Sage Journals: Discover world-class research

Abstract

While there are targeted treatments for triple positive breast cancers, lack of specific biomarkers for triple-negative breast cancers (TNBC) has hindered the development of therapies for this subset of cancers. In this study, we evaluated the anticancer properties of cardiac glycoside Digitoxin (Dtx) and its synthetic analog MonoD on breast cancer cell lines MCF-7 (estrogen receptor-positive breast cancer) and MDA-MB-468 (triple-negative breast cancer). Both cardiac glycosides, at concentrations within the therapeutic range, increased the fraction of cells in the G₀/G₁ phase of the cell cycle, decreased viability, and inhibited the migration of MCF-7 and MDA-MB-468 cells. Both cardiac glycosides increased production of superoxide and induced apoptosis in both cell types. Reduced protein levels of nuclear factor kappa B and IkappaB kinase-beta were found in cardiac glycoside-treated cells, indicating that the cellular effects of these compounds are mediated via nuclear factor kappa B pathway. This study demonstrates the cytotoxic potential of digitoxin, and more importantly its synthetic analog MonoD, in the treatment of triple-positive breast cancer and more importantly the aggressive triple-negative breast cancer. Collectively, this study provides a basis for the reevaluation of cardiac glycosides in the treatment of breast cancer and more importantly reveals their potential in the treatment of triple-negative breast cancers.

Keywords

Breast cancer triple negative estrogen receptor positive digitoxin MonoD apoptosis reactive oxygen species

Introduction

Breast cancer is a heterogeneous disease that can be subdivided into triple-positive breast cancer (TPBCs) or triple-negative breast cancers (TNBCs) on the basis of expression of molecular markers. TNBCs lack estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) and do not respond to hormonal therapies or to HER2-specific antibodies and inhibitors such as trastuzumab or lapatinib. TNBCs occur at a higher frequency in young premenopausal women with African ancestry and account for 15%–20% of all breast cancer cases.¹ TNBCs are aggressive with higher rates of relapse and shorter overall survival compared with other subtypes of breast cancer. Because of the lack of well-defined molecular targets, cytotoxic chemotherapy is currently the only treatment option for TNBCs.² For advanced disease, a number of clinical trials using drugs that target angiogenesis, poly-adenosine diphosphate (ADP)-ribose-polymerase (PARP), epidermal growth factor receptor (EGFR), phosoinositol-3 kinase (PI3K), mitogen-activated protein kinase (MAPK), checkpoint kinase (CHK), and histone-deacetylase (HDAC) pathways are ongoing, but preliminary data suggest that the clinical benefit from such therapies was still limited.² There is therefore an urgent need to identify new anticancer drugs that target breast cancer independent of their receptor status and against TNBCs in particular.

Digitoxin (Dtx), a cardenolide used to treat cardiac disorders, showed promise as an anticancer agent before concerns regarding its narrow therapeutic range prematurely tempered enthusiasm in relation to its tumoristatic potential. A few studies have evaluated the anticancer properties of Dtx alone or in combination with other potential anticancer drugs on breast cancers. Dtx alone can sensitize T47D breast cancer cells (ER⁺ and PR⁺) to anoikis³ and has potent antiproliferative effects on several breast cancer cell lines,⁴ and its effect can be potentiated with actein⁵ or paclitaxel.⁶ However, with regard to breast cancer subtypes, only one study compared the effect of Dtx on ER⁺ versus ER⁻ using three different cell lines isolated form Caucasian patients: MDA-MB-453 (ER⁻ and Her2 overexpressing), MCF-7 (ER⁺ and Her2 low), and BT474 (ER⁺ and Her2 overexpressing).⁶ While the study suggested that a combination of Dtx and Paclitaxel is a promising treatment for ER-negative breast cancer, there have not been any studies evaluating the effect of Dtx on TNBC from African descent.

In this study, we evaluated the anti-tumorigenic effect of the Dtx and its novel monosaccharidic analog MonoD on MCF-7 versus MDA-MB-468 cells. While the MCF-7 cell line was isolated from a pleural effusion of a 69-year-old Caucasian female patient from a metastatic site, the MDA-MB-468 cell line is a TNBC (ER⁻, PR⁻, and Her2⁻) cell line that was isolated from a pleural effusion of a 51-year-old black female patient with metastatic adenocarcinoma of the breast.⁷ Our data demonstrate that Dtx and MonoD inhibit proliferation and migration and induce oxidative stress, cell-cycle arrest, and apoptosis of both breast cancer cells independent of their receptor status at a concentration within the therapeutic range. We believe that further exploration of this class of drugs may hold the promise for a suitable therapeutic strategy against TPBCs and TNBCs in patients from both African and Caucasian origin.

