then treated each group of cells with PSZ and/or EHT 1864.Observing the changes of cells' morphology in each group at 4h,8h,12h,24h and 48h.Statistical analysis:Data were analyzed by IBM SPSS Statistics 20.0 and were expressed as mean±SD(standard deviation).A significance level of p<0.005 was used.Figures are completed with GraphPad Prism 6.0.Results:1.205g of alcohol extract was collected from 5kg of Platycarya strobilacea.The extract rate was 4.1%.The main compounds of PSZ alcohol extract were phenolic acids and flavonoids.2.SUNE1,CNE1,CNE2 and 5-8F cells were incubated for 24h with PSZ(1.0mg/ml).SUNE1,CNE1 and CNE2 cells had significant morphology changes such as cells merging,accumulate of large cytoplasmic vacuoles,membrane rupture and the nucleus had no dramatically changes.5-8F cells have no changes at all.3.According to the KEGG database,we speculated that the H-Ras/Arf6/Racl signal pathway may be associated with the occurrence of methuosis death.4.RT-qPCR results demanstrated that H-Ras,Racl and MAPK1 gene expression levels rise,Arf6,MAPK3 and FOS gene expression levels decreased.5.Western Blotting results revealed that H-Ras and Racl proteins are overexpressed in PSZ extract-treated cells.6.Methuosis vanished when added EHT 1864.Conclusion:1.Aalcohol refluxing method is a stable and potential technology for collecting effective components from Platycarya strobilacea.2.PSZ(1.0mg/ml)can induce methuosis in CNE1,CNE2 and SUNE1 while failed in 5-8F.3.The mechanism of methuosis which induced by PSZ extracts in NPC may be related to the stimulation of RAS/Arf6/Rac1 and MAPK signal pathway.4.Racl is one of the important targets in methuosis.