Mol Cell Oncol. ;2(4):e (-)-Oleocanthal rapidly and selectively induces cancer cell death via lysosomal membrane permeabilization. LeGendre . We investigated the effect of OC on human cancer cell lines in culture and found that OC induced cell death in all cancer cells examined as rapidly as J Cancer. Jul 5;8(11) doi: /jca eCollection of CTCF in Breast Cancer Cells Selectively Induces Cancer Cell Death via p
Cell Death Cancer Induces
Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside GEV out of 46 CGs for its low nanomolar anti-lung cancer activity.
GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy.
In the presence of GEV, markers of autophagic flux e. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors.
Interestingly, GEV triggered caspase-dependent apoptosis in U acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.
Cardenolides belong to the group of cardiac glycosides CG and attracted much interest in preclinical anticancer research. Cardiac glycosides like digoxin or digitoxin are clinically used for the treatment of heart failure and atrial arrhythmia. More recently, antiviral and anticancer activities were described Cerella et al. As anticancer agents, CGs triggered different cell death mechanisms including the intrinsic or the extrinsic apoptosis Juncker et al.
Furthermore, CG-induced autophagic cell death was described in breast Farah et al. More recently, the ability of CGs to trigger anoikis Pongrakhananon et al. The ability of CGs to trigger non-canonical cell death modalities constitutes an advantage, especially for cancer types that developed intrinsic resistance against apoptotic cell death when treated with a broad range of chemotherapeutic agents Diederich and Cerella, ; Diederich et al.
Also, the capacity of CGs to exert cell-type specific anti-cancer effects will allow personalized treatments against selected cancer subtypes Diederich et al. Considering the increased interest in non-canonical cell death modalities, new compounds presenting alternative mechanisms of cell death induction are essential and the aim of this report.
Based on a preliminary screening of 46 CGs, we focused our study on glucoevatromonoside GEV , a cardenolide isolated from a Brazilian cultivar of Digitalis lanata Castro Braga et al. We investigated the effect of GEV on a panel of lung cancer cell lines and selected A Schneider et al. In order to provide a proof of concept of the activity of GEV, we generalized our findings on a panel of cancer cell models from different tissues, including examples of other solid and hematological forms.
GEV exhibits a significant cytostatic and cytotoxic effect at nanomolar levels in adherent and non-adherent cancer cell types, without affecting healthy cell models.
Our results demonstrate the capacity of GEV to activate caspase-independent cell death in the lung cancer model, validated by 2D and 3D cell culture, spheroid and colony formation assays as well as by in vivo zebrafish xenografts. Furthermore, here we extended our mechanistic studies to an example of hematological cancer by selecting U cells, which exhibit a similar susceptibility to GEV compared to A cells to be within a comparable concentration range for the induction of cell death modalities.
Our results show in this instance the induction of a caspase-dependent apoptosis, indicating a cancer cell type-specific induction of different modalities of cell death induced by GEV. The origin of all tested cardenolides is indicated in Supplementary Table 1. Paclitaxel was from Sigma-Aldrich St. PBMCs were isolated by density gradient centrifugation from freshly collected buffy coats obtained from healthy adult human volunteers Red Cross, Luxembourg, Luxembourg. All healthy volunteers gave informed written consent.
The impact of the most efficient cardenolide in A cells glucoevatromonoside; GEV was tested at different concentrations after 24 and 48 h treatment. Trypan blue staining was used to evaluate the loss of plasma membrane integrity in A and U cells.
Data were normalized to the control and described as a percentage of Trypan blue-positive cells. After removing ligands in the original data, we performed computational docking using the Autodock Vina program Trott and Olson, with the protein and the compound as receptor and ligand, respectively.
PBMCs were isolated and maintained as described above Juncker et al. The experiments were performed on mixed mononuclear cells. To further identify a differential effect of GEV on lymphocytic vs. Analysis of monocytic marker expression confirmed the quality of the monocytic sub-population not shown. A and U cells were treated with different concentrations of GEV, at various time points. Cells were then washed twice with phosphate-buffered saline PBS, pH 7.
Percentages of cells with fragmented or shrunken nuclei were estimated as described Cerella et al. After treatment, samples were prepared as previously described Schnekenburger et al. Sections were stained with uranyl acetate and lead citrate and examined with a CM12 transmission electron microscope Philips, Eindhoven, The Netherlands. The assay was performed according to the manufacturer's instructions, and luminescence was measured using a well plate Orion Microplate Luminometer Berthold, Pforzheim, Germany.
