Tumor cell death visualization of renal cell carcinoma under the combined effect of the Gratiola officinalis extract and cyclophosphamide using fluorescent staining methods

Objective of the study: We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavonoid-containing extract of Gratiola officinalis and cytostatic (cyclophosphamide). Materials and methods: The dyes were propidium iodide and acridine orange, which were used in the “alive and dead” test. This test helped us to identify the total number of dead cells in the forms of necrosis and apoptosis and the number of cells in which apoptosis had started, it was characterized by the appearance of apoptotic bodies or nucleus pyknosis. Results: We found the most pronounced cytotoxic activity at the ratio of extract of Gratiola officinalis and cyclophosphamide concentrations of 1:1. The number of living cells decreased when exposed to the ratio of extract and cytostatic concentrations of 2:1. When the ratio of concentration of the extract relative to the cytostatic increased to 3:1, the cytostatic activity of the extract began to appear, the total number of tumor cells decreased. The number of cells with nucleus pyknosis and the number of cells with apoptosis signs significantly increased at a 3:1 ratio of extract and cytostatic concentrations, which confirms the presence of pro-apoptotic activity of the studied combination. This trend indicates the dependence of a certain form of cell death (apoptosis, necrosis) on the ratio of extract and cytostatic doses, and it also demonstrates the cytostatic and cytotoxic effects of this combination. Conclusion: Fluorescence methods of investigation in the “alive and dead” test allowed us to visualize the forms of cell death of human kidney carcinoma A498 by combined exposure to the flavonoid-containing extract of Gratiola officinalis and cytostatic (cyclophosphamide) 24 h after exposure. We found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect.

Download Full-text

Evaluation of the cytotoxic potential of extracts from the genus Passiflora cultived in Brazil against cancer cells

10.1101/337253 ◽

2018 ◽

Author(s):

Ricardo Guimarães Amaral ◽

Silvana Vieira Floresta Gomes ◽

Ângelo Roberto Antoniolli ◽

Maria Claudia dos Santos Luciano ◽

Cláudia do Ó Pessoa ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Cytotoxic Activity ◽

Cancer Cells ◽

Tumor Cells ◽

Cytotoxic Potential ◽

Leaf Extracts ◽

High Cytotoxic Activity ◽

Apoptosis And Necrosis

AbstractThis work aimed to evaluate the cytotoxic potential against cancer cells of Passiflora genus plant species cultivated in Brazil and identify the mechanism of cytotoxicity induced by the most promising extract. Leaf extracts from 14 Passiflora (P.) species were obtained ASE and in vitro cytotoxicity evaluated against cancer cell lines using MTT assay at a single concentration of 50 μg/mL. Additionally, the IC50 of the P. alata (ELPA) leaf extracts was determined against both tumor (HCT-116, SF-295, OVACAR-8, and HL-60), and non-tumor cells (PBMC). The ELPA flavonoids were identified by HPLC-DAD and UHPLC-MS/MS. The morphological analyses used light and fluorescence microscopy, and cell cycle and DNA fragmentation analyses used flow cytometry to determine the mechanism of cell death induced by ELPA in HL-60. Among the Passiflora leaf extracts evaluated; ELPA stood out with high cytotoxic activity, followed by P. capsularis and P. quadrangulares with varying high and low cytotoxic activity. ELPA presented high cytotoxic potency in HL-60 (IC50 19.37 μg/mL), yet without cytotoxic activity against PBMC, suggesting selectivity for tumor cells. The cytotoxic activity of ELPA may well be linked to the presence of ten identified flavonoids. Cells treated with ELPA presented the hallmarks typical of apoptosis and necrosis, with cell cycle arrest in the G2/M phase. Conclusion: From among the studied species, ELPA presented greater cytotoxic activity, possibly a consequence of synergistic flavonoid action which induces cell death by apoptosis and necrosis.

Download Full-text

Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death

Journal of Experimental Medicine ◽

10.1084/jem.20050915 ◽

2005 ◽

Vol 202 (12) ◽

pp. 1691-1701 ◽

Cited By ~ 730

Author(s):

Noelia Casares ◽

Marie O. Pequignot ◽

Antoine Tesniere ◽

François Ghiringhelli ◽

Stéphan Roux ◽

...

