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Clinical Chemistry 53: 1315-1322, 2007. First published May 10, 2007; 10.1373/clinchem.2006.082081
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(Clinical Chemistry. 2007;53:1315-1322.)
© 2007 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

Digoxin-Like Immunoreactive Factors Induce Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia

Kenneth Ihenetu1,3, Hassan M. Qazzaz1, Fabian Crespo1, Rafael Fernandez-Botran1 and Roland Valdes, Jr1,2,a

Departments of1 Pathology and Laboratory Medicine and 2 Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY.

aAddress correspondence to this author at: Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, MDR Bldg., Rm. 209, 511 South Floyd St., Louisville, KY 40202. Fax 502-852-1771; e-mail: rvaldes{at}louisville.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Plant-derived cardenolides reportedly possess anticancer properties in human leukemic cells via selective induction of apoptosis, cell cycle arrest, and differentiation. Selective induction of apoptosis with mammalian-derived digoxin-like immunoreactive factor (DLIF) could provide new strategies for anticancer drug development or the identification of biomarkers for cancer. We investigated whether DLIFs selectively induce apoptosis in human lymphoblastic leukemic cells.

Methods: We compared the relative potencies of digoxin, ouabain, and DLIF on induction of programmed cell death in Jurkat cells (an acute T-leukemic cell line), K-562 (a myelogenous leukemia cell line), and nonpathologic human peripheral blood mononuclear cells (PBMCs). Apoptosis was measured by flow cytometry with the annexin V/propidium iodide method.

Results: Digoxin and ouabain induced apoptosis in Jurkat cells [digoxin 50% inhibitory concentration (IC50), 24 nmol/L; ouabain IC50, 26 nmol/L]. Neither digoxin nor ouabain induced apoptosis in K-562 cells or PBMCs. DLIF was more potent (IC50, 1.9 nmol/L) and >2-fold more effective than digoxin or ouabain at inducing maximum apoptosis in Jurkat cells. The IC50 values in the apoptosis assays were >100-fold lower (DLIF) and 20-fold lower (digoxin and ouabain) than the IC50 required for Na+- and K+-dependent ATPase (DLIF, 200 nmol/L; digoxin, 910 nmol/L; ouabain, 600 nmol/L).

Conclusion: DLIF selectively induces apoptosis in a human acute T-cell lymphoblastic leukemia cell line but not in K-562 cells or PBMCs. These data suggest a new physiological role for these endogenous hormone-like factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Plant-derived cardiac glycosides such as digoxin are used for the treatment of congestive heart failure and other cardiac disorders (1). Their main pharmacologic actions are mediated through inhibition of the sodium pump, Na+- and K+-dependent ATPase (NKA) 1 (EC 3.6.3.9) (2). NKA, a ubiquitous membrane cationic transporter protein, controls normal membrane potential in all eukaryotic cells by maintaining high K+ and low Na+ concentrations. It consists of an {alpha} catalytic subunit and a ß glycoprotein subunit (3).

Studies have suggested that plant-derived cardiac glycosides regulate some cellular processes, such as proliferation and apoptosis, in a variety of cancer cells (4)(5)(6)(7). Of great interest is the identification of a family of endogenous mammalian compounds, the digoxin-like immunoreactive factors (DLIFs) (8)(9)(10)(11)(12)(13) and ouabain-like factors, which are secreted by the adrenal cortical glands and are believed to constitute a hormonal axis regulating the activity of NKA (8)(9)(14). Various groups have confirmed the existence of these mammalian cardenolides (7)(8)(9), and although their physiological roles and pharmacologic actions are not fully understood, these compounds and the plant-derived compounds have similar structures, including a steroid backbone to which a lactone ring and sugars are attached (8)(9)(10). The possibility that these mammalian compounds possess anticancer properties, via selective induction of apoptosis in transformed cells, has not been explored.