Materials and methods

Synthesis of MonoD

The β-D-Digitoxose (MonoD) form of Dtx was synthesized using a palladium-based de novo method as described previously by Iyer et al.⁸

Chemicals and reagents

Thiazolyl blue tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)), cisplatin, sodium dichromate (Na₂Cr₂O₇.2H₂O) (Cr(VI)), Triton X-100, DNAse-free RNAse, and propidium iodide were obtained from Sigma Aldrich (St. Louis, MO). The oxidative probes 4,5-diaminofluorescein diacetate (DAF-2DA), dichlorofluorescein (DCF) diacetate, dihydroethidium (DHE), and the apoptosis dye Hoechst 33342 were obtained from Molecular Probes (Eugene, OR). Antibodies for PARP, caspase-9, p-Akt (Ser 473), total-Akt, p27, cyclin D3, cyclin-dependent kinase 2 (CDK2), p21, nuclear factor kappa B (NF-κB), and IkappaB kinase-beta (IKK-β) and peroxidase-labeled secondary rabbit and mouse antibodies were purchased from Cell Signaling Technology (Denvers, MA). Antibody for Bcl-2 was procured from Santa Cruz Biotechnology (Dallas, TX). Bicinchoninic acid (BCA) and SuperSignal^® West Pico chemiluminescent substrate were purchased from Thermo Fisher Scientific (Waltham, MA).

Preparation of anticancer drugs

Dtx and MonoD were stored as stock solution (10 mM) in dimethyl sulfoxide (DMSO) in glass containers. Final dilutions were prepared freshly in culture media before use. The control experiment contained the highest concentration (0.001%) of DMSO only. Cisplatin was prepared as stock solution (100 mM) in DMSO and stored in aliquots at −20°C.

Cell culture

The human breast cancer cell lines MCF-7 and MDA-MB-468 were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/high glucose supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. All cells were cultured in a 5% CO₂ environment at 37°C.

MTT assay

Cells were plated in 96-well cell-culture microplates (Costar, IL, USA) at ~2500 cells per well and incubated overnight in cell-culture medium. The cells were subsequently exposed to the appropriate concentration of drug or vehicle for 72 h in serum-free media. Cell viability was evaluated by the MTT assay. The absorbance of solubilized formazan was read at 570 nm using the BioTek Synergy plate reader (BioTek, VT, USA). In all cases, the highest concentration of DMSO was used in the control, and this concentration was maintained below 0.001% (v/v). This DMSO concentration did not show any significant antiproliferative effect.

Cell-cycle synchronization and analysis

Cells were synchronized in G₀/G₁ phase by serum starvation. Briefly, 5 × 10⁵ cells were plated overnight in a six-well plate in medium containing 1% FBS before culturing them in serum-free medium for 72 h, followed by restimulation with complete medium for control samples, and addition of drugs in complete medium for test samples. Cells were harvested after 72 h serum starvation for a serum-free control. Other samples were re-stimulated in complete medium with and without Dtx/MonoD for additional 24 h.

Cell-cycle analysis based on DNA content was performed as follows. Cells were harvested and washed twice with ice cold phosphate-buffered saline (PBS). Cells were then fixed in 2 mL of ice cold 70% ethanol for at least 24 h at −20°C. Cells were centrifuged, resuspended in 500 µL of propidium iodide staining solution (0.1% (v/v) Triton X-100, 2 mg DNAse-free RNAse A, and 400 µL of 500 µg/mL PI in 10 mL of PBS), and incubated at room temperature for 30 min. The cells were then analyzed with BD FACSCalibur (Becton Dickinson Immunocytometry, Mountain View, CA), Acea Novocyte 2060 (Acea Biosciences, San Diego, CA), and ModFit LT™ cycle analysis software (Verity Software, Topsham, ME). The cell-cycle distribution is shown as the percentage of cells containing G0/G1, S, and G2/M DNA as identified by propidium iodide staining.

