The Effect Of Immune Activation Induced By Cinobufotalin On Inhibiting Bladder Cancer

BackgroundBladder Cancer(BC)is the most common malignant tumour of the urinary system,with a high mortality rate and recurrence rate.It is a major public health problem.Currently,radical cystectomy combined with chemotherapythe is still the standard treatment method for BC.The therapeutic goal for most anticancer drugs is to induce tumor cell death.A growing body of evidence indicates that the clinical success of conventional chemotherapy not only results from cytotoxicity,but also depends on the restoration of intratumoral immune surveillance.Immunogenic cell death(ICD)inducers,such as anthracyclines,can cause damaged dying tumor cells to release immune-stimulating danger signals,which activate dendritic cells and cytotoxic T cells,priming anti-tumor immune response.So far,however,a limited number of ICD inducers have been identified.In addition,the expression of programmed cell death ligand 1(PD-L1)in the tumor cells is usually up-regulated by ICD inducers and thus deactivtes T cells via binding to programmed death 1(PD-1).Therefore,the combination of ICD inducers and PD-1/PD-L1 immune checkpoint inhibitors is required to achieve therapeutic efficacy,which is bond to increase drug-related adverse reactions and cost of healthcare.Cinobufotalin(CBT)is a bufadienolide compound extracted from the skin secretion of the traditional Chinese medicine Bufo bufo,which belongs to type I ICD inducer cardiac glycosides.It has cardiotonic,hemostasis and anticancer effects,and can also enhance the therapeutic effect of chemotherapy drugs on malignant tumors.CBT has inhibitory effect on a variety of tumors,however,the effect of CBT on BC has not been reported at present.Recently,it has been proved that CBT can inhibit tumor growth by blocking signal transduction and activator of transcription 3(STAT3)signaling pathway,which is related to the expression of PD-L1.Therefore,we speculate that CBT may activate anti-tumor immune response by inducing ICD and blocking STAT3 signaling pathway to down-regulate the expression of PD-L1.ObjectiveTo explore whether CBT can activate immune response by inducing ICD and down-regulating PD-L1 expression simultaneously,and then inhibit the growth of BC,so as to find a new therapeutic strategy for BC patients in clinical practice.Methods1.BC cell lines 5637,T24 and MB49 were treated with different concentrations of cinobufotalin(CBT)for 24 h and 48 h,respectively,and the proliferation ability of the cells was detected by CCK-8.2.5637,T24 and MB49 cell lines were treated with different concentrations of CBT for 24 h,respectively.Flow cytometry and Western blot were utilized to detect the apoptosis rate and the expression level of apoptosis-related proteins in BC cells,respectively.3.After BC cells were treated with CBT,the expression levels of ICD markers p-e IF2? and CRT were detected by Western blot.The number of cells with positive CRT expression on the cell membrane surface was analyzed by flow cytometry,and the localization of CRT was detected by analysis of fluorescence intensity along the cell membrane using confocal microscopy.4.After BC cells were treated with CBT,the expression of autophagy marker protein LC3 in cells was assessed by means of Western blot analysis.GFP-LC3 plasmid was transfected into human BC 5637 cells and LC3 puncta were visualized using fluorescence microscope to assess CBT-induced autophagy.The autophagosome-like structures in cells were visualized using transmission electron microscope.ATP release by BC cells was measured with ATP Assay Kit.5.After BC cells were treated with CBT,the expression level of HMGB1 protein in cells was tested using Western blot.Culture supernatants of untreated and treated BC cells were used to measure extracellular HMGB1 using the HMGB1 ELISA Kit.6.The immunogenic property of CBT was evaluated using the gold-standard in vivo vaccination assay.First,murine MB49 cells were treated in vitro with CBT(10 ?M)for 24 h to initiate cell death,or cells were lysed by three cycles of freeze-thaw(-80 °C to room temperature).Next,either dying cells exposed to CBT or