We hypothesized that DLIF might have proapoptotic properties against malignant hematologic cell lines similar to those of digoxin (15). To test this premise, we investigated the proapoptotic actions of digoxin, ouabain, and DLIF in a human acute T-cell lymphoblastic leukemic cell line (Jurkat E6-1), a human myelogenous erythroblastoid leukemic cell line (K-562), and peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
reagents
All chemicals were of reagent grade and were purchased from Sigma-Aldrich. We dissolved digoxin in dimethyl sulfoxide and ouabain in doubly distilled water. Both solutions (10 mmol/L) were stored at –20 oC. They were diluted further in cell culture medium for in vitro studies. Phytohemagglutinin (PHA) was dissolved in cell culture medium to a concentration of 1 mg/L. DLIF was extracted and isolated from bovine adrenal cortical tissue and purified as previously reported (16). We estimated DLIF concentration as the digoxin-equivalent concentration by measuring cross-reactivity with previously reported antidigoxin antibodies (16) and calibrating cross-reactivity with known digoxin concentrations.

cell lines
Cell lines used in this study included Jurkat E6-1, an acute T-lymphoblastic leukemia cell line generated from a 14-year-old boy (17), and K-562, which is derived from Caucasian chronic myelogenous leukemia cells (18). All cell lines were maintained in RPMI 1640 medium (Invitrogen/Gibco BRL).

isolation and culture of pbmcs
We obtained heparinized blood from healthy volunteers and isolated PBMCs by density gradient centrifugation with Histopaque R-1077 (Sigma-Aldrich), as described previously (19). Informed consent was obtained by means of Institutional Review Board–approved protocols for sample collection; all samples were blinded for greater confidentiality.

nka catalytic activity
We assessed cardiac glycoside inhibition of NKA catalytic activity in porcine cerebral cortex (PCC) tissue by measuring the release of phosphate after ATP hydrolysis as described by Qazzaz et al. (20).

FASL production at the MRNA level
Jurkat and K-562 cells were treated with digoxin (100 nmol/L) or DLIF (10 nmol/L digoxin equivalent) for 12 h. In a separate experiment, Jurkat cells were treated with 1 mg/L PHA for 12 h as a positive control for apoptosis induction. We assessed the production of mRNA transcripts specific for FasL by means of semiquantitative reverse transcription–PCR and real-time PCR analyses. After treatment with digoxin (100 nmol/L) for 12 h, we isolated total RNA from cultured cells and PBMCs with the Ambion PARIS Kit according to the manufacturer’s instructions. The samples were reconstituted in ribonuclease-free water and stored at –80 oC until use. FasL mRNA production was quantified by PCR amplification after reverse transcription with a High-Capacity cDNA Archive Kit (Applied Biosystems). For PCR analysis, we used both target-specific primers and high-capacity cDNA (Applied Biosystems) according to the manufacturer’s protocol. The enzyme or RNA was omitted in reactions used as negative controls. Real-time PCR analyses used the Prism 7500 Sequence Detection System and SYBR Green 1 Dye reagents (Applied Biosystems). Primer3 software programs (21) were used in the design of the following specific primers for human cyclophilin (PPIA) 2 and FasL (FASLG) genes: cyclophilin right primer, 5'-CCC ACC GTG TTC TTC GAC AT-3'; cyclophilin left primer, 5'-CCA GTG CTC AGA GCA CGA AA-3'; FasL right primer, 5'-ACA CCT ATG GAA TTG TCC TGC-3'; FasL left primer, 5'-GAC CAG AGA GAG CTC AGA TAT ACG-3'. The threshold cycle was defined as the cyclophilin cycle number at which the fluorescence passed the threshold. We used the 2–{Delta}{Delta}Ct method (where Ct is the threshold cycle) in the experiment to analyze FASLG gene expression relative to that of PPIA.

cell viability studies
We measured cell viability via a modification of the thiazolyl blue tetrazolium bromide and trypan blue dye-exclusion methods (19). In the latter method, cell viability was expressed as the percentage of cells that excluded the dye.

measurement of cardiac glycoside–induced apoptosis
We cultured the cell lines (5 x 105 cells/well) and PBMCs (1 x 106 cells/well) for 48 h in 24-well plates containing 0–500 nmol/L DLIF, digoxin, or ouabain. The cells were harvested, washed twice in PBS, and analyzed for apoptosis induction by means of the fluorescein isothiocyanate–conjugated annexin V/propidium iodide method (BD Biosciences). We measured apoptosis by flow cytometry on a FACScan instrument (BD Biosciences). In experiments measuring the effect of PHA, we seeded cells in a 24-well plate and stimulated the cells with 1 mg/L PHA for a minimum of 24 h before treatment with different concentrations of DLIF (expressed as digoxin equivalents) (16), ouabain, or digoxin. As a positive control for apoptosis, cells were exposed to ultraviolet (UV) irradiation (2–6 Gy) for 48 h, and apoptosis was measured by means of the method described above.

