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. 2017 Jan 23:3:16102.
doi: 10.1038/cddiscovery.2016.102. eCollection 2017.

The plant defensin NaD1 induces tumor cell death via a non-apoptotic, membranolytic process

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The plant defensin NaD1 induces tumor cell death via a non-apoptotic, membranolytic process

Amy A Baxter et al. Cell Death Discov. .

Abstract

Cationic anti-microbial peptides (CAPs) have an important role in host innate defense against pathogens such as bacteria and fungi. Many CAPs including defensins also exhibit selective cytotoxic activity towards mammalian cells via both apoptotic and non-apoptotic processes, and are being investigated as potential anticancer agents. The anti-fungal plant defensin from ornamental tobacco, Nicotiana alata Defensin 1 (NaD1), was recently shown to induce necrotic-like cell death in a number of tumor cell types within 30 min of treatment, at a concentration of 10 μM. NaD1-mediated cell killing within these experimental parameters has been shown to occur via binding to the plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) in target cells to facilitate membrane destabilization and subsequent lysis. Whether NaD1 is also capable of inducing apoptosis in tumor cells has not been reported previously. In this study, treatment of MM170 (melanoma) and Jurkat T (leukemia) cells with subacute (<10 μM) concentrations of NaD1 over 6-24 h was investigated to determine whether NaD1 could induce cell death via apoptosis. At subacute concentrations, NaD1 did not efficiently induce membrane permeabilization within 30 min, but markedly reduced cell viability over 24 h. In contrast to other CAPs that have been shown to induce apoptosis through caspase activation, dying cells were not sensitive to a pancaspase inhibitor nor did they display caspase activity or DNA fragmentation over the 24 h treatment time. Furthermore, over the 24 h period, cells exhibited necrotic phenotypes and succumbed to membrane permeabilization. These results indicate that the cytotoxic mechanism of NaD1 at subacute concentrations is membranolytic rather than apoptotic and is also likely to be mediated through a PIP2-targeting cell lytic pathway.

