The Apoptosis Effects On Human Esophageal Cancer Cells By Soyasaponin Bb And Nanoliposomal Quercetin

The esophageal squamous epithelial carcinoma(ESEC),is developed from malignant squamous epithelial tissue.One of the highest incidences of ESEC is in China.The cancer cells are characterized with resistance to hypoxia,chemical drug and radiation.The tumor cells have strong proliferation capacity,high expression of Cyclin D1,inhibition of PTEN suppressor gene expression,which in turn,actives the PI3k/Akt signal transduction pathway. As the result,cell proliferation is inhibited,c-met and VEGF in human esophageal cancer cells is over expressed.Saponins extensively exist in the plants and ocean biophores.It has been reported that the saponins have multiple bioactive,including anti-oxidant,free radicle scaranging, anti-inflammation,anti-hypersensitivity and anti-bacteria,anti-viral,even anti-tumors; However report regarding the mechanisms rang.The Soyasaponin can be mainly classified into Soyasaponin A and Soyasaponin B subgroups,especially Soyasaponin Bb extract with stronger effects than Soyasaponin A equivalent.Quercetin(1,3,4,5.7-5 hydroxyflavone) is one kind of flavinoids.It is rich in various vegetables and fruits,as well as the component of some traditional Chinese medicines.It is difficult to be dissolved in water.Multiple effects exerted on normal cells/neoplasm associated with corresponding underlying mechanism have been reported as anti-oxidant,anti-inflammatory/anti-viral anti-tumor drugs,and blood pressure or lipids suppressor;however,the effects of nanoliposomal quercetin,water dissolvable,and the signaling mechanism remains to be explored.The anticancer effects induced by drugs,such as quercetin,could down-regulate gene expressions of oncogenesis,metastasis,up-regulate tumor suppressor and apoptosis-related genes.It has been found in further studies that the alteration of these gene expressions in tumors associated not only with gene mutation or altered coding sequence,but also modification of chromatin,namely epigenetic modification,including histone methylation, acetylation and so on.Such chromatin modification can affect a serious of cellular processes, such as differentiation,proliferation,survival or apoptosis.In this experiment,the water-soluble soysaponin Bb and nanoliposomes quercetin were studied in cultured esophageal cancer cells to explore whether the soyasaponin Bb or nanoliposome quercetin can be used as histone deacetylase(HDAC) inhibitor drug to reverse the esophageal cancer malignancy and its underlying mechanism.Aim:To explore the effect and potential mechanism of malignancy reverse and apoptosis in the Eca-9706 cells induced by soyasaponin Bb and nanoliposomal quercetinMethods1.Preparation of nanoliposomal quercetin:The quercetin and lipids mixture was dissolved in chloroform and DMSO(3:1),subsequently passed through ultrasound destructor,and followed by evolutionary evaporation under vacuum and finally filtered through 80 nm pore filter.The soyasponin Bb extract was purchased from Tongtian Company,China.2.Cell growth suppression rate detected by MTT:The cultured Eca-9706 cells were divided into four experimental groups,control(C) group and the soyasponin Bb(SSBb) group concentration were 26.5μmol/L,106μmol/L,424μmol/L,control(C) group of culture without drugs.The Eca-9706 cells were cultured 24h,48h,72h after the detection of OD values in each group;The cultured Eca-9706 cells were divided into three groups:(1) soyasponin Bb(SSBb) group:106μmol/L cocultured with 9706 cells for 48h;(2) nanoliposomal quercetin(nLQ) group:40μmol/L nLQ cocultured with 9706 cells for 48h, and(3) control(C) group:no drug was cocultured with the cells for 48h.3.Apoptotic rate:The cultured ECa-9706 cells were divided into three experimental groups, control(C) group,the soyasponin Bb(SSBb) group concentration was 106μmol/L,the nanoliposomal quercetin(nLQ) group concentration was 40μmol/L,control(C) group of culture without drugs.Apoptotic rate in each group was detected by TUNEL assay.4.Immunohistochemical staining:The expression level and location in each group of 9706 cells were immunostained on the 4%paraformaldehyde fixed slides with SP kits by HDAC1,NF-kB,PTEN,Cyclin D1,c-met,VEGF or caspase-3 antibody.Meanwhile,the primary antibody substituted for PBS was performed as the negative control.5.Immunoblotting:After the total protein extracted from each group of ECa-9706 cells, SDS-PAGE was performed,the immunoblotting was detected by NF-kB,PTEN,Cyclin D1,c-met,HDAC1 and VEGF(β-actin as internal reference).6.Statistical analysis:The data obtained from.image analyzer were statistically analyzed use SPSS 12.0 soft ware.The variance in normal distribution was shown as((?)+s) and ANOVA among groups was analyzed,andα=0.05 was considered as significance.Results1.MTT assay:There was significant difference in cell growth suppressing rate between the SSBb/nLQ group and C group,P＜0.05;while no significant difference was found between the SSBb group and nLQ group,P＞0.05.2.Immunohistochemical staining:The immunohistochemical reactivity(IR) appeared as brownish granules,activated IR signals were distributed in the cell nuclei detected by HDAC1,NF-kB,PTEN,Cyclin D1 or caspase-3;and c-met or VEGF IR signal distributed in the cell cytoplasm/nuclui.When compared with the C group,the IR signals of PTEN or Caspase-3 were enhanced,those of HDAC1,NF-kB,Cyclin D1,c-met or VEGF were attenuated in the SSBb or nLQ group,P＜0.01.When the SSBb group was compared with the nLQ group,no significant difference in HDAC1,PTEN or Cyclin D1 in IR signals,P＞0.05.3.Immunoblotting:When compared with the C group,the immunoblotting signals of PTEN or caspase-3 were enhanced,those of HDAC1,NF-kB,Cyclin D1,c-met or VEGF were decreased in the SSBb or nLQ group,P＜0.01.When the SSBb group was compared with the nLQ group,no significance was found in HDAC1,PTEN or Cyclin D1 blotting signals,P＞0.05.There was a positive correlation between immunostaining and immunoblotting,r=0.87～0.99.4.Apoptosis:TUNEL apoptotic signal was blue-violet in Eca-9706 cells.In the apoptotic cells,cell nucleus is shrinkage or nucleus located in cells around the crescent-shaped. some cells can be seen apoptotic bodies.C group,nLQ group and the group SSBb apoptosis rates were 5.0%,42%and 40%.Compared to the control group,nLQ group and SSBb group apoptosis rate is significant,P＜0.01.Conclusions1.SSBb and nLQ can suppress Eca-9706 cell growth,while there was no significant difference in cell growth suppression rate between SSBb and nLQ groups.2.SSBb or nLQ can exhibit reverse effects on over expression of c-met,VEGF and Cyclin D1 in Eca-9706 cells.3.Apoptotic rates:SSBb and nLQ can induce ECa-9706 cells apoptosis,apoptosis rates were 40%and 42%.4.Eca-9706 cell apoptosis can be induced by SSBb or nLQ through inhibiting HDAC1-NF-kB and activating PETEN and caspase-3 signaling pathways.