Immunization with a recombinant GnRH vaccine fused to heat shock protein 65 inhibits mammary tumor growth in vivo

Abstract

Gonadotrophin-releasing hormone (GnRH) is the prime decapeptide hormone in the regulation of mammalian reproduction. Active immunization against GnRH has been a good treatment option to fight against hormone-dependent disease such as breast cancer. We designed and purified a novel protein vaccine Hsp65-GnRH(6) containing heat shock protein 65 (Hsp65) and six copies of GnRH in linear alignment. Immunization with Hsp65-GnRH(6) evoked strong humoral response in female mice. The generation of specific anti-GnRH antibodies was detected by ELISA and verified by western blot. In addition, anti-GnRH antibodies effectively neutralized endogenous GnRH activity in vivo, as demonstrated by the degeneration of the ovaries and uteri in the vaccinated mice. Moreover, the growth of EMT-6 mammary tumor allografts was inhibited by anti-GnRH antibodies. Histological examinations have shown that there was increased focal necrosis in tumors. Taken together, our results showed that immunization with Hsp65-GnRH(6) elicited high titer of specific anti-GnRH antibodies and further led to atrophy of reproductive organs. The specific antibodies could inhibit the growth of EMT-6 murine mammary tumor probably via an indirect mechanism that includes the depletion of estrogen. In view of these results, the protein vaccine Hsp65-GnRH(6) appears to be a promising candidate vaccine for hormone-dependent cancer therapy.

Fig. 1

Expression and purification of the fusion protein Hsp65–GnRH₆. a Schematic diagrams of the construction process of the expression plasmid pETHG6. Upper the Hsp65 (represented by open box) gene was placed under the control of T7lac promoter (represented by cross box); middle DNA fragment encoding GnRH3-hinge-MVP was ligated to Hsp65 gene through the restriction enzymes NheI and HindIII; lower the fragment hinge-MVP (represented by bias box) was substituted by another DNA fragment encoding three copies of GnRH (represented by dark box). The resulting plasmid was designated as pETHG₆. b Expression level of the fusion protein Hsp65–GnRH₆. Total cell proteins were analyzed on a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250. Lane 1 marker proteins with molecular masses in kilodaltons indicated at left margin; lanes 2–9 total cell proteins from E. coli BL21 with plasmid pETHG₆ after induction 0, 1, 2, 3, 4, 5, 6 and 7 h, respectively. c SDS-PAGE analysis of partially purified HSP65–GnRH₆ by ammonium sulfate precipitation. Lane 1 marker proteins; lanes 2, 3 total cell proteins from E. coli BL21 with plasmid pETHG₆ after induction 7 h; lanes 4–8 the pellets precipitated by 20, 25, 30, 35 and 40% saturated ammonium sulfate, respectively. d SDS-PAGE analysis of purified HSP65–GnRH₆. Lane 1 marker proteins; lane 2 the flow-through of DEAE column; lane 3 partially purified HSP65–GnRH₆ precipitated by 40% saturated ammonium sulfate; lane 4 purified HSP65–GnRH₆ by further DEAE anion exchange chromatography

Fig. 2

Assay of specific anti-GnRH antibodies. a ELISA results of anti-GnRH antibodies in sera of the mice immunized by subcutaneous injection (n = 8, mean ± SD). Balb/c male mice were immunized four times at biweekly intervals. Sera samples were collected biweekly after initial immunization and subjected to ELISA (PBS dark bar; Hsp65 gray bar; Hsp65–GnRH₆ empty bar). b Endpoint titer of anti-GnRH antibody. Endpoint titers were represented as the reciprocal log2 of the last sample dilution giving an OD₄₅₀ value above 0.1. c Western blot analysis of anti-GnRH antibodies. Antibodies from the mice reacted with VEGF–GnRH rather than VEGF. Lane 1 prestained protein marker; lane 2 VEGF with DTT; lane 3 VEGF–GnRH with DTT; lane 4 VEGF without DTT; lane 5 VEGF–GnRH without DTT

Fig. 3

Effect of anti-GnRH antibodies on the reproductive organs. The morphology of ovaries and uteri of mice immunized with PBS (a), Hsp65 (b) or Hsp65–GnRH₆ (c). d Comparsion of the total weights of ovaries and uteri immunized with PBS, Hsp65 or Hsp65–GnRH₆. e–j The microscopic observation of H&E-stained sections of ovaries and uteri from mice treated with PBS (e, f), Hsp65 (g, h) and Hsp65–GnRH₆ (i, j)

Fig. 4

Effect of anti-GnRH antibodies on tumor growth. Solid tumors from mice were aligned and their photos were taken. a PBS; b Hsp65 and c Hsp65–GnRH₆. d The wet weights of tumor of mice treated with PBS, Hsp65 or Hsp65–GnRH₆ e–g The microscopic observation of H&E-stained sections of tumor from mice treated with PBS (e), Hsp65 (f) and Hsp65–GnRH₆ (g)