Oleandrin Induces Immunogenic Cell Death Via the PERK/elF2α/ATF4/CHOP Pathway in Breast Cancer

Abstract

Background Chemotherapeutic agents have been linked to immunogenic cell death (ICD) induction that is capable of augmenting antitumor immune surveillance. The cardiac glycoside oleandrin, which inhibits Na+/K+-ATPase pump (NKP), has been shown to suppress breast cancer growth via inducing apoptosis. However, the implications of oleandrin in antitumor immune response and potential ICD induction remain unexplored till now, which is investigated in the present study.Methods Calreticulin (CRT) exposure was detected by immunofluorescence and flow cytometry, and high mobility group protein B1 (HMGB1) and Adenosine Triphosphate (ATP) secretion was quantified by ELISA and both the intracellular and extracellular expression of Heat shock protein 70/90 (HSP70/90) was detected by western blotting in breast cancer cells treated with oleandrin. Dendritic cells (DCs) were co-cultured with oleandrin-treated breast cancer cells before the expressions of activation markers and cytokines were examined by quantitative real time polymerase chain reaction (qRT-PCR), ELISA, and flow cytometry. Immune activation effects of oleandrin were determined in murine breast cancer model using BALB/C mice. The differential mRNA expression incurred by oleandrin was investigated by mRNA sequencing and subsequently confirmed by qRT-PCR and Western blotting. Results Oleandrin treatment induced CRT exposure on cell surface and the release of HMGB1, HSP70/90 and ATP. The maturation and activation of DCs were increased by co-culturing with oleandrin-treated cancer cells, which subsequently enhanced CD8+ T cell cytotoxicity. In animal models, oleandrin inhibited tumor growth and increased tumor infiltrating lymphocytes including DCs and T cells. Mechanistically, oleandrin induced endoplasmic reticulum (ER) stress associated caspase independent immunogenic cell death (ICD) mainly through PERK/elF2α/ATF4/CHOP pathway. Activation of IRE1 pathway but not ATF6, the other two canonical sensors of ER stress, was also observed. Conclusion Oleandrin triggered ER stress and induced ICD-mediated immune destruction of breast cancer cells. Oleandrin combined with immune checkpoint inhibitors might improve the efficacy of immunotherapy.

Mechanisms providing effects of cardiotonic steroids in mammalian cells with special attention to blood cells

“…It was shown by Li et. al [113] that after administration of oleandrin to BALB/c mice with engrafted murine breast cancer cell line EMT6 and tumor-bearing mice intraperitoneally (0.3 mg/kg and 0.6 mg/kg) every day tumor growth was suppressed and the amount of tumor-infiltrating lymphocytes including dendritic cells and CD8+ T-cells were increased. In another study it was shown that ouabain (0.56 mg/kg with 140 mg/kg sodium succinate of hydrocortisone ) injected intraperitonially induced the death of immature double positive lymphocytes (CD4+CD8+) whereas CD69+ cells survived after both treatments [114].…”

“…Cellular and systemic effects of cardiotonic steroids on different blood cells. Inhibition of the growth of melanoma tumors in murine model[115] (2) Reversed the inability of Cisplatin to trigger calreticulin exposure, and HPMA copolymer-amplified Cisplatininduced ATP release in melanoma mice model[116] Oleandrin(1) Inhibite tumor growth and increase tumor-infiltrating lymphocytes including dendritic cells and T cells[113] B cells OBN (1) Decrease the level of B-cells in bone marrow, spleen and peripheral blood in 24 hours Immunobiology [117]. …”