Data were normalized to luminescence of untreated cells. Three brightness filter images were collected every three h up to 72 h. Spheroids were generated by the hanging drop method. After incubation for 96 h with GEV 0, 1, 10, 50, nM , spheroids were recovered in a 6-well plate in 4 mL culture media. Images were taken Nikon and further analyzed by ImageJ software http: For cancer xenograft assays, after mating, fertilized eggs were incubated in Danieau's solution with 0.
Micropipettes for injection and anesthesia were generated from a 1. Subsequently, zebrafish were incubated in well plates containing Danieau's solution with 0. Area of fluorescent tumors was quantified by ImageJ software. Blots were incubated with primary antibodies: After overnight incubation with primary antibodies, membranes were washed with PBS-Tween, followed by incubation for 1 h at room temperature with the corresponding secondary horseradish peroxidase-conjugated antibodies from Santa Cruz Biotechnology.
We evaluated the anticancer potential of a panel of 46 natural or hemisynthetic cardenolides on A cells Supplementary Table 1. The predicted docking modes of GEV are shown as stick models colored in magenta, and the coordinates of ouabain, bufalin, and digoxin are represented by green, orange, and blue sticks, respectively. The hydrogen and oxygen atoms are colored in white and red, respectively. We then further evaluated the anti-cancer potential and selectivity of GEV.
We generalized our findings with additional lung cancer cell lines and other types of solid tumor compared to non-adherent cancer models Table 1. After 48 h of treatment, GEV was fold and 8. The population of monocytes was affected by GEV after 48h of treatment only at concentrations beyond the range of this investigation nM Supplementary Figure 1A.
Considering the differential toxicity and selectivity of GEV toward lung cancer cell models, we selected the lung A cells for in vitro and in vivo investigations. Furthermore, we compared results to the acute myeloid leukemia cell line U, which exhibits a similar susceptibility to GEV as A cells. Predicted docking orientation of the disaccharide of GEV was different from that of ouabain as well as from those of bufalin and digoxin. GEV reduced the proliferation of A cells in a concentration and time-dependent manner Figures 2A,B concomitant with the accumulation of cell death after treatment with 50 and nM GEV 24 h: Observation by fluorescence microscopy of Hoechst-stained nuclei confirmed the progressive accumulation of cells displaying pyknotic nuclei, besides a minor additional fraction with nuclear fragmentation Figures 2C,D.
Analysis by transmission electron microscopy TEM further validated severe ultrastructural alterations of cytoplasm and nucleus in GEV-treated A cells. After 24 and 48 h, cells committed to death showed a disruption of the cytoplasmic architecture with an accumulation of large vacuoles, resembling intracellular edemas. Meanwhile, nuclei were shrunken but not fragmented, presenting locally condensed chromatin Figure 2E. Remarkably, no cells with intermediate levels of degeneration were observed, suggesting a rapid evolution after cell death commitment.
Real-time videomicroscopy of A cells treated with GEV at 50 nM confirmed a peculiar evolution of cellular alterations compared to etoposide VP16 , a canonical apoptosis inducer, used as a control Figure 2F and Supplementary Videos 1—3. Whereas, VP16 sequentially triggered shrinkage, fusiform morphology, detachment, and fragmentation, GEV induced increased cell volume and granularity as quantified by flow cytometry Figure 2G.
Altogether, these observations suggested that GEV induced a differential, non-apoptotic, cell death pathway. Glucoevatromonoside exerts cytostatic and cytotoxic effects on A cells. A GEV reduces proliferation of A cells in a concentration-dependent manner; B analysis of cell death by trypan blue staining after 24 and 48 h of treatment at 10, 50, and nM; C Hoechst staining; and D quantification of cell fractions presenting shrinking and fragmented nuclei white arrows in treated cells vs.
Images show nuclear and cytoplasmic alterations. Arrows represent cells with increased cell volume and granularity. Significance is reported as: Glucoevatromonoside induces cell death with hybrid characteristics. As a positive control PC , U cells were treated with etoposide 50 nM VP16, 3 h ; B Quantification of substrate cleavage using luminescence-based assays. Caspase activity was monitored after 12, 24, 36, and 48 h. All results excluded the induction of parossistic autophagy by GEV.
