Preparation And The Therapeutical Effect Of Liposomal Curcumin Combined With Irradiation On Endometrial Carcinoma:A Primary And Preclinical Study

1.The preparation and identification of liposomal curcuminObjective:The purpose of this study is to prepare and identify a formulation of liposomal curcummin.Methods:(1)Liposomal curcumin(LC)was prepared using liposome encapsulation technology.(2)Content and purity determination was assayed by High-performance liquid chromatography(HPLC)according to the general principle.(3)The measurement of the particle size distribution and Zeta potential of LC was done through a particle size potentiometer.(4)The appearance and dispersion of LC was observed macroscopically.Results:(1)Under the chromatography,standard curcumin and LC showed a peak at 11.835th and 11.831th min respectively,whereas the lipids and accessorily chemical reagents did no peak.(2)The concentration of LC was 1mg/ml and its purity was excellent.(3)The mean particle size of LC was approximately 126.8nm and the final zeta potential was-8.88mV.(4)LC could be distributed uniformly,without no solids and precipitates observed and the appearance of its emulsion was half-transparent.Conclusions:LC was prepared successfully,which could be further attempted to the treatment of endometrial carcinoma(EC).2.The Therapeutical Effect of Liposomal Curcumin On Endometrial Carcinoma:A Primary And Preclinical Study Objective:The purpose of this study is to investigate the therapeutical effect of LC on EC and its potential mechanism,which could provide a theoretical basis for clinical use of LC targeting EC.Methods:(1)Human endometrial carcinoma cell lines Ishikawa and HEC-1 were treated for 24 hours with various concentrations of LC.Cell viability was determined by MTT assay.(2)Under the treatment of IC15-20 or IC50 of LC,the apoptosis analysis of Ishikawa and HEC-1 cells were performed by Hoechst 33258 staining assay and Flow assay.(3)Under the treatment of lower than IC15-20 of LC,the invasion analysis of Ishikawa and HEC-1 cells were performed using wound healing and transwell assay.(4)Under the treatment of no more than IC50 of LC,the mRNA level of NF-κB was detected by RT-qPCR assay.(5)Under the treatment of no more than IC50 of LC,the protein expression of NF-κB followed with Caspase-3 and MMP-9 were detected by Western Blot Analysis.(6)Toxicity Assay of LC was performed based on zebrafish model.(7)Under the treatment of IC 15-20 or IC50 of LC,the tumor volume of zebrafish tumor model of EC was examined by fluorescence detection and the mRNA level of NF-κB was detected by RT-qPCR assay.Results:(1)MTT assay exhibited LC could result in a dose-dependent proliferation inhibition for EC cells in vitro.After regression fit analysis,IC 15-20 and IC50 were 15.50μM for Ishikawa,30、80μM for HEC-1 respectively.(2)Hoechst 33258 fluorescence assay showed LC could lead to morphological change of apoptosis for EC cells.Under the treatment of IC 15-20 or IC50 of LC,flow assay further showed the apoptosis rates were 15.12±0.59%,43.25%±1.48%for Ishikawa cells and 9.67%±0.79%,38.79%±4.55%for HEC-1 respectively,significant higher than control.(3)Cell migration assay indicated cells motility was significantly reduced following the treatment with 5,10μM of LC on Ishikawa or 10,20μM on HEC-1 respectively.With the same treatment,cell invasion assay further indicated these cells’invasiveness was reduced by 34.88%,81.93%for Ishikawa and 41.3%,89.57%for HEC-1 respectively.(4)With the LC treatment,the mRNA level of NF-κB was down regulated in a concentration-dependent manner in both Ishikawa(from 10 to 50μM)and HEC-1(from 20 to 80μM)cells compared to the levels in untreated cells.(5)Accordingly,the nuclear protein level of NF-κB was down regulated in both cell lines,also in a concentration-dependent manner(from 0 to 50uM for Ishikawa and from 0 to 80μM for HEC-1).At the same time,the protein level of Caspase-3(uncleavcd-form)and MMP-9 was found to be significantly reduced in Ishikawa and HEC-1 cells treated with LC and the Caspase-3(cleaved-form)increased significantly.(6)Treatment up to 80μM LC for 6 hours did not significantly caused embryo death and the length of embryo was also not reduced after exposure up to 80μM LC for 6 hours.