Platycarya Strobilacea Sieb.et Zucc Alcohol Extracts(PSZ)Inhibited The Proliferation And Induced Methuosis In Human Nasopharyngeal Carcinoma Cells Via ARF6/ERK1/2/c-Fos/DUSP1 Pathway

Background:Nasopharyngeal carcinoma(NPC)arising from the epithelial cells that cover the surface and line the nasopharynx.80%of the world’s nasopharyngeal cancer occurs in 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,searching for more effective anti-NPC drugs with less toxic side effects are urgently needed.Platycarya Strobilacea Sieb.et Zucc alcohol extracts(PSZ)belongs to Platycarya and Juglandaceae.Its infructescence,which has been reported to have various pharmacological effects,such as anti-inflammation,anti-oxidant,and anti-angiogenesis properties.heat-evil,expel superfidal evils,activate blood circulation,and dispel wind-evil.It has been used as a useful drug for treating nasal sinusitis and nasopharyngeal carcinoma in Chinese people.The main active components of PSZ were extracted by ethanol extraction and identified.Our previous study found that PSZ inhibited the proliferation and induced accumulation of large phase-lucent cytoplasmic vacuolesin in NPC CNE1 and CNE2 cells,followed by a loss of metabolic capacity,plasma membrane integrity,and finally lead to cell death.The majority of degenerating cells do not exhibit cell shrinkage,chromatin condensation and nuclear fragmentation associated with apoptosis.According to the morphological classification proposed by the Nomenclature Committee on Cell Death(NCCD)(2009),We suppose PSZ induces a new cell death which previously described as Methuosis.Our previous studies showed that PSZ induced methuosis through the continuous activation of RAS and RAC1.Moreover,PSZ induced methuosis death can be reversed by using selective RAC1 inhibitor EHT1864.In this study,we further studied the anti-tumor effect of PSZ in vitro and in vivo.At the same time.we found that the expression of ARF6,p-ERK,c-Fos,and p-DUSP1 protein was reduced after the action of PSZ on CNE1 and CNE2 in nasopharyngeal carcinoma cells.In addition,we found that c-Fos and DUSP1 inhibitors can inhibit cell proliferation and induce a large number of vacuoles accumulate in cytoplasm,which produce a phenomenon similar to methuosis death.When ARF6 and c-Fos are expressed,it can also reverse the accumulation of vacuoles induced by PSZ,suggesting ARF6,c-Fos and DUSP1 play an important role in PSZ induced Methuosis.Our present study showed that PSZ might exert the tumour suppression function and induce methuosis by inactivated ARF6 and inhibited ERK1/2/c-Fos/DUSP1 pathway.Objectives:To clarify the anti-tumor activity of Platycarya Strobilacea Sieb.et Zucc alcohol extracts(PSZ)in vitro and in vivo experiments.and investigate the related signal pathways of Methuosis in nasopharyngeal carcinoma cells.In addition,clarify the target points,Expound its mechanism,promote the further research and clinical application of PSZ.Methods:1.PSZ inhibits the survival and proliferation in CNE1 and CNE2 cells and induced Methuosis,a non-apoptosis cell death1.1 Cell Culture and treatments1.2 MTT assay were used to determine the proliferation inhibitory effect of PSZ1.3 Colony formation assay1.4 Inverted microscope were used to observe the morphological and structural changes of PSZ-treated CNE1/2 cells1.5 Uptake of Fluid-Phase Tracers2.PSZ induces nasopharyngeal carcinoma cell Methuosis by regulating ARF6/ERK1/2/c-Fos/DUSP12.1 High-throughput RNA sequencing and transcriptome analysis2.2 Qunantitative Real-time qPCR to detect the expression of related genes2.3 Pulldown assays2.4 Western Blot Analysis2.5 Immunofluorescence(IF)staining2.6 CCK8 test of cell proliferation3.Depressive effect of PSZ on the growth of transplanted tumor in nude mice3.1 Xenograft NPC tumors in nude mice and drug administration3.2 Calculation of the rate of tumor formation and drug inhibition in nude mice3.3 Immunohistochemical staining(IHC)Results:1.PSZ inhibits the survival and proliferation in CNE1 and CNE2 cellsInverted microscopy showed that NPC cells became filled with lucent cytoplasmic vacuoles after treatment with PSZ(1 mg/mL)for 24 h/48h,the