Preliminary Evaluation Of Active Immunization With Recombinant Human HMGB1 Protein On Breast Cancer In Mice

Background:HMGB1 is a highly conserved non-histone nucleosomal protein in mammalian cells, which has been shown to be involved in DNA repair, recombination, replication, and transcription. During the development of tumors, HMGB1 could be released actively or passively into the extracellular milieu. Recent studies demonstrated that extracellular HMGB1 promotes the proliferation, invasion and metastasis of malignant tumors via binding to its corresponding receptors such as TLRs or RAGE on the target cells, while HMGB 1 antagonist could inhibit the growth of tumors. Based on these findings, we hypothesized that active immunization using HMGB1 as immunogen may influence the proliferation or metastasis of tumor. In the present study, we used a vaccine prepared with purified recombinant human HMGB1 protein to inoculate mice and evaluate the growth of transplanted breast cancer on the immunized mice.Method:First, a previously constructed plasmid pET28a-HMGB1 containing human HMGB1 coding region was transformed into Escherichia coli BL21; the expression of HMGB1 was induced with lactose and identified by SDS-PAGE and Western blotting. The recombinant HMGB1 protein was purified by Nickel column affinity chromatography. Next, the purified HMGB1 was mixed with freund’s adjuvant to prepare vaccine, and then female BALB/c mice were immunized by subcutaneous injection with the vaccine on the back of which at different points; equal volumes of PBS were given to the control mice similarly. Anti-HMGB1 antibody titer in serum of mice was detected by indirect ELISA. After the titer of Anti-HMGB1 antibody reaches to a steady state, mice were transplanted subcutaneously with a certain density of 4T1 breast cancer cells to establish transplantation tumor model. The relative tumor volumes and relative tumor growth rate were determined by calculating the longest diameter and shortest diameter of the tumor with vernier caliper at a two days interval. The levels of HMGB 1 in the serum of mice were measured by double antibody sandwich ELISA assay. The effects of active immunization with HMGB1 on the growth of transplanted breast tumor were assessed by comparison of the relative tumor volume, tumor weight, histology and anti-tumor rate between the immunized mice and the control mice.Results:Recombinant human HMGB1 protein was successfully purified as evidenced by SDS-PAGE and Western Blotting examinations. After immunization, anti-HMGB1 antibody was detectable in the serum of HMGB1 immunized mice, and the antibody titer became elevated with the enhancement of immunization; after the sixth immunization was performed, the antibody titer came to a steady state. By comparison, the anti-HMGB1 antibody in the serum of control mice was undetectable. When 4T1 breast cancer cells were inoculated to mice, visible tumor masses were observed at the inoculation sites both in HMGB1 immunized mice and control mice, and the tumor sizes increased as time pass by. In comparison, the tumors in HMGB1 immunized mice grow faster than that in the control mice; the average tumor weights were 2.337±0.3122g and 1.997±0.2401g in immunized mice and control mice, respectively. The anti-tumor rate of immunized mice was-17%. Results of double antibody sandwich ELISA assay showed that HMGB1 levels in the sera of tumor-bearing immunized mice were much lower than that in the control mice.Conclusion:The prepared vaccine with recombinant HMGB1 protein could effectively provoke humoral immune responses in BALB/c mice. Active immunization with HMGB1 protein could significantly reduce the HMGB1 level in the serum of tumor-bearing mouse but has no influences on the growth of tumor. Our results suggested that inhibitory strategy targeting HMGB 1 may be futile in treatment of malignant tumors; in addition, the relationship between HMGB1 and cancers needs further investigations.