| Objective: To investigate the synergistic anti-breast cancer effect of Toll-like receptor 7 agonist T7-ethacrynic acid conjugation(T7-EA) in combination with receptor-tyrosine-kinase-like orphan receptor 1(ROR1).Methods: Syfpeithi epitope prediction software was employed to predict the ROR1 cytotoxic T lymphocyte(CTL) epitope. Mouse Spleen lymphocytes were stimulated with different concentration of T7-EA. ELISA assay was used to measure the levels of interferon-γ(IFN-γ) and interleukin 12(IL-12). Mouse spleen lymphocytes and bone marrow dendritic cells(DC) were stimulated with 4 μmol/L T7-EA, 4 μmol/L ROR1 or 4 μmol/L T7-EA combined with ROR1, respectively. ELISA assay was used to measure the levels of IFN-γ, IL-12 and tumor necrosis factor-α(TNF-α). Xenograft model was established via subcutaneously injection of mouse breast cancer 4T1 cells. Mice were treated weekly through intraperitioneally administration of 3 mg/kg T7-EA, 15 mg/kg ROR1 or combination of T7-EA and ROR1, respectively. After four round of treatment, tumor tissues were analyzed. Specific CTL activities was detected by lactate dehydrogenase(LDH) assay. Serum level of anti-4T1 tumor protein IgG was measured by ELISA.Results: The peptide PYCDETSSV was chosen as an antigen epitope of breast cancer. The T7-EA highly activated in vitro lymphocytes in a dose-dependent manner, which wasn’t affected by other relavant peptides. Combination of T7-EA and ROR1 stimulated the secretions of IFN-γ and IL-12 by lymphocytes and TNF-α by bone marrow DCs. The growth of tumor was significantly inhibited in T7-EA+ROR1 than that in T7-EA or ROR1. The specific CTL activity triggered by T7-EA+ROR1 was much stronger than that triggered by T7-EA or ROR1. The titer of anti-4T1 tumor protein IgG induced by T7-EA+ROR1 was higher than that induced by T7-EA or ROR1.Conclusion: The combination of T7-EA and ROR1 has a highly detrimental effect to anti-breast cancer. |
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