Induced Expression Of HSP70 On C6 Cells And The Anti-tumor Effect Of HSP70 Enriched Tumor Cell Vaccine In Vitro

Objective To establish the optimum conditions for inducing the expression of membrane protein HSP70 of rat C6 glioblastoma cells whereby to study the anti-tumor effect and mechanisms of the SD rat splenic cells vaccinated with MMC inactivated, HSP70 enriched C6 cells in vitro.Methods Flow cytometry was applied to determine the kinetic expression of membrane HSP70 of rat C6 glioblastoma cells under different heat shock conditions and/or the treatment with tumoricidal agents 3 -elemene, Kang-Lai-Te injection or mitomycin C. The survival of C6 cells was determined by MTT assay. Mitomycin C-inactivated C6 cells withenriched membrane HSP70 obtained under optimum heat shock conditions thus established were used as the stimulants for their co-incubation with rat splenic cells. The resulting effector cells were incubated with either heat-shock treated C6 cells or their wild-type originals. The cell death was evaluated by MTT assay. Apoptosis of the target cells was analyzed by flowcytometry and electronic microscopy.Results The cell survival rate were not significantly changeable subjected to heat-shock treatment at 42 or 43 "C within 12 hours.The expression rate of membrane HSP70 on C6 cells at 43 for 1, 2, 4, 6, 8, 10, 12, 14, 16, 20, 24 hours were 17.57%, 28.19%, 37.35%, 61.38%, 73.19%, 88.61%, 89.15%, 70.18%, 49.00%, 40.17% and 38.85%, respectively. These expression percentages were significantly higher than those detected in control group (4.72% in average, P < 0.05). The expression rate of membrane HSP70 on C6 cells with P -elemene and heat shock treated at 43 for 2h in combination was highest than those with other drugs and/or heat shock treated. Heat shock treatment at 43 for 12h was observed as the optimum condition and the cells thus treated were used to stimulate the generation of splenic effector cells. In vitro, the specific cytotoxicity of effector cells elicited by HSP70-rich C6 cells were shown to be significantly higher than that of fresh splenic cells. On the other hand, heat-treated C6 cells, as compared with their wild-type originals, were more sensitive to the effector cell killing. The peak level of cytotoxicity of splenocytes stimulated by heat-treated C6 cells(12 hrs,43) for 6 days against the same heat-treated C6 cells reached 99.25%. This figure was higher than those seen in the killing of wild type cells as the targets (63.10%). The evidence of HSP70 as the stimulating molecule was given by an antibody blocking experiment: when C6 cells were blocked with anti-HSP70 McAb, no enhancement of thecytotoxicity was induced. Further analysis using FCM revealed that apoptosis of target cells was the key form of the cell killing resulted from HSP70 stimulation. The rate of apoptotic heat-shock type and wild-type C6 cells reached 52.07 and 27.15%, respectively, when the splenocytes were stimulated by heat-treated C6 cells for 6 days. No significant apoptosis were observed when fresh splenocytes were used as the effector cells.Conclusions Proper heat shock treatment and/or some drugs can increase the expression of HSP70 on C6 cells and hence improve the immunogenicity of the tumor cells. The anti-tumor effect of the splenic cells stimulated with MMC inactived, heat-shocked C6 cells can be greatly enhanced possibly by the apoptosis-mediated mechinasm.