Wogonin Induced Calreticulin/Annexin A1 Exposure Dictates the Immunogenicity of Cancer Cells in a PERK/AKT Dependent Manner

Abstract

In response to ionizing irradiation and certain chemotherapeutic agents, dying tumor cells elicit a potent anticancer immune response. However, the potential effect of wogonin (5,7-dihydroxy-8-methoxyflavone) on cancer immunogenicity has not been studied. Here we demonstrated for the first time that wogonin elicits a potent antitumor immunity effect by inducing the translocation of calreticulin (CRT) and Annexin A1 to cell plasma membrane as well as the release of high-mobility group protein 1 (HMGB1) and ATP. Signal pathways involved in this process were studied. We found that wogonin-induced reactive oxygen species (ROS) production causes an endoplasmic reticulum (ER) stress response, including the phosphorylation of PERK (PKR-like endoplasmic reticulum kinase)/PKR (protein kinase R) and eIF2α (eukaryotic initiation factor 2α), which served as upstream signal for the activation of phosphoinositide 3-kinase (PI3K)/AKT, inducing calreticulin (CRT)/Annexin A1 cell membrane translocation. P22/CHP, a Ca2+-binding protein, was associated with CRT and was required for CRT translocation to cell membrane. The releases of HMGB1 and ATP from wogonin treated MFC cells, alone or together with other possible factors, activated dendritic cells and induced cytokine releases. In vivo study confirmed that immunization with wogonin-pretreated tumor cells vaccination significantly inhibited homoplastic grafted gastric tumor growth in mice and a possible inflammatory response was involved. In conclusion, the activation of PI3K pathway elicited by ER stress induced CRT/Annexin A1 translocation (“eat me” signal) and HMGB1 release, mediating wogonin-induced immunity of tumor cell vaccine. This indicated that wogonin is a novel effective candidate of immunotherapy against gastric tumor.

Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8

“…1 Nonetheless, at least some anticancer agents display an optimal efficacy in the presence of an intact immune system, but not when mice lack T cells, dendritic cells (DCs), several other effectors of innate and adaptive immunity or when they are treated with a CD11b-specific antibody that blocks the extravasation of DC precursors. [2][3][4][5][6] These results point to a hitherto poorly understood role for anticancer immune responses in determining the long-term success of chemotherapy, a notion that is in line with abundant clinical data indicating that tumor infiltration by cytotoxic T lymphocytes influences disease outcome. [7][8][9][10] Cancer cells respond to some chemotherapeutics such as anthracyclines and oxaliplatin by undergoing an immunogenic form of cell death, meaning that such dying cells become able to induce a potent cellular immune response on subcutaneous injection into immunocompetent mice.…”

Consensus guidelines for the detection of immunogenic cell death

“…Several assays are currently available for the detection of the different arms of the ER stress response. 136,[254][255][256] For instance, the phosphorylation state of EIF2A and/or of the major EIF2A kinases, including EIF2A kinase 1 (EIF2AK1, best known as HRI), 257 EIF2AK2 (best known as PKR), 258 and EIF2AK3 (best known as PERK), [259][260][261] can be assessed by immunoblotting, flow cytometry or immunofluorescence microscopy with phosphoneoepitope-specific antibodies. 260 The splicing status of X-box binding protein 1 (XBP1) mRNA, reflecting the activation of the ER stress sensor endoplasmic reticulum to nucleus signaling 1 (ERN1, best known as IRE1a), can be monitored by quantitative real-time RT-PCR, 262 as well as by flow cytometry or fluorescence microscopy, either in cells that express a fluorescently-tagged version of XBP1 263 or upon the administration of a self-quenched RNA probe that can be cleaved by IRE1a.…”

Danger signalling during cancer cell death: origins, plasticity and regulation

“…How exactly these two signalling components should operate to efficiently induce ICD is still elusive. ER stress could be secondary to mitochondria dysfunction and ROS production ((i.e., cardiac glycosides, UVC, wogonin 1,12,23,24 ) or to the local ROS generation at the ER ((i.e., hypericin-based photodynamic therapy (Hyp-PDT) 7,11,25 ) and be propagated by lipid peroxidation signalling ((i.e., doxorubicin, mitoxantrone, ionizing irradiation, Hyp-PDT 7,11,12 ) or be secondary to the activation of the unfolded protein response ((i.e., shikonin, bortezomib and CVB3 virus 12 ).…”