Keyword(s):

Immune Response ◽

Cell Death ◽

Tumor Cell ◽

Tumor Cells ◽

Tumor Cell Death ◽

Anticancer Chemotherapy ◽

Caspase Inhibition ◽

Induced Apoptosis

Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.

Download Full-text

Cellular cytotoxicity is a form of immunogenic cell death

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2019-000325 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000325 ◽

Cited By ~ 3

Author(s):

Luna Minute ◽

Alvaro Teijeira ◽

Alfonso R Sanchez-Paulete ◽

Maria C Ochoa ◽

Maite Alvarez ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

Dendritic Cells ◽

Tumor Cell ◽

Tumor Cells ◽

Cd8 T Cells ◽

Immunogenic Cell Death ◽

Cell Debris ◽

Tumor Cell Death ◽

Mhc Multimer

BackgroundThe immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or natural killer (NK) lymphocytes, is actually immunogenic currently remains unknown.MethodsIn this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated NK cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I major histocompatibility complex (MHC)-multimer stainings. Mice deficient inBatf3,Ifnar1andSting1were used to study mechanistic requirements.ResultsWe observe in cocultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. Ovalbumin (OVA)-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100-specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA+EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient inBatf3-dependent dendritic cells (conventional dendritic cells type 1, cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8+T lymphocytes.ConclusionThese results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death.

Download Full-text

Apoptosis and Tumor Cell Death in Response to HAMLET (Human α-Lactalbumin Made Lethal to Tumor Cells)

Advances in Experimental Medicine and Biology - Bioactive Components of Milk ◽

10.1007/978-0-387-74087-4_8 ◽

2007 ◽

pp. 217-240 ◽

Cited By ~ 40

Author(s):

Oskar Hallgren ◽

Sonja Aits ◽

Patrick Brest ◽

Lotta Gustafsson ◽

Ann-Kristin Mossberg ◽

...

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Tumor Cells ◽

Tumor Cell Death

Download Full-text

O8 Gemcitabine induces pro-apoptotic BH3 only proteins and sensitizes pancreatic ductal adenocarcinoma cells for RLH-triggered immunogenic cell death

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.13 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A7.1-A7

Author(s):

P Metzger ◽

HT Bourhis ◽

M Stieg ◽

D Böhmer ◽

S Endres ◽

...

Keyword(s):

Cell Death ◽

Mouse Model ◽

Tumor Cell ◽

Single Agent ◽

Ductal Adenocarcinoma ◽

Immunogenic Cell Death ◽

Tumor Cell Death ◽

Antigen Uptake ◽

Cross Presentation

BackgroundDespite tremendous effort, the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) remains poor and therapy options are limited. Recent advances in chemotherapeutic schemes have increased the survival of PDAC patients by a few months only. So far, the success of immunotherapy seen in other cancer types could not be transferred to PDAC. Our group has demonstrated that single agent RIG-I-like helicase (RLH)-targeting immunotherapy induces an anti-tumoral immune response and improves survival in a PDAC mouse model dependent on the induction of immunogenic cell death. In addition, we and others were able to show that tumor cell death induction by RLH ligands is partially dependent on the induction of the pro-apoptotic BH3-only proteins PUMA and NOXA. In the current study we aim at improving therapy response using a combinatorial chemo-immunotherapy (CIT) approach.MethodsTumor cell death induction by gemcitabine, oxaliplatin and 5-fluoruracil (5-FU) alone or in combination with RLH ligands was evaluated in the murine cell line Panc02. The induction of PUMA and NOXA was measured by real-time PCR. The capability of chemo-immunotherapy -induced tumor cell death to activate splenic CD8a+dendritic cells (DC) as well as to induce antigen uptake and cross-presentation was investigated in vitro. Therapeutic efficacy was evaluated in vivo using an orthotopic PDAC mouse model.ResultsGemcitabine, oxaliplatin and 5-FU induced dose-dependent tumor cell death in vitro. however, only gemcitabine lead to an induction of the pro-apoptotic proteins PUMA and NOXA. Simultaneous treatment with gemcitabine and RLH-ligand increased cell death induction without affecting the cytokine secretion substantially. CD8a+ DC activation upon RLH-therapy was not affected by chemotherapy. Of note, antigen uptake as well as T cell priming was increased by chemo-immunotherapy. Most importantly, the survival of orthotopic PDAC bearing mice was significantly prolonged in the chemo-immunotherapy group compared to single agent treatment.ConclusionsGemcitabine treatment of PDAC induces PUMA and NOXA expression which leads to mitochondrial priming and sensitization towards RLH-induced cell death. chemo-immunotherapy increases the cross-presentation capability of DC in vitro and prolongs the survival of PDAC bearing mice. chemo-immunotherapy is therefore an attractive combinatorial therapeutic approach in PDAC.FundingThe project was supported by the Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 179062510 and 329628492 - SFB 1321 as well as the Förderprogramm für Forschung und Lehre (FöFoLe) funded by the Ludwig-Maximilians-Universität München.Disclosure InformationP. Metzger: None. H.T. Bourhis: None. M. Stieg: None. D. Böhmer: None. S. Endres: None. P. Düwell: None. L.M. König: None. M. Schnurr: None.

