Frese S, Frese-Schaper M, Andres AC, Miescher D, Zumkehr B, Schmid RACardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5. Cancer Res 66(11): 5867-5874

Department of Clinical Research, Universität Bern, Berna, Bern, Switzerland
Cancer Research (Impact Factor: 9.33). 07/2006; 66(11):5867-74. DOI: 10.1158/0008-5472.CAN-05-3544
Source: PubMed


Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

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Available from: Steffen Frese, Oct 03, 2014
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    • "The persistent depolarization is observed not only in human leukemia cells such as Jurkat cells and U937 cells but also in human primary T cells, although primary cells are more resistant than leukemia cells to the effect. The persistent depolarization is pro-apoptotic, because some cardiac glycoside inhibitors of Na+–K+-ATPase such as ouabain and oleandrin, can sensitize human leukemia cells and NSCLC cells to apoptosis induced by anti-Fas, TRAIL, and mitochondrial toxins (14, 15, 18). These observations suggest that Na+–K+-ATPase is a key player connecting death ligands, depolarization, and apoptosis. "
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    ABSTRACT: Conventional genotoxic anti-cancer drugs target the proliferative advantage of tumor cells over normal cells. This kind of approach lacks the selectivity of treatment to cancer cells, because most of the targeted pathways are essential for the survival of normal cells. As a result, traditional cancer treatments are often limited by undesirable damage to normal cells (side-effects). Ideal anti-cancer drugs are expected to be highly effective against malignant tumor cells with minimal cytotoxicity toward normal cells. Such selective killing can be achieved by targeting pathways essential for the survival of cancer cells, but not normal cells. As cancer cells are characterized by their resistance to apoptosis, selective apoptosis induction is a promising approach for selective killing of cancer cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising tumor-selective anti-cancer drug. However, the congenital and acquired resistance of some cancer cell types, including malignant melanoma cells, currently impedes effective TRAIL therapy, and an innovative approach that can override TRAIL resistance is urgently required. Apoptosis is characterized by cell shrinkage caused by disruption of the maintenance of the normal physiological concentrations of K(+) and Na(+) and intracellular ion homeostasis. The disrupted ion homeostasis leads to depolarization and apoptosis. Recent evidence suggests that depolarization is an early and prerequisite event during TRAIL-induced apoptosis. Moreover, diverse natural products and synthetic chemicals capable of depolarizing the cell membrane exhibit tumor-selective killing and TRAIL-sensitizing effects. Here, we discuss the role of depolarization in selective killing of cancer cells in connection with the emerging concept that oxidative stress is a critical mediator of mitochondrial and endoplasmic reticulum dysfunctions and serves as a tumor-selective target in cancer treatment.
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    • "In addition, adjuvant effects at the cellular level have also been observed. For example, oleandrin can independently sensitize cancer cells to radiation,[8] or to pro-apoptotic ligands such as apo2 L/TRAIL (TNF alpha Related Apoptosis-Inducing Ligand).[9] In mice with intact immune systems, equivalent carcinotoxic effects have been observed for chemically induced skin papillomas and pulmonary tumors with either digitoxin[6] or oleandrin.[7] "
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    ABSTRACT: Cardiac glycosides such as digitoxin have been shown to directly cause apoptotic death of cancer cells both in vitro, and in vivo. However, the mechanism connecting cardiac glycoside action to apoptosis is not known. It has been reported that compounds resembling digitoxin are able to reduce c-MYC expression. Furthermore, it has been previously shown that the transcription of c-MYC depends on nuclear factor of activated T-cells (NFAT) binding sites in the c-MYC promoter. We have therefore hypothesized that NFAT might mediate digitoxin effects on c-MYC mRNA message. We have chosen to study this process in HeLa cells where structurally intact c-MYC genes in 8q24 co-localize with human papilloma virus 18 at all integration sites. Here we show that within the 1(st) h following treatment with digitoxin, a significant reduction in c-MYC mRNA occurs. This is followed by a precipitous loss of c-MYC protein, activation of caspase 3, and subsequent apoptotic cell death. To test the NFAT-dependence mechanism, we analyzed the effects of digitoxin on NFAT isoform-dependent auto-activation of a NFAT-luciferase expression system. Drug dependent effects on expression varied according to each of the four canonical NFAT isoforms (1, 2, 3 or 4). The most digitoxin-sensitive NFAT isoform was NFAT1. Using c-MYC chromatin immune precipitation, we find that digitoxin inhibits interaction of NFAT1 with the proximal c-MYC promoter. These results suggest that the carcinotoxic activity of digitoxin includes suppression of NFAT-driven c-MYC expression.
    No preview · Article · May 2013 · Journal of Carcinogenesis
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    • "Multiplex gene expression analysis demonstrated a decade ago that various cardiotonic steroids inhibit prostate target genes [37]. Several studies have demonstrated that various cardiotonic steroids are able to sensitize apoptosis-resistant cancer cells to pro-apoptotic stimuli [38-40] and directly induce apoptosis in lymphoma [41] and leukemia [42] cells. Cardiotonic steroids can also induce cancer cell death through Src- or MAPK-mediated inhibition of p53 expression [43], the inhibition of general protein synthesis [44], the inhibition of HIF-1a synthesis [45], sustained and irreversible autophagy [10,46] and lysosomal membrane permeabilization [30]. "
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    ABSTRACT: Background Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK. Methods The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. Results Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. Conclusions Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth.
    Full-text · Article · Apr 2013 · Molecular Cancer
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