caspase-3 activity assay
We measured caspase-3 activity with a Caspase 3 Assay Kit (Sigma-Aldrich). Digoxin at 100 nmol/L was used to test the activation of caspase-3 enzyme in Jurkat cells, K-562 cells, and PBMCs. Jurkat cells (1 x 1010 cells/L) and PBMCs (1 x 1010 cells/L) were exposed to UV light or digoxin (100 nmol/L) for 12 h, and caspase-3 activity was measured according to the manufacturer’s instructions.

statistical analysis
The Student t-test was used for statistical evaluations. Differences with a P level ≤0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We hypothesized that endogenous mammalian cardenolides (DLIF) and plant-derived cardenolides (digoxin and ouabain) would induce greater apoptosis in human malignant hematologic cell lines than in nonpathologic nontransformed cells. Typical flow cytometric analyses of Jurkat cells stained with annexin V and propidium iodide after treatment with UV light, digoxin, or DLIF are shown in Fig. 1 . The percentages of cells in the lower-right and upper-right quadrants indicate cells in early- and late-phase apoptosis, respectively; viable cells are in the lower-left quadrant.


Figure 1
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  		Figure 1. Flow cytometric analysis of apoptosis induction.

Top 2 panels show Jurkat cells unexposed and exposed to UV irradiation (see Materials and Methods) for 48 h. The middle 3 panels show cell responses to increasing digoxin concentrations. The bottom 3 panels show cell response to increasing DLIF concentration. Note progressive increase in apoptotic cells (increases in upper- and lower-right quadrants) with increasing digoxin or DLIF concentration. PI, propidium iodide; FITC, fluorescein isothiocyanate; de, digoxin equivalent.

effect of plant cardenolides on apoptosis of malignant hematologic cell lines and pbmcs
We exposed Jurkat cells, K-562 cells, and PBMCs to UV irradiation, digoxin (10–500 nmol/L), or ouabain (10–500 nmol/L) for 48 h and stained the cells with annexin V/propidium iodide. Treatment with digoxin significantly increased the percentage of apoptotic cells in a dose-dependent manner in Jurkat cells (P <0.05), but not in K-562 cells or PBMC cultures, compared with the untreated controls (Fig. 2 ). Ouabain similarly induced apoptosis in Jurkat cells but not in K-562 cells or PBMCs (data not shown). Additional increases in the digoxin or ouabain concentration beyond the 500 nmol/L dose did not significantly increase apoptosis in Jurkat cells.


Figure 2
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  		Figure 2. Effect of digoxin on induction of apoptosis in lymphoblastic cancer cells.

Jurkat cells, K-562 cells, and PBMCs were exposed to UV irradiation or digoxin for 48 h at the indicated concentrations. The percentage of apoptosis (percentage of cells in early and late apoptosis relative to controls) was measured by flow cytometry (as in Fig. 1Up ). Data are expressed as the mean (SE) of 4 separate experiments. Asterisks denote significant difference (P <0.05) from the untreated control (Student t-test).

effect of dlif on apoptosis induction in malignant hematologic cell lines
We investigated the effect of DLIF (1–100 nmol/L) on apoptosis induction in a cell line that responded to plant cardenolide–induced apoptosis (Jurkat) and a cell line that resisted apoptosis (K-562). Exposure to DLIF significantly increased the mean (SE) rate of apoptosis in Jurkat cells (P <0.05) in a dose-dependent manner [1 nmol/L, 41.5% (3.6%); 10 nmol/L, 79% (3.6%); 100 nmol/L, 97.8% (2.9%)] compared with the untreated controls but not in K-562 cells [1 nmol/L, 4.2% (2.2%); 10 nmol/L, 9.4% (2.5%); 100 nmol/L, 8.6% (1.6%); Fig. 3A ]. DLIF at equivalent concentrations did not induce apoptosis in PBMCs (data not shown). The potency of DLIF-induced apoptosis in Jurkat cells appeared to be 10 times higher than the potencies of the plant-derived cardenolides: DLIF 50% inhibitory concentration (IC50), 1.9 nmol/L (95% CI, 1.7–2.4 nmol/L); digoxin IC50, 24 nmol/L (CI, 11–56 nmol/L); ouabain IC50, 26 nmol/L (CI, 19–48 nmol/L; Fig. 3B ).