Rather, the robust autophagic flux of A cells was abrogated by this treatment Figure 4B and Supplementary Figure 2. Effect of glucoevatromonoside on autophagic flux. A Representative transmission electron microscopy of A cells treated with GEV 50 nM with and without bafilomycin A1 10 nM after 12 and 24 h of treatment. The blots shown are representative of three independent experiments.
Results were confirmed by real-time imaging Supplementary Figure 3 and Supplementary Videos 6— This result confirms caspase-dependency involved in PS exposure and points at two possible alternative scenarios where apoptotic and non-apoptotic features of cell death might co-exist in different cell subpopulations or the same cell. Figure 5A shows a reduction of the proliferation of U cells in a concentration-dependent manner, similarly to A cells.
GEV-treated U cells undergo changes in their nuclear morphology in a concentration-dependent manner leading to apoptotic nuclear condensation and fragmentation Figures 5C,D. Altogether, these results indicate the ability of GEV of triggering a caspase-dependent apoptosis in U cells. Glucoevatromonoside impairs cell growth and induces caspase-dependent apoptosis in U A GEV reduces proliferation of U cells in a concentration-dependent manner; B Analysis of cell death performed by trypan blue staining after 24 and 48 h of treatment at 10, 50, and nM; C GEV induces apoptotic nuclear morphology.
Arrows show fragmented nuclei; D Quantification of cell death by Hoechst staining of fragmented nuclei in treated cells vs. It also down-regulated p53 at all concentrations tested as earlier as 12 h Figure 6. Glucoevatromonoside induces cell cycle arrest. Cell cycle analysis of cells treated with GEV for 24 and 48 h. DMSO served as vehicle control. We then confirmed the potential of GEV to impair the ability of A cells in in vitro and in vivo 3D tumor formation assays in the presence of increasing concentrations of the compound.
To extend our colony formation assays to an in vivo situation, we assessed the capacity of GEV to abrogate A tumor formation in a zebrafish xenograft model.
Our results confirmed a strong abrogation of tumor formation after injection of 50 nM GEV-pretreated, fluorescently stained A, validating our in vitro results Figure 7C. Glucoevatromonoside abrogates tumor formation in 3D culture and zebrafish xenografts. Quantification of tumor area and volume are shown; B A colony formation assays in the presence of increasing concentrations of GEV 10 days. Cancer cells not only multiply uncontrollably, they successfully dodge this natural mechanism of death.
Cancer cells hijack the cell machinery and stop apoptosis by blocking the activity of a particular gene — p This protector gene first stops the division of abnormal cells, and then initiates either their repair or destruction if the damage is severe.
Normally, the p53 gene activates specific apoptotic proteins from the Bcl2 family and the Caspases enzymes that initiate and continue the process of apoptosis. The malfunctioning of the p53 gene results in rapidly growing and aggressive cancers. Restoration of the normal process of apoptosis is one of the avenues being explored for anticancer treatments. Several drugs, including Aspirin and other non-steroidal-anti-inflammatory-drugs NSAIDs , are being researched in cancer cells for their usage as apoptosis inducers.
We explored the potential of micronutrients in inducing apoptosis in different cancer cell lines. Cancer cells are typically immortal, but our results show that a specific micronutrient combination is capable of triggering metabolic and genetic changes that kill cancer cells by inducing their natural cell death cycle.
The pharmaceutical drugs with similar pro-apoptotic action act indiscriminately, inducing apoptosis in cancer cells and healthy cells alike.
These drugs can cause brutal side effects including drug-resistance and fatal liver failure. To assess the safety of the micronutrient combination we also tested it on normal cells. Although apoptosis is vital for destroying abnormal cells it is also important in maintaining optimum health, thus requiring a precise balance between cell formation and destruction. Based on our research, the micronutrient combination is a safe and effective way to achieve this balance.
The Benefits of Micronutrients in inducing Cancer Cell Death
Interestingly, GEV triggered caspase-dependent apoptosis in U acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. In this study, we demonstrate that a combination of quinacrine and SAHA is potent in inducing cancer cell death and abrogating tumor growth. Amicoumacin A is an antibiotic that was recently shown to target bacterial ribosomes. It affects translocation and provides an additional contact.