The E7 oncoprotein of human papillomavirus (HPV) is an ideal target for developing immunotherapeutic strategies against HPV-associated tumors. However, because protein-based immunogens alone are poor elicitors of the cytotoxic T-lymphocyte (CTL) responses, they have been difficult to exploit for therapeutic purposes. In this study, we report that a recombinant lipoprotein consisting of inactive E7 (E7m) biologically linked to a bacterial lipid moiety (rlipo-E7m) induces the maturation of mouse bone marrow-derived dendritic cells through toll-like receptor 2 (TLR2), skews the immune responses toward the Th1 responses and induces E7-specific CTL responses. We further studied the ability of rlipo-E7m to provide protection against a TC-1 tumor cell challenge in an animal model. Mice prophylactically immunized with two 10-µg doses of rlipo-E7m were found to be free of TC-1 tumor growth. Experiments in a therapeutic immunization model showed that the tumor volume in mice receiving a single dose of rlipo-E7m was less than 0.01 cm
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on day 40, whereas the tumor volume in mice treated with rE7m was 2.28±1.21 cm
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. The tumor volume of the entire control group was over 3 cm
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. In addition, we demonstrated that the CD8+ T cells play a major role in anti-tumor immunity when administration of rlipo-E7m. These results demonstrate that rlipo-E7m could be a promising candidate for treating HPV-associated tumors.
BackgroundAlthough cytotoxic T lymphocytes (CTLs) play a major role in eradicating cancer cells during immunotherapy, the cancer-associated immunosuppressive microenvironment often limits the success of such therapies. Therefore, the simultaneous induction of cancer-specific CTLs and reversal of the immunosuppressive tumor microenvironment may be more effectively achieved through a single therapeutic vaccine. A recombinant lipoprotein with intrinsic Toll-like receptor 2 (TLR2) agonist activity containing a mutant form of E7 (E7m) and a bacterial lipid moiety (rlipo-E7m) has been demonstrated to induce robust CTL responses against small tumors. This treatment in combination with other TLR agonists is able to eliminate large tumors.MethodsMouse bone marrow-derived dendritic cells (DCs) were employed to determine the synergistic production of pro-inflammatory cytokines upon combination of rlipo-E7m and other TLR agonists. Antigen-specific CTL responses were investigated using immunospots or in vivo cytolytic assays after immunization in mice. Mice bearing various tumor sizes were used to evaluate the anti-tumor effects of the formulation. Specific subpopulations of immunosuppressive cells in the tumor infiltrate were quantitatively determined by flow cytometry.ResultsWe demonstrate that a TLR9 agonist (unmethylated CpG oligodeoxynucleotide, CpG ODN) enhances CTL responses and eradicates large tumors when combined with rlipo-E7m. Moreover, combined treatment with rlipo-E7m and CpG ODN effectively increases tumor infiltration by CTLs and reduces the numbers of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment.ConclusionThese findings suggest that the dramatic anti-tumor effects of the recombinant lipoprotein together with CpG ODN may reflect the amplification of CTL responses and the repression of the immunosuppressive environment. This promising approach could be applied for the development of additional therapeutic cancer vaccines.
β-amyloid (Aβ)-mediated neuronal apoptosis contributes to the pathogenesis of Alzheimer's disease (AD). This study aimed to investigate whether astragalosides (AST) could inhibit Aβ-induced apoptosis in vivo and in vitro and to explore the underlying mechanisms. Amyloid β-protein fragment 25-35 (Aβ) was administered to cerebral lateral ventricle of rats to make the AD models in vivo. AST was able to attenuate both cortical cell degeneration and memory deficits in the AD rats. AST also inhibited Aβ-induced cytotoxicity (e.g., decreased cell viability); apoptosis (e.g., increased caspase-3 expression, increased DNA fragmentation, and Tau hyperphosphorylation); synaptotoxicity (e.g., increased loss of both a dendritic marker, microtubule-associated protein 2 (MAP-2) and synaptic proteins, synaptophysins); and mitochondrial dysfunction (e.g., increased mitochondrial membrane potential) in cultured primary rat cortical cells. The beneficial effect of AST in reducing Aβ-induced cytotoxicity, apoptosis, and mitochondrial dysfunction in cortical cells were blocked by inhibition of phosphoinositide 3-kinase (PI3K)-dependent protein kinase B (PKB, as known as AKT) activation with LY294002. In addition, inhibition of extracellular protein kinase (ERK) with U0126 shared with the AST the same beneficial effects in reducing Aβ-induced apoptosis. Our data suggest that the cortical PI3K/AKT and MAPK (or ERK) pathways as appealing therapeutic targets in treating AD, and AST may have a positive impact on AD treatment via modulation of both PI3K/AKT and ERK pathways.
