The Effect And Mechanism Of Oleandrin Induces Immunogenic Cell Death Through Endoplasmic Reticulum Stress In Breast Cancer Cells

Abjective:Breast cancer is the most common malignant tumor in China and around world.In recent years,the incidence of breast cancer has increased year by year.In 2018,the number of women who died of breast cancer in the world ranked first in the number of malignant tumor deaths,has been seriously affecting women’s health.At present,the comprehensive treatments with surgery,chemotherapy,radiotherapy,endocrine therapy and targeted drug treatment have shown good effects on early stage breast cancer and effectively prolonged the overall survival.However,metastatic breast cancer,especially triple negative breast cancer which is a lack of effective treatment shows poor prognosis.In recent years,immunotherapy represented by immune-checkpoint inhibitors has made remarkable achievements in the field of tumor therapy.For advanced triple negative breast cancer cell,programmed death ligand 1(PD-L1)antibody(Atezolizumab)combined with paclitaxel-albumin group significantly prolonged the progression free survival and total survival compared with paclitaxel-albumin group.In the course of immunosuppressive therapy,the number and function of dendritic cells(DC)are closely related to the prognosis.Therefore,the key to tumor immunotherapy is to increase the number of tumor infiltrating DC cells and improve the function of DC cells.Oleandrin which is the monomer compound extracted from oleander belongs to cardiotonic glycosides(CG)from plants.Recently,it has been reported that cardiac glycosides can induce tumor cell apoptosis,autophagy and inhibit migration.It plays an anti-tumor role at a low concentration.In addition,cardiac glycosides can also trigger immunogenic cell death(ICD).It has been proved that oleandrin can inhibit the tumor cell proliferation and apoptosis,but whether it can cause ICD and its possible mechanism has not been reported.The initial stimulating factors induce cell death,make the dead cell have immunogenicity,and can further activate the adaptive immune response,then this way of death is called ICD.The main characteristics of ICD are as follows: the turnover of CRT to cell membrane,the release of ATP and HMGB1,and the secretion of HSP70/90.The above death related molecular models(damps)can attract the enrichment of DC cells,promote the maturation of DC cells,release related cytokines,and then activate the anti-tumor immune response of the body.This study focused on the three points.1 Whether Oleandrin can induce ICD in breast cancer,especially in triple negative breast cancer? 2 Whether Oleandrin can promote the activation of DC cells,increase the secretion of cytokines and tumor infiltrating immune cells and has the anti-tumor effects in vivo? 3 This study will investigate the mechanism of oleandrin inducing breast cancer cell death.The results will provide theoretical support for the application of oleandrin in the field of breast cancer treatments.Methods:1.Tumor cell viability was detected by using xCELLigence RTCA TP system.2.Apoptotic cells were double stained by Annexin-V / PI and detected by using flow cytometry.3.Cell nuclear morphology was stained with Hoechst33342.4.CRT expression was observed by immunofluorescence microscopy.5.Secretory protein expression level was detected by ELISA.6.ATP expression was detected by Chemiluminescence.7.DC cell was cultured in vitro.8.Tumor cells co-cultured with DC cells were separated by cell sorter.9.Cell surface markers were detected by flow cytometry.10.QRT-PCR was used to detect the expression of mRNA.11.Western blot was used to detect the expression of protein.12.EMT-6 cells were implanted into BALB/C mice mammary gland.Tumor bearing mice were divided into groups according to the treatments.Tumor size was measured.13.The experiment was repeated three times in each group.SPSS 23.0 statistical software was used to conduct t-test or one-way ANOVA.The results were expressed as mean ± standard deviation((?) ± s),p < 0.05,which was statistically significant.Results:MCF7(Luminal A subtype),SK-BR-3(Her2 subtype),MDA-MB-231(TNBC)cells were treated with Oleandrin at several concentrations.Cell viabilities were detected by xCELLigence and showed that Oleandrin had a dose / time-dependent killing effect on three groups of breast cancer cell lines.However,Oleandrin had no effects on human mammary epithelial cell MCF10 A cells.In order to further study the possible mechanisms of death,MCF7 and MDA-MB-231 cell line were selected for follow-up study.The concentration of Oleandrin was chosen as the median lethal concentration(IC50).Annexin-V / PI double staining flow cytometry was used to detect cell apoptosis.The results showed that oleandrin could induce apoptosis of breast cancer cell lines.The karyopyknosis and fragmentation were also observed by hoechst33342 staining.Each group of cells were treated with Oleandrin at the concentration of IC50.