Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML)

<div><p>Cardiac glycosides (CGs), inhibitors of Na⁺/K⁺-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli.</p></div

CGs effects on PML SUMOylation, cell survival and anti-viral effects are mediated by NKA α1.

<p><b>A,</b> HEK293T cells were transfected with plasmids encoding HA-Sumo-1, HA-Sumo-2 and Flag-PM IV together with either empty vector or rat NKAα1. The next day the cells were treated as indicated for 24h, followed by lysis and IP using anti-Flag (PML) antibody. The total lysates before immunoprecipitation and the IP-ed complexes were separated on SDS-PAGE followed by immunoblotting with anti-Flag (PML), anti HA (Sumo-1 and Sumo-2) or anti-ACTIN antibodies. <b>B,</b> Human NKA subunit α1 contains residues Q118 and N129, which are essential for CG binding. Rat NKAα1 lacks these critical residues, rendering it insensitive to CG. HEK293T cells were transfected with either empty vector or rat NKA α1. The following day the cells were treated as indicated for 48h, followed by cell viability determination using Cell Titer Glo assay. Data are means of three replicates and the error bars are standard deviations. <b>C,</b> HEK293T cells were transfected with either NKAα1 or pcDNA. The following day, the cells were recovered and dispensed into 24-well plates and the day after they were pre-treated with 0, 25 nM, 50 nM or 100 nM ouabain for 5h, followed by infection with HSV-1 KOS for 24 hrs. The produced virus was harvested for infection of naïve cells in D, and was detected by immunoblotting with anti-VP16 antibody. <b>D,</b> Phase-contrast image of HEK293T cells 48h after infection with HSV-1 virus produced in cells as described in C. Less virus was produced in cells treated with ouabain, as evident from less VP16 expression (C) and less cell killing (D). The scale in D represents 800 μm. The effect of ouabain was reversed with overexpression of rat NKAα1.</p

Loss of PML does not attenuate cell death induced by apoptotic stimuli.

<p>Two <i>PML</i> knockout HEK293T cell clones (PML KO c1 and c2) were generated by CRISPR/Cas9 approach. <b>A,</b> PML protein levels were determined by immunofluorescence using anti-PML antibody in the control and 2 μM arsenic trioxide (As₂O₃)-treated wild type HEK293T (WT), as well as in two PML knockout clones (PML KO c1 and c2). <b>B,</b> Wild-type and knockout clones were cultured in 96 well plates at a density of 15,000/well. The next day, the cells were treated with 60 μM C2-ceramide, 300nM ouabain or 5 μM arsenic trioxide for 48h. Cell viability was determined using Cell Titer Glo assay. Data are mean ± SEM (n = 3).</p

High content screening identifies cardiac glycoside as an inducer of PML nuclear body formation.

<p><b>A,</b> Scatterplot of the data obtained from a total of 1,008 384-well library plates that were screened, imaged, and analyzed, yielding an average Z’ of 0.50 and a hit rate of 0.3%. IFN 10 U/μl (positive control, dark gray), DMSO (negative control, light gray), compounds (black) <b>B and C,</b> HeLa cells were treated with increasing concentrations of gitoxigenin as well as DMSO as a negative control for 18h. PML NB formation was determined by immunofluorescence with anti-PML antibody and was analyzed using an algorithm described in Materials and Methods. The images are representative PML NB staining of cells treated with DMSO and 0.04 μM, 1.25 μM and 20 μM gitoxigenin for 18h (B). The graphs show fitted curves for PML NB (black diamonds) and cell count (grey circles) over the full dose range of gitoxigenin (C). PML NB values at high concentrations are shown with open diamonds and are excluded from the fitted curve due to the cytotoxicity as evident from dramatic decrease in cell counts at these concentrations.</p

CGs do not induce PML expression or association with nuclear matrix.