Figure 3
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  		Figure 3. Induction of apoptosis in Jurkat and K-562 cell lines and the potency of DLIF relative to digoxin for inducing apoptosis in Jurkat cells.

Jurkat and K-562 cells were exposed to UV irradiation or digoxin for 48 h at the indicated concentrations. Apoptosis was measured by flow cytometry as the percentage of cells in early and late apoptosis. Data are expressed as the mean (SE) of 4 separate experiments. (A), asterisks indicate P <0.05 vs untreated control (Student t-test). (B), note differences between the effects of DLIF and digoxin on the induction of apoptosis in Jurkat cells.

comparison of the potencies of mammalian and plant cardenolides for inducing apoptosis and for inhibiting nka catalytic activity
We investigated the inhibitory potencies of DLIF, digoxin, and ouabain on PCC tissue, a standard system that expresses the 3 {alpha} NKA subunits ({alpha}1, {alpha}2, and {alpha}3) (22). Table 1 summarizes the IC50 values obtained in the 2 assays. The plant and mammalian cardenolides exhibited statistically significant differences (P <0.05) with respect to both the effect of apoptosis induction in Jurkat cells and the inhibition of NKA catalytic activity in PCC tissue. DLIF has at least a 100-fold greater potency for inducing apoptosis in Jurkat cells than for inhibiting NKA catalytic activity in PCC tissue. Digoxin and ouabain have at least a 20-fold greater potency for inducing apoptosis in Jurkat cells than for inhibiting NKA catalytic activity in PCC tissue.


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  		Table 1. Relative inhibitory potencies of plant and mammalian cardenolides on NKA catalytic activity in PCC and on the induction of apoptosis in lymphoblastic cell lines.1

effects of digoxin on caspase-3 activity
Exposure of Jurkat cells to UV irradiation or digoxin (100 nmol/L) increased the caspase-3 activity in these cells (2- and 8-fold, respectively) relative to that of untreated control cells (Fig. 4 ). Consistent with the annexin V/propidium iodide results, no increases in caspase-3 activity were observed in K-562 cells or PBMCs.


Figure 4
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  		Figure 4. Effect of cardiac glycoside on Asp-Glu-Val-Asp–dependent caspase-3 activity.

Tumor cell lines (1 x 1010 cells/L) and PBMCs (1 x 1010 cells/L) were exposed to UV irradiation or digoxin (100 nmol/L) for 12 h. Caspase-3 activity is expressed relative to that of untreated controls. The results of 3 independent experiments differed by <10%. C, control; UV, UV irradiation; dig, 100 nmol/L digoxin.

effect of digoxin and dlif on production of FasL mRNA
Jurkat cells showed at least a 10-fold increase in FasL mRNA compared with the untreated control (Fig. 5 ). No such change was observed in the apoptosis-resistant K-562 cell line. No increase in FasL mRNA production was evident in PBMCs after treatment with 100 nmol/L digoxin or 10 nmol/L DLIF (data not shown).


Figure 5
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  		Figure 5. FasL mRNA production in Jurkat and K-562 cells relative to controls.

Jurkat cells (1 x 109/L) and K-562 cells (1 x 109/L) were treated with 100 nmol/L digoxin or stimulated with 1 mg/L PHA for at least 12 h. FasL mRNA production was quantified by real-time reverse transcription–PCR analysis. Results are presented as the mean (SE) of 4 independent experiments. *, P <0.05 compared with the control.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study provides evidence that DLIFs selectively and specifically induce apoptosis in a human acute T-lymphoblastic leukemia cell line (Jurkat) but not in a human chronic myelogenous erythroblastoid leukemia cell line (K-562) or in human PBMCs. Previous reports have suggested that some plant-derived cardenolides induce apoptosis at nontoxic concentrations in a variety of human cancer cells, including those of hematologic origin (15)(23) and those described in this study. To our knowledge, mammalian cardenolides such as DLIF or ouabain-like factor have not been reported to possess proapoptotic properties against cancer or nonpathologic cells. The mammalian DLIF used in this study not only is selectively effective in inducing apoptosis in an acute T-cell leukemia but also appears to be more potent than a plant-derived counterpart, digoxin.

