| BACKGROUNDAutophagy is a process of cell death discovered in recent years,namely type Ⅱprogrammed cell death,and involved in a series of biochemical processes in cell characterized by" autophagic bodies in cytoplasm with the bilayer structure package of long-lived proteins and organelles of self-digestion".Autophagy is commonly regarded as a double-edged sword.When the cell is in some specific conditions such as starvation,hypoxia and other nutritional stress,autophagy is a protective factor for the cell through the moderate autophagic degradation to meet its basic survival material and energy demand.But in the stimulation with some drugs,excessive autophagy may probably lead to cell autophagy death.Platycarya Strobilacea Sieb.et Zucc belongs to Platycarya and Juglandaceae.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 acute or chronic rhinitis in Chinese people.Moreover,used clinically to treat Nasopharyngeal Carcinoma.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.Moreover,NPC is a highly malignant tumor with typical cervical lymph node metastases frequently in its early stage.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.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 recent years,great progresses have been made in exploring the role and mechanism of autophagy antitumor drugs,especially in some Chinese traditional medicine and their effective components such as curcumin,matrine and triptolide,etc,so autophagy becomes a new hot spot in cancer research.In thegiven references,Platycarya strobilacea exact 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.NMETHODExtraction of effective components from infructescence of Platycarya strobilacea sieb.et Zucc by alcohol refluxing method;Methyl Thiazolyl Tetrazolium(MTT)assay in vitro were used to measure the inhibition rate of drugs on growth of NPC cells;Cell morphology were observed by optical microscope and photographed;Cell cycle was detected by flow cytometry;Apoptosis was detected by apoptosis detection kit;MDC fluorescence staining was observed by fluorescence microscope to observe the formation of autophagic bodies;transmission electron microscopy;The protein expression of LC3,Akt and PTEN was detected through western blot;Transplantation tumor model of human naspharyngeal carcinoma was established on nude mice.After stomach perfusion,HE stained transplantation tumor tissues were observed under optical microscope;microtubule-associated protein light chain 3(MAPLC3)expression was tested by immumohistochemistry.RT-PCR was applied to detect the expression of mTOR,Akt,PTEN and Beclinl mRNA.RESULTSPart I Extraction of the effective components from Platycarya strobilacea1.1 Extraction rate of Platycarya strobilacea sieb.et Zucc extractsTotally 180g of extract was obtained from 4.5kg of Platycarya strobilacea,so the extract rate was 4.0%.1.2 Effective components of Platycarya strobilacea extractsPlatycarya 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,may contain terpenoids and volatile oil.1.3 Extraction rate of Platycarya strobilacea sieb.et Zucc essential oil The essential oil rate was 0.12%.1.4 Effective components of Platycarya strobilacea essential oilThe main compounds of Platycarya strobilacea volatile oil were sesquiterpenes,such as C-eudesmol(18.92%)and B-eudesmol(18.74%).There are also more long chain hydrocarbons,fatty acids and fatty amides,tetrapentacontane(8.64%),Palmitic acid(7.87%),palmitamide(5.04%),octadecanamide(4.84%),aromadendrene(4.23%)and dotriacontane(3.99%).Part 2 Autophagy induced by Platycarya strobilacea sieb.et Zucc extract on human nasopharyngeal carcinoma cells2.1 Effect of Platycarya strobilacea sieb.et Zucc extract on the proliferation of CNE1 and CNE2 cellsResults:Extract of Platycarya strobilacea sieb.et Zucc significantly inhibited the growth and proliferation of CNE1 and CNE2 cells in time and dose dependent.When the extract’s concentration was 25mg/ml,the proliferation was most obviousiy inhibited with the IC50 being 8.78g/ml and 15.56g/ml at 48h in CNE1 and CNE2 cells repectively.2.2 Cell morphology observationAccording to the results of MTT,we treated the human nasopharyngeal carcinoma cell line CNE1 and CNE2 with 25mg/ml extract repectively.After Oh,4h,8h,12h,24h and 48h,cell morphology was observed under the light microscope.With the drug action time prolonged,cells swelled after shrinking,became round,refraction less visible,cytoplasmic vacuolization,cell gap widened,cell lysis and cell colony reduction phenomenon,verifying cytotoxic effect of the infructescence of Platycarya strobilacea sieb.et Zucc extract on CNE1 and CNE2 cells.2.3 Effect of Platycarya strobilacea sieb.et Zucc extract on cell cycleThe nasopharyngeal carcinoma cell lines were treated with Platycarya strobilacea sieb.et Zucc extracts at various