(1)CRT protein expression was detected with immunofluorescence and showed that CRT turned from cytoplasm to the cell membrane.The expression of CRT on cell surface was also confirmed by flow cytometry and showed that the proportion of PI-/CRT+ cells increased significantly after 6 hours of oleandrin treatment.(2)Breast cancer cells were treated with Oleandrin and HMGB1 secretion in the culture supernatant was detected by ELISA.The results showed that the expression level of HMGB1 was significantly increased in the culture supernatant 24 hours after oleandrin treatment.(3)The secretion of ATP was detected by chemiluminescence method.The results showed that after 6 hours of oleandrin treatment,ATP was detected in the cell culture supernatant,and reached the peak value at 12 hours.(4)The cell culture supernatant and cells were collected.The culture supernatant was concentrated,and HSP70/90 expression was detected by Western blotting.The results showed that the expression of HSP70/90 was detected in the supernatant 48 hours later.There was no significant change in the expression of these two proteins.Oleandrin induced ICD of breast cancer cells and activated anti-tumor immune response.The HLA phenotypes of tumor cells and healthy volunteers were confirmed by flow cytometry.The tumor cell line MDA-MB-231 expressing HLA-A2 was selected for follow-up experiment with HLA-A2 volunteers’ PBMC.Monocytes from the volunteers PBMC was induced by cytokines and differentiated into DC cells.MDA-MB-231 cells were pretreated with Oleandrin and then co-culture with DC cells in vitro for 48 h.DMSO treatment was used as control group.DC cells were then stained with CD45 and sorted by cell sorter.The mRNA expressions of CD86,IL-2,IL-10 and IFN-γ were detected by qRT-PCR.Compared with the control group,the expressions of CD86,IL-2 and IFN-γ in DC cells were increased but IL-10 was decreased.CD86 expression on the cell surface was further confirmed by flow cytometry.Although the level of CD86 mRNA was significantly increased,there was no significant difference on the cell surface expression between control group and Oleandrin treatment group,which both had reached a high level,the percentage of CD80 was increased and HLA-DR was not changed.Consistent with the qRT-PCR results,IL-2 and IFN-γ detected by ELISA was increased,but IL-10 was decreased in the Oleandrin group.To observe the effect of oleandrin on immune response in the mouse model,BALB / c mice were inoculated with breast cancer cell EMT-6 in situ,and oleandrin 0.3mg/kg or 0.6mg/kg was injected intraperitoneally once a day.The tumor growth was inhibited in 0.3mg/kg group and decreased significantly in 0.6mg/kg group.Primary cells were collected,the spleen and tumor infiltrating lymph cells were analyzed by flow cytometry.The results showed that oleandrin increased the percentages of CD3+CD4+ and CD3+CD8+ T cells in spleen,however,there was no significant change in the percentage of DC cells.Oleandrin increased the number of DC,CD4+ T cells and CD8+ T cells in tumor,especially CD8 + T cells.Oleandrin triggered endoplasmic reticulum stress,and then activated mitochondrial apoptosis pathway to induce cell apoptosis in breast cancer.To further explore the possible mechanism of oleandrin induced ICD and its relationship with apoptosis,zVAD-fmk,an apoptosis inhibitor,was used to observe its effects on oleandrin induced apoptosis and CRT translocation.The results showed that z-VAD-fmk could inhibit the cell apoptosis induced by oleandrin but could not inhibit the overturn of CRT.Western blotting results showed that the expression of p-perk,e IF2 α,p-eif2 α,ATF-4 and CHOP in ER stress pathway was up-regulated,but there was no significant change in the expression of perk.As mitochondrial apoptosis pathway was regulated by CHOP,the expression of Bcl-2 family protein was detected.Oleandrin inhibited the expression of Bcl-2 protein but upregulated the expression of Bim and Bax.Conclution:1.Oleandrin induced cell apoptosis and ICD in breast cancer.2.Oleandrin induced breast cancer cell ICD to activate antitumor immune response in vitro and in vivo.3.Oleandrin induced ICD through endoplasmic reticulum stress,and then activated mitochondrial apoptosis pathway to induce cell apoptosis.