(7)Under the treatment of IC 15-20 or IC50 of LC,the tumor growth in zebrafish was dramatically delayed in a concentration dependent manner(0-50μM for Ishikawa or 0-80μM for HEC-1).RT-qPCR assay further showed the mRNA level of NF-κB in zebrafish was dose-dependently reduced by LC treatment(0-50μM for Ishikawa or 0-80μM for HEC-1).Conclusions:LC exhibited the biological roles of resisting EC probably through negatively regulating the NF-κB pathway in vitro and in vivo and had potential therapeutic effect on EC.3.The Therapeutical Effect of Liposomal Curcumin Combined With Irradiation On Endometrial Carcinoma:A Primary And Preclinical StudyObjective:The purpose of this study is devoted to investigate the possibility of LC combined with irradiation in the treatment of EC and to analyze its potential mechanism,which could provide a theoretical basis for clinical strategy targeting EC radiotherapy.Methods:(1)A total of 5 EC patients who received radiotherapy in Jiangsu Cancer Hospital during 2015 and 2017 were collected.The changes of nuclear expression of NF-κB before and after radiotherapy were analyzed by immunohistochemistry assay.(2)The Ishikawa cells were treated with different doses of radiation.The mRNA level and activity of NF-κB before and after irradiation were detected by RT-qPCR and ELISA respectively.(3)Clone formation assay was used to detect the clonogenic rate of Ishikawa cells treated with LC ± irradiation.(4)Flow cytometry assay was used to detect the apoptosis of Ishikawa cells treated with LC ± irradiation.(5)RT-qPCR and Western Blot were used to detect the changes of NF-kB mRNA or protein level in Ishikawa cells treated with LC ± irradiation.(6)Fluorescence detection method was used to evaluate the anti-tumor effect of LC ± irradiation treatment on zebrafish tumor model of EC.The corresponding mRNA level and activity of NF-κB were detected by RT-qPCR and ELISA respectively.Results:(1)Immunohistochemistry assay showed that the nuclear expression of NF-κB in endometrial carcinoma increased significantly after radiotherapy,especially for P65 subunit protein.(2)RT-qPCR assay indicated that the mRNA level of NF-κB in Ishikawa cells was significantly increased in a dose-dependent manner after irradiation(3-12Gy)compared with the control group.The results of ELISA also indicated that the NF-κB activity of Ishikawa cells was significantly increased after irradiation(3-12Gy)as compared with the control group,still with a dose-dependent manner.(3)Clone formation assay exhibited that after 15μM or 50μM of LC combined with 2Gy or 4Gy irradiation treatment,the cloning rate of Ishikawa cells was significantly lower than that in control group,irradiation group and LC treatment group.(4)Flow cytometry showed that the apoptotic rate of Ishikawa cells treated with 15μM of LC combined with 2Gy irradiation was significantly higher than that in the control group,the LC treatment group and the irradiated group.(5)The results of RT-qPCR indicated that the mRNA level of NF-κB in Ishikawa cells treated with 15μM of LC combined with 2Gy irradiation was significantly lower than that in control group,irradiation group and LC treatment group.Western Blot results showed that the expression of NF-κB in Ishikawa cells treated with 15μM of LC combined with 2Gy irradiation was significantly lower than that in the control group and the irradiation group.But compared with the LC treatment group,no significant difference was found between these two groups.(6)The results of zebrafish tumor model of EC showed that after the treatment of 15μM of LC combined with 2Gy irradiation,the growth of xenografts was significantly inhibited compared with the control group,the irradiation group and the LC treatment group.The results of RT-qPCR and ELISA assay indicated that the mRNA level or the activity of NF-κB was significantly lower than that in the control group and the irradiation group,but not significantly different to the LC treatment group.Conclusions:(1)Radiation therapy can activate NF-κB signaling in endometrial cancer cells.(2)By inhibition of NF-κB signaling pathway,LC combined with radiotherapy can significantly inhibit the proliferation and induce apoptosis of EC cells in vitro.In vivo this combination treatment can significantly inhibit the growth of EC xenografts.(3)LC has the effect of radiation sensitization,proposing the potential value of combination with radiotherapy in the treatment of EC.