lysed by freeze-thaw were injected subcutaneously into C57BL/6 mice as vaccines in the left axilla(n = 6 mice per vaccine group).RPMI 1640 was used as the control vaccine.Seven days post-vaccination,mice were challenged with live MB49 cells via subcutaneous inoculation on the right axilla.The mice were routinely monitored for palpable tumor lesions.7.Subcutaneous MB49 tumor-bearing C57BL/6 mice were constructed and randomly divided into two groups: the control group and treatment group,with 6 mice in each group.Tumor volume and body weight of mice were monitored every 3 days during the treatment.When the tumor volume exceeded 1500 mm3,the mice were sacrificed and the tumor weight was measured.The expression levels of Ki-67,Cleaved Caspase-3,CRT and HMGB1 in tumor tissues were detected by immunohistochemical staining.8.Hematoxylin and eosin(HE)staining was performed to detect the infiltration of lymphocytes in tumor tissues.Flow cytometry was utilized to detect the infiltration proportion of CD4+ and CD8+ T cells in tumor tissues and tumor draining lymph nodes.Immunohistochemical staining was performed to assess the expression levels of CD4,CD8,Foxp3,TNF-? and granzyme B in tumor tissues.9.After treatment with different concentrations of CBT,the expression levels of STAT3,p-STAT3 and PD-L1 in cells and tissues were assessed by means of Western blot and immunohistochemical analysis,respectively.Results1.CBT inhibited the proliferation of BC cells in a time-dose dependent manner.After 24 h treatment,the IC50 values of CBT on 5637,T24 and MB49 cell lines were 1.59 ± 0.863,8.19 ± 0.135 and 10.4 ± 0.847 ?M,respectively.At 48 h,the IC50 decreased to 0.17 ± 1.740,3.93 ± 0.363 and 4.42 ± 0.462 ?M,respectively.2.For these three BC cells,the apoptosis rate of cells increased with the increase of CBT concentration.Compared with the control group,the expression levels of PARP and Bcl-2 were decreased in the CBT-treated group,while the expression levels of Cleaved PARP,Cleaved Caspase-3 and Bax were increased.3.CBT up-regulated the expression of p-e IF2? and promoted the translocation of CRT from endoplasmic reticulum to cell surface.Compared with the control group,the expression of CRT protein on the cell membrane of CBT treatment group was increased,the number of cells expressing CRT was increased using flow cytometry,and the fluorescence intensity was enhanced using laser scanning confocal microscopy.4.CBT markedly increased the levels of LC3-II protein and the number of LC3 fluorescent puncta and autophagosome structures.Moreover,exposure of T24 and MB49 cells to CBT for 10 h markedly provoked the release of ATP into the extracellular culture supernatant.5.Exposure of bladder cells to CBT for 24 h did not significantly affect the expression of HMGB1 protein in the lysate,but markedly facilitated the release of HMGB1 into the extracellular culture supernatant.6.In vivo vaccination assay,mice that were vaccinated with either RPMI 1640 or freeze-thaw-treated MB49 cell vaccines developed palpable tumors within 15 days post challenge.Remarkably,mice that received the CBT vaccination displayed a significantly robust antitumoral response,with hindered tumor formation.7.CBT treatment significantly delayed tumor growth,reduced tumor volume and weight,but had no significant effect on the weight of mice.8.CBT promoted the exposure of CRT and release of HMGB1 in tumor tissues and increased the infiltration ratio of CD8+ T cells and production of TNF-? and granzyme B by CD8+ T cells.Conversely,CBT reduced the proportion of Foxp3+ Treg cells in tumor tissues.9.CBT inhibited the phosphorylation of STAT3 and expression of PD-L1.Colivelin,an activator of STAT3,reversed the inhibitory effect of CBT on the expression of PD-L1.Conclusions1.CBT could inhibit the proliferation,induce cell apoptosis,and reduce tumor growth in vivo.2.In vitro,CBT can induce the exposure or release of DAMPs in BC cells and has the potential to be an ICD inducer.3.In vivo,CBT can enhance the immunogenicity of BC cells,effectively induce ICD,and trigger an effective systemic immunotherapy.4.CBT could inhibit the growth of BC by blocking STAT3/PD-L1 signaling pathway.