cells,and nascent macropinosomes to merge with each other,resulting in the formation of large phase-lucent vacuoles and rupture of the cell.The cell viability of PSZ against CNE1 and CNE2 cells was investigated in vitro by treating CNE1 and CNE2 cells with increasing doses of PSZ(0,0.25,0.5,1,1.5,and 2mg/mL)for 24 and 48h.We then measured the proliferation of PSZ-treated cancer cells using the MTT assay.The findings indicated that PSZ could inhibit proliferation of CNE1 and CNE2 cells according to concentration with time.This suggests that PSZ had significant toxicity to CNE1 and CNE2 cells.the survival and proliferation of the CNE1 and CNE2 cells decreased with an increasing PSZ dose.The long-term consequences of PSZ for cell survival are evident in colony-forming assays where addition of PSZ resulted in an 85%to 90%reduction in the number of colonies.2.PSZ induced Methuosis cell death in nasopharyngeal carcinoma cells via ARF6/ERK1/2/c-Fos/Duspl pathwayTo test the potential of targeting c-Fos,DUSP1 for therapy,we performed experiments using small molecule inhibitors T-5224(c-Fos inhibitor)and R031-8220(DUSP1 inhibitor).The result show that treatment with T-5224 or R0 31-8220 can both induce vacuoles in CNE1/2 cells,indicating that inhibite c-Fos or DUSP1 can mimic the effect of PSZ.Nest,we Overexpress ARF6 and c-Fos by tranfected CNE2 cells with ARF6 Plasmid or c-Fos Plasmid before treating CNE2 cells with PSZ for 24h,the results show a decrease of vacuoles in CNE2 cells afer treated with 1mg/ml PSZ for 24h.Which indicating that the overexpression of ARF6 or c-Fos can both block methuosis induced by PSZ.And,inhibiting c-Fos or DUSP1 inhibite cell proliferation.We found that PSZ downregulated the expression of c-Fos in CNE1 and CNE2 cells.Since ARF6 and C-fos both participates in the methuosis induced by PSZ,we wondered whether ARF6 could regulate PSZ-induced methuosis Via ERK inactivation.Wetern blottiong assays showed that the levels of phospho-ERk and c-Fos were significantly increased after overexpression of ARF6,whereas the total levels of ERK remained unchanged.Downregulation of ARF6 by specific siRNA largely inhibited ERK activity.These results suggest that ARF6 act as upstream effectors of ERK/c-Fos.We hypothesized that PSZ induce methuosis in CNE1 and CNE2 cells via modulating H-RAS/RAC1/ARF6 signaling pathway and inhibiting ERK1/2/c-Fos/DUSP1.3.Depressive effect of PSZ on the growth of Xenograft tumor in nude miceAt the end of the experiment,nude mice were Dissected and The tumor volume and tumor weight were measured.The nude body weight of both control group and the experimental group were increased,the difference was not statistically significant(P>0.05).The average tumor volume of high concentration group compared with the control group was significantly(P<0.05).The average tumor volume of high concentration group compared with the control group was significantly(P<0.05).On the thirtieth day after implanting tumor cells,the tumor volumes were significantly(p<0.05)larger in the mice implanted with CNE2 cells than in CNE2 cell xenograft mice treated with PSZ.During the test period,the body weights increased at a similar rate across all animal groups.The results show PSZ inhibit the growth of tumor.4.The expression of c-Fos and p-DUSP1 was significantly down regulated in nude mice xenograft tissuesThe transplanted tumor tissues of nude mice were taken and paraffin sections were made in the control group and the drug treatment group.The expression of the target protein was observed under the microscope by the immuno histochemical experiment,and the score was scored according to the standard.The results showed that compared with the control group,the expression of c-Fos and P-DUSP1 decreased significantly,and the results were consistent with those of cells.Conclusion:In summary,we are now able to draw the following conclusions:(1)Platycarya Strobilacea Sieb.et Zucc alcohol extracts(PSZ)inhibited theproliferation and induced Methuosis in human nasopharyngeal carcinomacells.(2)PSZ induces NPC cells Methuosis by ARF6/ERK1/2/c-Fos/DUSP1 pathway.(3)PSZ alcohol extracts has anti-tumor activity in vivo.