The Experimental Research On Calreticulin Exposure In Mouse Melanoma Cell Line B16 By Using Epirubicin,Mitoxantone And 5-fluorouracil

Objective: To investigate the immunogenicity associated protein of calreticulin (CRT) exposure in mouse melanoma cell line B16 by using the chemotherapeutants Epirubicin (EPI),mitoxantone (MIT) and 5-fluorouracil (5-FU). To explore the optional concentrations with which the chemotherapeutants can induce CRT translocation to the cell surface effectively. To supply basic experimental data and theoretical supports for the tumor immunotherapy induced by chemotherapy.Methods: The inhibition efficacy of EPI,MIT and 5-FU to the B16 cell growth was evaluated by using CCK-8 (Cell Counting Kits-8) assay. The CRT exposure was measured by flow cytometry (FCM) after a coculturing with different chemotherapeutants for 24 hours.Results:1. The results of CCK-8 assay: The inhibition rates to the B16 cell growth was significantly increased when concentrations of each chemotherapeutant going higher. The median inhibition concentration (IC50) of chemotherapeutants for 24h was: EPI 1.6ug/ml, MIT 1.0ug/ml, and 5-FU 50ug/ml, respectively. 2. CRT expression on surface of B16 cells treated with IC50-concentrations of three chemotherapeutants: B16 cells were respectively treated with 0.01mol/L PBS, 1.6ug/ml EPI, 1.0ug/ml MIT, and 50ug/ml 5-FU for 24 hours, then the CRT expressions on cell surface were determined by FCM and resulted as: 58.27±3.66 in PBS group, 250.98±9.9 in EPI group, 110.76±6.76 in MIT group, and 155.96±6.95 in 5-FU group. The CRT expression of each experimental group was significantly higher than that of the control group (P<0.001). The CRT exposure was effectively induced by three chemotherapeutants, in which the strongest inducer was EPI, the second was 5-FU, and the third was MIT.3. CRT expression on surface of B16 cells treated with different concentrations of three chemotherapeutants:①CRT expression in EPI group was significantly higher than that in the control group (P<0.001), and was significantly increased along with the increasing of EPI concentrations (P<0.001, respectively).②CRT expression was significantly increased along with the increasing of MIT concentrations, however, it was no longer increasing when the concentration reached 8×IC50. CRT expression in each concentration group was significantly higher than that of the control group (P<0.001), furthermore, a statistical difference was found among all the concentration groups (P<0.01, respectively), except the groups 4ug/ml and 8ug/ml (P=0.377>0.05).③CRT expression in 5-FU group was significantly higher than that in the control group (P<0.001), furthermore, a statistical difference was found among all the concentration groups (P<0.001, respectively), except the groups 200ug/ml and 400ug/ml (P=0.189>0.05).Conclusions:1. Previous studies suggest only anthracyclins, for example EPI and MIT can induce CRT exposure to stimulate the immunogenicity of tumor cell death. Present results indicated an increased CRT expression on surface of B16 cells induced by 5-FU. These findings implied that other cytotoxic drugs might also be functioned in inducing CRT exposure, which can stimulate the immunogenicity of tumor cell death. Further exploration of these specific drugs will benefit for improving anticancer immune response in cancer immunotherapy.2. CRT expression on cell surface was correlated with the concentrations of chemotherapeutants; however, low dose chemotherapy, which is strong enough to induce better CRT expression on cell surface, might be much valued clinically, though it should be further estimated in the animal experiments.Present study suggested that some chemotherapeutants, including anthracyclins and 5-FU, can effectively induce CRT exposure on cancer cell surface, although the definite mechanism is still a mystery. It has been verified in experiments that CRT exposure stimulated the APC to phagocytose apoptotic or necrotic tumor cells, and to induce anticancer immune response effectively. So that we can further explore an effective chemotherapy with eligible doses to induce CRT exposure of the cancer cells, which is possible to provide new evidence and valuable reference for more effective antitumor immunotherapy.