concentrations.After 24h and 48h,we found G0/G1 phase cells decreased and S pgase cells increased;However,typical apoptosis peaks before G1 phase did not appear,indicating that apoptosis was not obvious in this case.2.4 Apoptosis rate was evaluated by flow cytometryCNE1 and CNE2 were cultured in vitro and treated with various concentrations of Platycarya strobilacea sieb.et Zucc extracts.Apoptosis was analyzed by flow cytometry.Results showed that,with the increase of drug concentration and action time,apoptosis rate did not increase significantly,which was verified by the results of cell cycle analysis;the infructescence of Platycarya strobilacea sieb.et Zucc extracts induced cells death not by apoptosis.2.5 Autophagy was induced by extract of Platycarya Strobilacea Sieb.et Zucc in Nasopharyngeal Carcinoma cellsMDC staining and immunofluorescence microscopywere performed to observe the alterations of autophagy.The expression of LC3 in the level of protein was detected through western blot.The results showed that Platycarya Strobilacea Sieb.et Zucc extracts could induce autophagy in vitro in nasopharyngeal carcinoma cell lines.Part 3 In vivo induction of autophagy by Platycarya strobilacea sieb.et Zucc extract in nasopharyngeal carcinoma3.1 The general growth status of nasopharyngeal carcinoma CNE2 in nude miceAt the end of the experiment,different groups of nude mice bearing tumors grew in good condition,and did not die.The weight and tumor volume of each group were increased,but the growth rates were not identical.Throughout the experiment,we did not observe significant side effects,such as diarrhea,retardation,anorexia.Transplanted tumors were spherical and irregular in shape,with clear and tenacious boundary.3.2 Effect of Platycarya strobilacea sieb.et Zucc extract on mouse weightDuring the experiment,the mouse weight was increased and not significant different in both control group and experimental group(P>0.05).3.3 Depressive effect of Platycarya strobilacea sieb.et Zucc extract on the growth of transplanted tumor in nude miceAt the end of experiment,the average tumor volumes were 2055.19± 876.93 mm3,1504.68±310.76 mm3,1165.62±72.46 mm3 and 1338.49±275.10 mm3 in the control,high,moderate and low drug concentration group respectively.Likewise,the tumor weights were 1.88±0.24 g,1.22±0.18 g,0.76 ± 0.26 g,1.13 ± 0.29 g in the control,high,moderate and low drug concentration group respectively.The significant difference of tumor weight and volume was found between the control,group and moderate drug concentration group(P<0.05).Futhemore,inhibition ratio was 35.11%in the high drug concentration group,59.57%in the moderate concentration drug group and 39.89%in the low concentration drug group.This suggesting Platycarya strobilacea sieb.et Zucc extract could inhibit NPC cell growth in vivo,and the effiency of the moderate drug concentration is the most obvious.3.4 HE staining examined the morphology of transplated tumors in miceMorphological characteristics of tumor in both groups were observed using HE staining under microscope.In the control group,tumor cells showed typically malignant features;the nucleus was enlarged,engrained and irregular.The nuclear fission was clear.In drug treatment group,tumor cells showed similar morphological features,but necrosis became more obvious.3.5The expression of LC3 protein was detected by using immunohistochemical method.Immunohistochemical staining displayed that LC3 positive cells showed dense-point staining in the cell and cytoplasm,which is a sign of cell autophagy.Results showed that the expression of LC3 were 16.67%,83.33%,66.67%,33.33%in the control,high,moderate and low drug concentration group respectively.The expression of LC3 has significant difference(X2=4.889,P=0.027).Part 4 Preliminary study on the mechanism of autophagy induced by Platycarya strobilacea sieb.et Zucc extracts.Autophagy-related genes induced by Platycarya strobilacea sieb.et Zucc extracts were detected by real-time PCR and Western Blotting.Compared to the control group,Platycarya strobilacea sieb.et Zucc extracts could reduce the expression of autophagy-related genes such as mTOR,Aktl,and increase the expression of BECN1 and PTEN,suggesting Akt/mTOR signal transduction pathway was blotked in this case.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 autophagy of nasopharyngeal carcinoma cells in vitro,and thereby inhibit the growth of tumor cells.The extent of autophagy 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(200g/kg group)has the strongest inhibition effect.(4)The mechanism by which autophagy was induced by Platycarya strobilacea sieb.et Zucc extracts in nasopharyngeal carcinoma may be related to blocking of Akt/mTOR signal transduction pathway. |
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