Immunogenicity Of Early Apoptsis A549 Cells Induced By Adriamycin And Cisplatin And It's Mechanism

Objective:The conventional idea deems that chemotherapy could suppress the immune function. But it's still unclear that if the apoptosis or death of tumor cells induced by chemotherapy drugs such as adriamycin could stimulate the immune cells,such as dendritic cells(DC),and furture arose the immune response to kill cancer cells? In this paper,we used the apoptosis A549 cells induced by adriamycin and cisplatin as antigen to stimulate dendritic cell and to sensitise T cells and observe it's killing effects to cultured A549 cells in vitro and further to explore it's mechanism. So as to provide laboratory evidence for research and application of chemotherapy combined with dendritic cells-based immunotherapy for cancer.Methods:The apoptosis ratio of A549 cells were detected by acridine orange / ethidium bromide double stain method. MTT method was used to detect the quantity of cells. DC cells were preparated from peripheral blood monocytes by some cytokines such as GM-CSF, IL-4. At the 3th and 10th day,using flow cytometry to detect the CD80 and CD83 moleculars on the cultured cells surface. Using confocal microscopy to observe the exposure states of calreticulin at the apoptsis A549 cells induced by adriamycin and cisplatin.Result:The apoptosis ratio of A549 cells treated by adriamycin for 12 h at the concentration of 1 mg/L,3 mg/L and 5 mg/L were respectively 17.6±1.77%,62.93±1.70% , 16.8±0.65%. The apoptosis ratio of A549 cells treated by cisplatin for 12 h at the concentration of 9 mg/L,13 mg/L and 21 mg/L were respectively52.99±8.39%,62.44±3.41%,47.14±8.99%. The rusults showed that both adriamycin and cisplatin could induce A549 cells apoptosis. , high apoptosis ratio were exhibit at the concentration of 3 mg/L for adriamycin and 13 mg/L for cisplatin and the concentration was separately used for the following-up experiments.The apoptosis A549 cells were divided into 2 groups : group A was induced by 3 mg/L adriamycin, group D by 13 mg/L cisplatin, group N was normal cultured A549 cells in vitro. Apoptosis A549 cells from 2 groups and normal A549 cells from N group were respectively used to stimulate cultured dendritic cells (DC) in vitro, after that, the DC were used to sensitize T cells and to kill culturecd A549 cells. The results showed that: OD values in group A was 0.6245±0.031715, group D 0.8745±0.032569 and group N 0.922627±0.041501. Group A had significant kill effects to A549 cells compare to the group D and group N(P<0.01). It sugeested that A549 cells treated by adriamycin showed stronger immunogenicity and could trigger stronger immune response to kill more tumor cells in vitro.On the 3th day, the membrane surface of the cultured monocyte which treated by IL-4 and GM-CSF expressed the CD80 molecule (24.67%) and CD83 molecule (31.13% ). On the 10th day, the CD80,CD83 molecules expressing ratio in the group A was 81.48% and 77.40% separately, in the group D was 80.82%, 91.61% separately and in the group N was 83.89%, 94.71% separately. CD80,costimulatory molecules can induce T cell proliferation and activation CD83 is a specific marker for DC which participating antigen-presenting and T-cell activation. By morphology in early stage, cultured cells had branch.The cells sticked to the wall. In later stage cells significantly increased in size, The shape of cells were in dendritic These dates revealed that the cultured cells were DC and it were in maturity state on the 10th day after cultured.The state of calreticulin on A549 cell surface was tested by immunology technology and confocal microscopy. The results showed that calreticulin appeared on the surface of apoptosis A549 cells which induced by adriamycin in group A, but disappeared in the group D and group N. It indicated that adriamycin could induce exposure of calreticulin on the surface of A549 cells and it might be relevanted to it's immunogenicity. Furture, using the blocking peptide of calreticulin to inhibite DC receptor binding of calreticulin on the surface of A549 cells induced by adriamycin (group Ca-A) would decrease it's immunogenicity and secondary kill effects to A549 cells. OD value in the group Ca-A (0.770727±0.006191) was significantly high than the group A ( 0.6245±0.031715) (p<0.01), it suggested that Calreticulin might play a key role in immunogenicity of apoptosis A549 cell induced by adriamycin.Conclusion:1. Both adriamycin and cisplatin could induce A549 cell apoptosis in the manner of none doses-dependment..2. Apoptosis A549 cells induced by adriamycin had stronger immunogenicity than by cisplatin and it might be relevanted to the exposure of calreticulin on the surface of apoptosis A549 cells induced by adriamycin. 3. The above results are benefit for explore a novel treatment pattern of chemotherapy combined with dendritic cells-based immunotherapy for lung cancer.