The pharmacology of cardiac glycosides has been extensively studied [for reviews, see Refs. (2)(24)]. NKA has been implicated as a target for the cardiac glycosides (digitalis) and their related congeners (1). They induce increases in intracellular calcium concentrations, particularly in myocardial cells (6). Whether these actions underlying the cardiotonic properties seen in myocardial cells also play significant roles in nonexcitable tissues is not known. Cardenolide-induced increases in intracellular calcium do not necessarily explain apoptosis induction in susceptible malignant cell lines (6). Our investigations raise questions about the specific targets of the plant and mammalian cardenolides with respect to their proapoptotic activities.

Our study compared the relative potency and efficacy of DLIF and digoxin on apoptosis induction in Jurkat cells and on inhibition of NKA catalytic activity in PCC tissue (Table 1Up ). PCC tissue produces the 3 NKA {alpha} subunits ({alpha}1, {alpha}2, and {alpha}3) (22), whereas Jurkat cells produce at least the {alpha}1 and {alpha}2 subunits (25). Whether the {alpha}3 subunit is also produced in this cell line is not known and needs to be evaluated in future studies. Interestingly, the potency of DLIF for inducing apoptosis in Jurkat cells was 100-fold greater than for inhibiting NKA catalytic activity in the PCC; the relative potency of digoxin was at least 20-fold greater in the same system (Table 1Up ). This result suggests that these compounds may have alternative binding sites and modes of action in these 2 model systems, but additional studies are required to confirm these apparent differences. Our results are consistent with recent work by Kometiani et al. (26), who demonstrated that nontoxic concentrations of ouabain activated a variety of proapoptotic signals in a breast cancer cell model. Collectively, our data are consistent with reports of antineoplastic activity induced by plant-derived cardenolides in malignant hematologic cell lines in vitro (5).

The mechanism by which cardiac glycosides induce apoptosis in transformed cells remains unclear, but several recent studies point to diverse mechanisms. These mechanisms include increases in intracellular calcium concentration, activation of the MAPK pathway, and altered expression of proapoptotic proteins such as caspase-3, antiapoptotic proteins such as Bcl-2, and proteins involved in reactive-oxygen generation (6)(23)(27)(28)(29)(30). A study by Repke et al. (15) suggested that the increased antineoplastic activity of cardiac glycosides in cancer cells stems from the inhibition of NKA activity by interrupting the flow of ADP and inorganic phosphate, increased turnover of glycolysis, and interrupting macromolecular biosynthesis. The net effect in highly proliferating cells such as cancer cells would be increased susceptibility to cell death via apoptosis. If this hypothesis were true, the antineoplastic effects of the plant and mammalian cardenolides would also be evident in other rapidly proliferating cells. We did not observe any such effect in K-562 cells. For example, the human myelogenous erythroblastoid cells (also a rapidly proliferating cell line) used in our studies resisted the proapoptotic effects of both the mammalian and plant cardenolides. Thus, it is possible that the increased antineoplastic activities of cardiac glycosides reported elsewhere (23) and those of the mammalian cardenolides found in this study may stem from a modification of the production of some proapoptotic proteins, such as FasL.

We explored this possibility and found that digoxin specifically upregulated FasL production in apoptosis-susceptible Jurkat cells but not in the resistant cell line K-562, suggesting that the increased antineoplastic effects of the cardenolides we observed may be due, at least in part, to activation of the Fas/FasL apoptosis pathway. This finding is particularly exciting because specific upregulation of a proapoptotic factor, FasL, by a cardiac glycoside has not previously been described. On the other hand, specific downregulation of other antiapoptotic proteins, such as Bcl-2, by cardiac glycosides is well recognized (28). Another intriguing finding stems from the fact that agents capable of specifically inducing apoptosis through upregulating FasL production are currently being explored as a novel treatment for a variety of neoplastic conditions, as well as for transplant rejection (31)(32). A number of these agents are currently available or in various stages of clinical trials [for a detailed review, see Ref. (33)]. We are currently exploring the possibility that the mammalian cardenolides can specifically modify FasL production in a variety of hematologic malignancies, including those of lymphoblastic origin.