<p><b>A,</b> HeLa cells were dispensed into 384-well pates at a density of 2000 cells/wells. The next day the cells were incubated with 2 μM gitoxigenin, 200 nM ouabain and 16 u IFNϒ for 18h. RNA was isolated and RT-qPCR was performed for PML and GUSB. PML expression was normalized to GUSB internal control. Error bars are SEMs of 6 replicates. <b>B,</b> HEK293T were transfected with Flag-PML IV. The next day the cells were treated as indicated for 24h, followed by lysis in RIPA buffer and centrifugation. RIPA supernatants were immunoprecipitated using anti-Flag antibody. The total lysates before immunoprecipitation, the immunoprecipitated complexes and the RIPA-insoluble pellets were separated on SDS-PAGE followed by immunoblotting with anti-Flag (PML) or anti-ACTIN antibody.</p

Diverse cardiac glycosides (CGs) and non-steroidal NKA inhibitors induce PML-NB formation.

<p><b>A,</b> Structures of diverse cardiac glycosides (CGs). All CGs share a common structural motif comprised of a steroidal core adorned with an unsaturated lactone at ring position 17. CG lactone head-groups come in two varieties, unsaturated butyrolactones, such as gitoxigenin, ouabain and uzarin, and α-pyrones such as proscillaridin A. With the exception of the aglycones (e.g. gitoxigenin), the core is double substituted to contain a sugar moiety at ring position 3. <b>B and C,</b> HeLa cells were treated with increasing concentrations of CGs for 18h. PML NB formation and cell count was determined as described in Materials and Methods. <b>D,</b> Structures of non-steroidal NKA inhibitors are shown. <b>E and F,</b> HeLa cells were treated with increasing concentrations of non-steroidal NKA inhibitors for 18h. PML NB formation and cell count was determined as described in Materials and Methods. The data are means of four replicates and the error bars are SEMs. PML NB values at high concentrations are shown with open symbols in B and E and are excluded from the fitted curve due to the cytotoxicity as evident from dramatic decrease in cell counts at these concentrations (C and F).</p

Characterization of Potent SMAC Mimetics that Sensitize Cancer Cells to TNF Family-Induced Apoptosis - Fig 4

<p>Condensed structures (A,B, and C) of the compounds series described in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#pone.0161952.t003" target="_blank">3</a>. (D) Structure of P₂ substituent of compound <b>18</b>.</p

SMAC mimetic 38 induces degradation of cIAP1.

<p>MDA-MB-231 cells were seeded at 40,000 per well of 12 well plates and cultured overnight. The next day, cultures were either left untreated (No trtm) or were treated for 6 h. with DMSO, 5 μM of SMAC mimetic <b>38</b> or 5 μM of inactive analogue compound <b>40</b>. Cells were lysed in SDS-sample buffer and lysates were analyzed by SDS-PAGE/immunoblotting using antibodies specific for cIAP1 and beta-actin. Molecular weight markers are indicated in kilo-Daltons (kDa).</p

Binding affinities of naphthyl series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2^a.

<p>Binding affinities of naphthyl series SMAC mimetic compounds for BIR domains of cIAP1 and cIAP2<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161952#t002fn002" target="_blank">^a</a>.</p

SMAC mimetics inhibit cIAP1 and cIAP2 binding to RIP1.

<p>HEK293T cells were transfected with myc-RIP1 plasmid and 24h later, cell lysates were prepared and divided into equal aliquots to which 7 μg of either GST-cIAP1-BIR3 or GST-cIAP2-BIR3 was added. Then, aliquots of either DMSO control or various concentrations of SMAC mimetics <b>37</b>, <b>38</b> or inactive analogue <b>40</b> were added. After overnight incubation, GST-cIAP1-BIR3 or GST-cIAP2-BIR3 proteins were recovered using glutathione-sepharose beads and the bound myc-RIP1 protein was detected by SDS-PAGE/immunoblotting using anti-myc antibody for detection of myc-RIP1 and anti-GST antibody for detection of GST-fusion proteins.</p