Dataset Information

G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells.

ABSTRACT: BACKGROUND:DNA G-quadruplex (G4) structures represent potential anti-cancer targets. In this study, we compared the effect of two G4-targeting compounds, C066-3108 and the gold standard BRACO-19. METHODS:In breast and prostate cancer cells, cytotoxicity induced by both molecules was measured by a sulforhodamine B assay. In breast cancer cells, cycle, apoptosis, the formation of G4 structures, calreticulin and high mobility group box 1 (HMGB1), as well as T cell activation, were analyzed by flow cytometry and adenosine triphosphate (ATP) by luminescence. RESULTS:Both ligands inhibited cell survival and induced DNA damage. In MCF-7 cells, G4 ligands increased the subG0/G1 phase of the cell cycle inducing apoptosis and reduced intracellular ATP. In untreated MCF-7 cells, we observed a slight presence of G4 structures associated with the G2/M phase. In MDA-MB231 cells, G4 ligands decreased the G1 and enhanced the G2/M phase. We observed a decrease of intracellular ATP, calreticulin cell surface exposure and an increase of HMGB1, accompanied by T cell activation. Both compounds induced G4 structure formation in the subG0/G1 phase. CONCLUSIONS:Our data report similar effects for both compounds and the first evidence that G4 ligands induce the release of danger signals associated with immunogenic cell death and induction of T cell activation.

SUBMITTER: Di Somma S

PROVIDER: S-EPMC6895816 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells.

Di Somma Sarah S Amato Jussara J Iaccarino Nunzia N Pagano Bruno B Randazzo Antonio A Portella Giuseppe G Malfitano Anna Maria AM

Cancers 20191115 11

<h4>Background</h4>DNA G-quadruplex (G4) structures represent potential anti-cancer targets. In this study, we compared the effect of two G4-targeting compounds, C066-3108 and the gold standard BRACO-19.<h4>Methods</h4>In breast and prostate cancer cells, cytotoxicity induced by both molecules was measured by a sulforhodamine B assay. In breast cancer cells, cycle, apoptosis, the formation of G4 structures, calreticulin and high mobility group box 1 (HMGB1), as well as T cell activation, were an ...[more]

PMID: 31731707

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Project description:Cordyceps militaris has been widely used as a traditional medicine in East Asia. Its effects against breast cancer have been reported previously. However, whether C. militaris-induced breast cancer cell death is immunogenic remains unelucidated. This study aimed to determine whether ethanolic extracts of C. militaris (CM-EE) could induce immunogenic cell death (ICD) in breast cancer immunotherapy to improve the efficacy of immune checkpoint inhibitors. Human and mouse breast cancer cells were treated with various concentrations of CM-EE for 72 h, and cytotoxicity was measured using the sulforhodamine B assay. Flow cytometry was used to assess cell death with annexin V/7-AAD staining and measure the surface exposure of damage-associated molecular pattern (DAMP) molecules including calreticulin, HSP70, and HSP90. Western blot for cleaved poly (ADP-ribose) polymerase (PARP) was used to confirm apoptotic cell death. The immunogenicity of CM-EE-induced dead cells was evaluated using the CFSE dilution assay. CM-EE reduced the viability of human (MCF7, MDA-MB-231, HS578T, and SKBR3) and mouse (4T1-neu-HA, TUBO-HA, and TUBO-P2J-HA) breast cancer cells. The IC50 was 25-50 µg/ml in human breast cancer cells and 10-50 µg/ml in mouse breast cancer cells at 72 h. CM-EE-treated breast cancer cells were positively stained by annexin V, cleaved PARP, and cleaved caspase 3/7 which were increased upon CM-EE treatment. Surface exposure of DAMP molecules was increased in dose- and time-dependent manners. The CFSE dilution assay revealed that dendritic cells fed with CM-EE-treated breast cancer cells successfully stimulated tumor-specific T cell proliferation without inhibiting DC function and T cell proliferation. The expression of PD-L1 mRNA and protein level was increased in dose-dependent manners. In addition, CM-EE also potentiated the cytotoxic activity of tumor-specific T cells. CM-EE can induce immunogenic and apoptotic cell death in breast cancer cells, and it is a good candidate for cancer immunotherapy and may improve the efficacy of immune checkpoint inhibitors.

Project description:Immunogenic cell death (ICD) is clinically relevant because cytotoxicants that kill malignant cells via ICD elicit anticancer immune responses that prolong the effects of chemotherapies beyond treatment discontinuation. ICD is characterized by a series of stereotyped changes that increase the immunogenicity of dying cells: exposure of calreticulin on the cell surface, release of ATP and high mobility group box 1 protein, as well as a type I interferon response. Here, we examined the possibility that inhibition of an oncogenic kinase, anaplastic lymphoma kinase (ALK), might trigger ICD in anaplastic large cell lymphoma (ALCL) in which ALK is activated due to a chromosomal translocation. Multiple lines of evidence plead in favor of specific ICD-inducing effects of crizotinib and ceritinib in ALK-dependent ALCL: (i) they induce ICD stigmata at pharmacologically relevant, low concentrations; (ii) can be mimicked in their ICD-inducing effects by ALK knockdown; (iii) lose their effects in the context of resistance-conferring ALK mutants; (iv) ICD-inducing effects are mimicked by inhibition of the signal transduction pathways operating downstream of ALK. When ceritinib-treated murine ALK-expressing ALCL cells were inoculated into the left flank of immunocompetent syngeneic mice, they induced an immune response that slowed down the growth of live ALCL cells implanted in the right flank. Although ceritinib induced a transient shrinkage of tumors in lymphoma-bearing mice, irrespective of their immunocompetence, relapses occurred more frequently in the context of immunodeficiency, reducing the effects of ceritinib on survival by approximately 50%. Complete cure only occurred in immunocompetent mice and conferred protection to rechallenge with the same ALK-expressing lymphoma but not with another unrelated lymphoma. Moreover, immunotherapy with PD-1 blockade tended to increase cure rates. Altogether, these results support the contention that specific ALK inhibition stimulates the immune system by inducing ICD in ALK-positive ALCL.

Dataset Information

G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells.

Publications

G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells.

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