Download Full-text

Parvovirus H-1-Induced Tumor Cell Death Enhances Human Immune Response In Vitro via Increased Phagocytosis, Maturation, and Cross-Presentation by Dendritic Cells

Human Gene Therapy ◽

10.1089/hum.2005.16.ft-92 ◽

2005 ◽

Vol 0 (0) ◽

pp. 050701034702004

Author(s):

Markus H. Moehler ◽

Maja Zeidler ◽

Vanessa Wilsberg ◽

Jan J. Cornelis ◽

Thomas Woelfel ◽

...

Keyword(s):

Immune Response ◽

Cell Death ◽

Dendritic Cells ◽

Tumor Cell ◽

Tumor Cell Death ◽

Cross Presentation ◽

Human Immune Response ◽

Human Immune ◽

Immune Response In Vitro

Download Full-text

Dominant-negative ATF5 rapidly depletes survivin in tumor cells

Cell Death and Disease ◽

10.1038/s41419-019-1872-y ◽

2019 ◽

Vol 10 (10) ◽

Author(s):

Xiaotian Sun ◽

James M. Angelastro ◽

David Merino ◽

Qing Zhou ◽

Markus D. Siegelin ◽

...

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Cancer Cells ◽

Dominant Negative ◽

Tumor Cell Death ◽

Cell Penetrating ◽

Selective Death ◽

Tumor Types

Abstract Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin.

Download Full-text

Mitoxantrone-Loaded Nanoparticles for Magnetically Controlled Tumor Therapy–Induction of Tumor Cell Death, Release of Danger Signals and Activation of Immune Cells

Pharmaceutics ◽

10.3390/pharmaceutics12100923 ◽

2020 ◽

Vol 12 (10) ◽

pp. 923

Author(s):

Teresa Ratschker ◽

Laura Egenberger ◽

Magdalena Alev ◽

Lisa Zschiesche ◽

Julia Band ◽

...

Keyword(s):

Cell Death ◽

Immune System ◽

Immune Cells ◽

Checkpoint Inhibitors ◽

Tumor Therapy ◽

Superparamagnetic Iron Oxide ◽

Dependent Manner ◽

Tumor Cell Death ◽

Immune Therapies

Stimulating the patient’s immune system represents a promising therapeutic strategy to fight cancer. However, low immunogenicity of the tumor cells within an immune suppressive milieu often leads to weak anti-tumor immune responses. Additionally, the immune system may be impaired by accompanying aggressive chemotherapies. We show that mitoxantrone, bound to superparamagnetic iron oxide nanoparticles (SPIONs) as the transport system, can be magnetically accumulated in adherent HT-29 colon carcinoma cells, thereby inducing the same cell death phenotype as its soluble counterpart, a chemotherapeutic agent and prototypic inductor of immunogenic cell death. The nanoparticle-loaded drug induces cell cycle stop, apoptosis and secondary necrosis in a dose- and time-dependent manner comparable to the free drug. Cell death was accompanied by the release of interleukin-8 and damage-associated molecular patterns (DAMPs) such as HSP70 and ATP, which fostered chemotactic migration of monocytes and maturation of dendritic cells. We furthermore ensured absence of endotoxin contaminations and compatibility with erythrocytes and platelets and investigated the influence on plasma coagulation in vitro. Summarizing, with magnetic enrichment, mitoxantrone can be accumulated at the desired place, sparing healthy peripheral cells and tissues, such as immune cells. Conserving immune competence in cancer patients in the future might allow combined therapeutic approaches with immune therapies (e.g., checkpoint inhibitors).