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Figures

Figure 1
Figure 1
NaD1 inhibits growth of mammalian tumor cells over 24 h at subacute concentrations that do not efficiently induce rapid membrane permeabilization. The ability of NaD1 to inhibit growth of MM170, HeLa, Jurkat and U937 cells was determined using the MTT/MTS cell viability assay. Cells were treated with increasing concentrations of NaD1 (1.25, 2.5, 5 and 10 μM) for 24 h, with determination of cell viability calculated as the percentage of 100% viability (untreated control samples). Half-maximal inhibitory concentration (IC50) values (μM) were extrapolated using Excel (annotated by black dashed arrows). Data in (a) is representative of three independent experiments. (b) The release of LDH from mammalian tumor cells following 30 min treatment with NaD1 at subacute concentrations (1.25, 2.5 and 5 μM) was measured via the LDH cytotoxicity assay. Data in (b) are representative of at least two independent experiments, error bars represent S.E.M, n=3.
Figure 2
Figure 2
NaD1 induces membrane permeabilization of mammalian tumor cells at subacute concentrations over 24 h. The release of LDH from MM170 (a) and Jurkat (b) cells following 6, 16 and 24 h treatment with NaD1 at subacute concentrations (1.25, 2.5 and 5 μM) was measured via the LDH cytotoxicity assay. Data are representative of at least two independent experiments, error bars represent S.E.M, n=3.
Figure 3
Figure 3
NaD1-mediated loss of mitochondrial membrane potential in MM170 cells occurs simultaneously to plasma membrane permeabilization. (a) The effect of NaD1 on the mitochondrial membrane potential in MM170 cells was determined by live cell imaging using confocal laser scanning microscopy (CLSM). Cells were pre-treated with MTR before being treated with 1.25 μM NaD1 and imaged at 30 s intervals over 4 h in growth medium containing FITC-dextran (4 kDa). Images of key time points (taken from Supplementary Video S1) display uptake of FITC-dextran and loss of MTR fluorescence in a single cell (white arrows). Relative time is indicated in top left of each panel (h:m:s:ms). Data are representative of three independent experiments. (b) Kinetic analysis of mean fluorescence intensity of FITC-dextran versus MTR fluorescence was plotted for individual cells from 1 min before to 7 min after final baseline reading of FITC-dextran before observable uptake into the cell (Time ‘0’, indicated by red dashed line). Data in (b) represent average fluorescence across all cells. n=27, error bars represent S.E.M.
Figure 4
Figure 4
NaD1-mediated tumor cell killing at subacute concentrations is not caspase-dependent. (a) The ability of NaD1 to induce caspase-dependent apoptosis in MM170 and Jurkat cells was determined using the Caspase-Glo 3/7 Assay. Caspase activity expressed as bioluminescence was detected at 6, 16 and 24 h following treatment with NaD1 at 1.25, 2.5 and 5 μM. NaD1 exposed to ultraviolet (UV) radiation for 4 h was used as a control. (b) Fragmentation of chromosomal DNA over 6, 16 and 24 h was determined by agarose gel electrophoresis following 1.25 μM (MM170) or 2.5 μM (Jurkat) treatment with NaD1, or 4 h after exposure to UV radiation. Data represent two independent experiments. (c) The ability of the pancaspase inhibitor, Q-VD-OPH, to inhibit the cytotoxic activity of NaD1 towards MM170 and Jurkat cells was determined over 6, 16h and 24 h following 1.25 μM (MM170) or 2.5 μM (Jurkat) treatment with NaD1. For MM170 cells – 6 h, P=0.686; 16 h, P=0.375; 24 h, P=0.491. For Jurkat cells – 6 h, P=0.977; 16 h, P=0.092; 24 h, P=0.485, unpaired Student’s two-tailed t-test. (d) To test directly the ability of Q-VD-OPH to inhibit apoptosis, phosphatidylserine exposure as determined by FITC-Annexin V staining of Jurkat cells 4 h after exposure to UV radiation was detected via flow cytometry. P=0.011, unpaired Student’s two-tailed t-test. Data in (a), (c) and (d) are representative of at least two independent experiments, error bars represent S.E.M, n=3. *P<0.05.
Figure 5
Figure 5
MM170 cells display a necrotic phenotype following subacute treatment of NaD1 over 24 h. Morphological changes in MM170 cells following treatment with NaD1 (1.25 μM) or ultraviolet (UV) radiation were examined via DIC live cell imaging by spinning disk confocal microscopy. (a) NaD1-treated cells were imaged over 24 h, with images recorded at 0, 6, 16 and 24 h time points. Scale bars in (a) represent 20 μm. (b) MM170 cells exposed to UV radiation were imaged 6 h after exposure. Data are representative of three independent experiments.
Figure 6
Figure 6
The cytotoxic activity of NaD1 at subacute concentrations over 24 h is significantly inhibited by neomycin but not necrostatin-1. The ability of the PIP2-sequestering agent, neomycin, to inhibit NaD1-mediated LDH release from MM170 and Jurkat cells was determined using the LDH cytotoxicity assay. MM170 (a) and Jurkat (b) cells were pre-treated with 10 mM neomycin for 1 h before the addition of NaD1 at 1.25 μM (MM170) or 2.5 μM (Jurkat) for 24 h. MM170, P=0.011, Jurkat, P=0.001, unpaired Student’s two-tailed t-test. Treatment with 10 μM NaD1 over 30 min was also performed on both cell lines as a comparison. MM170, P=0.000, Jurkat, P=0.000, unpaired Student’s two-tailed t-test. (c) To test directly the ability of neomycin to inhibit apoptosis, phosphatidylserine exposure as determined by FITC-Annexin V staining of Jurkat cells 4 h after exposure to ultraviolet (UV) radiation was detected via flow cytometry. P=0.910, unpaired Student’s two-tailed t-test. Data are representative of two independent experiments, error bars represent S.E.M, n=3. (d) The ability of necrostatin-1 (Nec-1) to inhibit NaD1-mediated membrane permeabilization was investigated. Cells pre-treated with 10 μM necrostatin-1 (Nec-1) were subjected to 24 h treatment with 2.5 μM NaD1 and analyzed by flow cytometry, with percentage of cell death indicated by ToPro3-positive staining. P=0.437, unpaired Student’s two-tailed t-test. Data are representative of three independent experiments, error bars represent S.E.M, n=3. *P<0.05, **P<0.01, ***P<0.001.

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