Objective. To explore the ability of asiatic acid to interfere with the invasion and proliferation of breast cancer cells by inhibiting WAVE3 expression and activation through the PI3K/AKT signaling pathway. Methods. The MDA-MB-231 cells with strong invasiveness were screened by transwell assay, and plasmids with high expression of WAVE3 were constructed for transfection. The transfection effect and protein expression level of plasmids were verified by PCR and WB. The effects of asiatic acid on cell proliferation and invasion were investigated by flow cytometry. The xenografted tumor models in nude mice were established to study the antitumor activity of asiatic acid. Results. Asiatic acid significantly inhibited the activity of MDA-MB-231 cells, and the expression level of WAVE3 increased significantly in the tissue of ductal carcinoma in situ and was lower than that in the metastasis group. After plasmid transfection, the mRNA and protein expression of WAVE3 increased significantly in the cells. Asiatic acid at different concentrations had an impact on cell apoptosis and invasion and could significantly inhibit the expression of WAVE3, P53, p-PI3K, p-AKT, and other proteins. The T/C(%) of asiatic acid (50 mg/kg) for MDA-MB-231(F10) xenografted tumor in nude mice was 46.33%, with a tumor inhibition rate of 59.55%. Asiatic acid could significantly inhibit the growth of MDA-MB-231 (F10) xenografted tumors in nude mice (p<0.05). Conclusions. Asiatic acid interferes with the ability of breast cancer cells to invade and proliferate by inhibiting WAVE3 expression and activation and the mechanism of action may be related to the PI3K/AKT signaling pathway.
Flagellin has the capacity to activate both Toll-like receptor 5 (TLR5) and Nod-like receptor C4 (NLRC4)/neuronal apoptosis inhibitory protein 5 (NAIP5) inflammasome signaling. We fused E7m (the inactivated E7 of human papillomavirus) to either end of the flagellin protein, and the resulting recombinant flagellin-E7m proteins (rFliCE7m and rE7mFliC) were used as immunogens. Both fusion proteins activated receptor signaling to different degrees. rE7mFliC-induced TLR5 activity was 10-fold higher than that of rFliCE7m, whereas rFliCE7m activated the NLRC4/NAIP5 pathway more strongly. Therefore, these recombinant proteins provided a tool to investigate which signaling pathway is critical for the induction of antigen-specific T cell responses and anti-tumor immunity. We demonstrated that rFliCE7m induced higher levels of E7-specific IFN-gamma-secreting cells and cytotoxic T lymphocytes (CTLs) than rE7mFliC, and a single injection with rFliCE7m but not rE7mFliC inhibited E7-expressing tumor growth in vivo. Furthermore, we confirmed that CD8+ T cells played a major role in the anti-tumor immunity induced by rFliCE7m. These findings suggested that the NLRC4/NAIP5 intracellular signaling pathway was critical for the induction of anti-tumor immunity. These observations provide important information for the rational design of flagellin-based immunotherapy.
The goal of therapeutic HPV vaccines is the induction of cytotoxic T lymphocyte immunity against HPV-associated cancers. Recombinant proteins and synthetic peptides have high safety profiles but low immunogenicity, which limits their efficacy when used in a vaccine. Self-adjuvanting lipid moieties have been conjugated to synthetic peptides or expressed as lipoproteins to enhance the immunogenicity of vaccine candidates. Mono-, di- and tri-palmitoylated peptides have been demonstrated to activate dendritic cells and induce robust cellular immunity against infectious diseases and cancer. Recently, a platform technology using the high-yield production of recombinant lipoproteins with Toll-like receptor 2 agonist activity was established for the development of novel subunit vaccines. This technology represents a novel strategy for the development of therapeutic HPV vaccines. In this review, we describe recent progress in the design of therapeutic HPV vaccines using lipoimmunogens.
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