Download Full-text

Soluble Asialoglycoprotein Receptors Reflect the Apoptosis of Hepatocytes

Cell Transplantation ◽

10.3727/000000002783985756 ◽

2002 ◽

Vol 11 (5) ◽

pp. 407-415 ◽

Cited By ~ 5

Author(s):

Tetsuji Kakegawa ◽

Hirohiko Ise ◽

Nobuhiro Sugihara ◽

Toshio Nikaido ◽

Naoki Negishi ◽

...

Keyword(s):

Cell Death ◽

Liver Tissue ◽

Liver Diseases ◽

Culture Medium ◽

Apoptotic Cell ◽

Rabbit Serum ◽

Mouse Serum ◽

Apoptosis And Necrosis

Cell death is thought to take place through at least two distinct processes: apoptosis and necrosis. There is increasing evidence that dysregulation of the apoptotic program is involved in liver diseases. However, there is no method to simply evaluate apoptosis in the liver tissue at present. It has been reported that the expression of asialoglycoprotein receptors (AGPRs) increases with apoptosis, but there is no report until now that investigates the influence of soluble AGPRs on apoptosis of hepatocytes. Soluble AGPRs have been reported to be present in human serum under physiological conditions. In the present study, in order to investigate the correlation between apoptosis of hepatocytes and soluble AGPR, mouse soluble AGPRs were detected using SDS-PAGE and Western blot analysis was conducted using anti-extracellular mouse hepatic lectin-1 (Ex-MHL-1) antiserum (polyclonal rabbit serum). The mouse soluble AGPRs were present in culture medium and mouse serum when hepatocytes were damaged. The soluble AGPRs increased proportionately, as the number of dead hepatocytes increased. In addition, soluble AGPRs existed more when apoptotic cell death was observed in in vitro and in vivo than when necrotic cell death was observed. The extracellular moiety of MHL-1 exists in the culture medium and mouse serum as a soluble AGPR, but the detailed mechanism of releasing soluble AGPR from hepatocytes has not been revealed yet. We described the first evidence for the relation between quantity of soluble AGPRs with two kinds of cell death: necrosis and apoptosis. Based on the results of our study, soluble AGPRs might become a new marker of apoptosis in the liver tissue and be useful for clinical diagnosis and treatment for liver diseases.

Download Full-text

Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid–based proteasome inhibitors

Blood ◽

10.1182/blood-2008-07-171389 ◽

2009 ◽

Vol 113 (23) ◽

pp. 5927-5937 ◽

Cited By ~ 158

Author(s):

Encouse B. Golden ◽

Philip Y. Lam ◽

Adel Kardosh ◽

Kevin J. Gaffney ◽

Enrique Cadenas ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Tumor Cell ◽

Green Tea ◽

Boronic Acid ◽

Proteasome Inhibitors ◽

Green Tea Polyphenols ◽

Tumor Cell Death

Abstract The anticancer potency of green tea and its individual components is being intensely investigated, and some cancer patients already self-medicate with this “miracle herb” in hopes of augmenting the anticancer outcome of their chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for multiple myeloma. Here, we investigated whether the combination of these compounds would yield increased antitumor efficacy in multiple myeloma and glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that various green tea constituents, in particular (-)-epigallocatechin gallate (EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced antagonistic function of EGCG was evident only with boronic acid–based proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non–boronic acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted with BZM and blocked its proteasome inhibitory function; as a consequence, BZM could not trigger endoplasmic reticulum stress or caspase-7 activation, and did not induce tumor cell death. Taken together, our results indicate that green tea polyphenols may have the potential to negate the therapeutic efficacy of BZM and suggest that consumption of green tea products may be contraindicated during cancer therapy with BZM.

Download Full-text