Investigation On Immunogenic Cell Death Induced By Combined Elemene And Mitomycin C And Mechanisms

Objective:Elemene combo tumor cell vaccine（EC-TCV) is a kind of cell-basedtumor vaccines prepared through combined treatment of tumor cells with elemene andmitomycin C(MMC).EC-TCV immunization can induce stronger anti-tumor immunityin human and mice. This experiment was designed to explore effects of immunogeniccell death and spliceosome associated protein62(SAP62) in enhanced immunogenicityof EC-TCV.Methods:1.Preparation of EC-TCV: Hca-F is a high lymphatic metastatic subline of murineheptocarcinoma H22.Hca-F single cell suspension was sterilely prepared and dividedinto4groups:①EC-TCV group: Hca-F cells were treated with elemene and MMC,percent of trypan blue dyed cells was about （65±3）%；②control group: no drug wasadded；③elemenegroup：Hca-F cells were treated with elemene；④M MCgroup：Hca-F cells were treated with MMC. In all of groups the last concentration of drugs was50μg/mL and the time of treatment was3hours.2.Immunoprotective test of EC-TCV：Healthy SPF Balb/c mice were divided into3groups, each was composed by5male and5female.①EC-TCV immunization：Micewere subcutaneously injected with EC-TCV cells0.2mL(5×107/mL) for3times，interval of6days；②Challengecontrol：Mice were intraperitoneally injected withnormal saline，③Combined with cytokines(EC-TCV+CKs)：Mice were intraperitoneallyinjected with0.1μg GM-CSF and0.1μg IL-4and subcutaneously injected EC-TCVsimulataneously. All of mice were challenged with1×106Hca-F cells/mouse on10thday after last immunization (0thday after challenge).Tumor growth and survival time ofmice were observed. Serum IFN-λ3was measured with ELISA kit.3.Cell proliferation was determined by MTT method，Morphological changes wereobserved with DAPI staining and Giemsa staining. Expression of SAP62in Hca-F cells was detected with Western blot method. The datas were analyzed by statistical softwareSPSS. P<0.05was used to show statistical significance of difference.Result:1.Immunoprotective effect of EC-TCV：No tumor was seen at injection site ofEC-TCV after immunization. On15thdays after Hca-F challenge，the tumor volume was1.22cm~3in challenge control group，the value was0.24cm~3in EC-TCV alone group，the value was0.34cm~3in EC-TCV+CKs group，the difference in tumor volume wassignificant between challenge control group and either of experiment groups (P<0.05)，the difference was not significant statistically between the experiment groups (P>0.05).All of challenge control mice had died till43thday，7/10and6/10mice survived over60days in EC-TCV group and EC-TCV+CKs group，respectively. Serum IFN-λ3concentration was(14.12±9.20)pg/mL in normal group，that was (19.32±9.76)pg/mLin challenge control group，that was (24.72±19.7)pg/mL in EC-TCV+CKs group，thedifference was not significant (P>0.05) statistically.2.Proliferation and death of Hca-F cell：the inhibitory rate of cell proliferation wasdetermined by MTT method：proliferation of Hca-F cells was inhibited by elemene，mitomycin C alone or combined treatment，the difference was significant (P<0.05)compared with control group. Results of DAPI staining and Giemsa staining showedthat both apoptotic cells and necrotic cells may be seen in EC-TCV cell suspension.3.Changes of SAP62expression：Results of western blot showed that SAP62expression was detected in Hca-F cells of all groups. Elemene or mitomycin Cdown-regulated SAP62expression，SAP62expression of EC-TCV cell was the lowestamong4groups，the difference in SAP62expression amount was significant betweenuntreated and treated with elemene，MMC or combined group (P<0.05).Conclusion:1.EC-TCV immunization induced protective effects against high metastatic sublineHca-F of MHC-Ⅰ-murine hepatocarcinoma H22in mice.GM-CSF and IL-4did notobviously enhance active immune protective effect of EC-TCV，induction of IFN-λ3was not the main mechanism of EC-TCV immune protective effects.2.The cell death observed in EC-TCV cells had the characteristics of immunogeniccell death，which is one of the mechanisms by which tumor immunogenicity ofEC-TCV was enhanced.3.Combination of elemene with and mitomycin C resulted in decreased SAP62expression in tumor cells，thereby affect the maturation of mRNA and alternative splicing to exert anti-tumor effect.