Although the cardiac glycosides used in our studies induced apoptosis in malignant lymphoblastic cell lines, no such effects were seen in the myelogenous proerythroblastoid cell line or in PBMCs (Figs. 2Up and 3Up ). Of particular note, however, is the result that the resistant cells (proerythroblastoid cells and PBMCs) were also resistant to apoptosis induction by the same dose of UV irradiation required to induce apoptosis in Jurkat cells (Figs. 2Up and 3Up ). These observations raised questions about the specificity of plant and mammalian compounds and the possibility of upregulating some antiapoptotic genes (e.g., BCL2) in apoptosis-susceptible cells, as has been demonstrated elsewhere (34). To address some of these issues, we compared the effect of digoxin on apoptosis induction in Jurkat cells and PBMCs after stimulating the cells with and without PHA. PHA promotes activation-induced cell death by apoptosis in T lymphocytes through activation of the Fas/FasL apoptosis pathway (35). We treated Jurkat cells and PBMCs with digoxin for 48 h, either alone or after a 24-h pretreatment with PHA, and measured apoptosis as described above (compared with apoptosis induced with PHA alone). Interestingly, digoxin synergistically increased the percentage of apoptosis in the acute T-cell lymphoblastic cell line but not in the PBMCs, indicating that digoxin-induced apoptosis was specific to the T-cell leukemic cell line (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue7). These data are consistent with previous studies (35) that showed activation-induced cell death in mature T lymphocytes and transformed T cells to be mediated by interaction with the Fas/FasL apoptosis pathway. Thus, resting T lymphocytes constitutively produce Fas but not FasL. On stimulation with PHA, a T-cell receptor ligand, FasL, is induced, and Fas/FasL interactions lead to apoptosis (35)(36).

Of significant relevance to the data we have presented is the fact that DLIFs are synthesized by the adrenal glands; DLIFs have thus been speculated to constitute a hypothalamic-pituitary-adrenal hormonal axis regulating NKA activity (8). The physiological roles of these mammalian compounds remain unclear, at least in cancer, particularly because the reported concentrations of these cardenolides in the blood appear to be 10–100 times lower (37) than the therapeutic blood concentrations of digoxin (1–2 nmol/L) (26) used in cardiac diseases. If these endogenous compounds in fact possess the same proapoptotic activities as the plant-derived compounds, then it is likely that an endogenous mechanism exists that regulates apoptosis by selectively destroying transformed cells in vivo. Thus, DLIFs may play a protective role as natural adjuncts to cancer therapy by selectively sensitizing neoplastic cells for physiological removal or destruction by chemotherapeutic agents. Interestingly, these events occur at nontoxic (nanomolar) concentrations, well below the concentrations of any reported toxic complications encountered with therapeutic cardiac glycoside doses. In any event, further experiments are required to confirm the molecular mechanisms underlying mammalian cardenolide–induced apoptosis in malignant hematologic cell lines.

The search for new anticancer drugs that selectively induce apoptosis in cancer cells and spare nonpathologic cells constitutes an important and powerful approach for cancer prevention and treatment. The data presented here indicate that the mammalian DLIFs selectively induce apoptosis in human leukemic cancer cells. A number of plant-derived agents are presently in various stages of clinical trials (38)(39)(40). Our study adds to the evidence suggesting anticancer properties for cardiac glycosides and introduces a potential new role for the endogenous mammalian cardenolide-like compounds DLIF and ouabain-like factors.


   Acknowledgments
 
Grant/funding support: Partial support for this study was provided by grants from the University of Louisville School of Medicine and the Department of Pathology and Laboratory Medicine Research Development Fund.

Financial disclosures: None declared.


   Footnotes
 
3 Current affiliation: Department of Life Science, Division of Biomedical and Molecular Sciences, Texas A&M University, Corpus Christi, TX.

1 Nonstandard abbreviations: NKA, Na+- and K+-dependent ATPase; DLIF, digoxin-like immunoreactive factor; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; PCC, porcine cerebral cortex; UV, ultraviolet; IC50, 50% inhibitory concentration.

2 Human genes: FASLG, Fas ligand (TNF superfamily, member 6); PPIA, peptidylprolyl isomerase A (cyclophilin A); BCL2, B-cell CLL/lymphoma 2.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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