Apoptosis assay

Apoptotic cells were detected by Hoechst 33342 and counting the percentage of cells with apoptotic nuclei (cells having intensely condensed and/or fragmented nuclei). The apoptotic index was calculated as the percentage of cells with apoptotic nuclei over total number of cells. Cells were incubated with 10 µg/mL of Hoechst 33342 for 30 min and visualized under EVOS^® FL Cell Imaging System fluorescence microscope. Apoptotic cells were quantified by creating a grid using ImageJ software and using a cell counter. Approximately 50 nuclei from five random fields were analyzed for each sample.

Detection of reactive oxygen species and nitric oxide

Intracellular nitric oxide (NO), hydrogen peroxide (H₂O₂), and superoxide ( $\overset{\cdot\cdot}{Y} O_{2}^{-}$ ) production was determined by fluorometric analysis using specific probes DAF, DCF, and DHE, respectively, as described earlier.⁹ Briefly, cells were incubated with the respective probe (10 µM) for 30 min at 37°C and analyzed for fluorescence intensity using a multi-well plate reader at the excitation/emission wavelengths of 495/515, 485/535, and 485/610 nm for DAF, DHE, and DCF, respectively.

Western blotting

Preparation of cell lysates and western blotting were performed as described previously.¹⁰ Briefly, equal amount of protein per sample (40 µg) was resolved on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a 0.45-mm nitrocellulose membrane (Pierce, Rockford, IL) using Trans-Blot Turbo™ Transfer System from Bio-Rad (Hercules, CA). The transferred membranes were blocked for 1 h in 5% non-fat dry milk in Tris buffered saline with Tween 20 (TBST; 0.05% Tween-20 in TBS) and incubated with the appropriate primary antibodies (dilution 1:1000) and horseradish peroxidase–conjugated isotype-specific secondary antibodies (dilution 1:5000). The immune complexes were detected by chemiluminescence imaged using myECL Imager (Thermo Fisher Scientific, Waltham, MA) and quantified by imaging densitometry using ImageJ software. Mean densitometry data from independent experiments were normalized to the control where indicated.

Wound healing assay (scratch assay)

Cells were plated at 3 × 10⁵ cells/well in a 24-well plate and allowed to grow to 85%–90% confluence as a monolayer. Gently, the cells were scratched with a 1 mL sterile tip across the center of the well. Cells were washed thrice with PBS to remove the detached and loosely adhered cells and phase-contrast images were photographed as control. Cells were treated with Dtx and MonoD in medium containing 5% FBS for 24 h and photographed. The cell migration within 24 h was calculated by measuring the area of the scratch using ImageJ.

Statistic analysis

The IC₅₀ (drug concentrations inhibiting cell growth by 50%) were determined by interpolation from the dose-response curves using a sigmoidal logistic three-parameter equation. Curve fitting was performed with Sigmaplot (V.11.0) software. The data represent mean ± SD from three or more independent experiments with triplicates or quadruplicates. Level of significance between different treatment groups relative to control was determined by one-way and two-way analyses of variance (ANOVAs); p < 0.05 was considered statistically significant.

Results

Digitoxin and MonoD inhibit viability of MCF-7 and MDA-MB-468 cells

Cells were seeded at 2500 cells/well and allowed to adhere overnight. Then, cells were treated with Dtx and MonoD (0, 10, 50, or 100 nM) in serum-free media for 24, 48, or 72 h. Cell viability was determined by the MTT assay. Figure 1 shows that both cardiac glycosides (CGs) inhibit the viability of MCF-7 and MDA-MB-468 breast cancer cells. The decrease in viability after 24 h in MCF-7 with 50 nM Dtx was comparable with 10 nM MonoD; and similarly, the decrease in viability with 100 nM Dtx was comparable with the viability with 50 nM MonoD.

Figure 1.

Dtx and MonoD inhibit the viability of (a) MCF-7 and (b) MDA-MB-468 breast cancer cells. Cells were incubated with the indicated concentrations of Dtx or MonoD for 24, 48, or 72 h. Cell viability was evaluated by the MTT assay. Cisplatin was used as a positive control. Data (mean ± SD) are representative of three independent experiments performed in triplicates.

Digitoxin and MonoD arrest MCF-7 and MDA-MB-468 cells in G₀/G₁

Cells were seeded at 5 × 10⁵ cells/well in a six-well plate and allowed to adhere overnight. Synchronized cells were treated with Dtx (25 nM) or MonoD (25 nM) in complete medium for 24 h. Figure 2 shows that both CGs increase the percentage of cells in the G₀/G₁ phase of the cell cycle. Cell-cycle synchronization was evident from the serum-free control harvested 72 h post serum starvation which had the cell-cycle distribution of 76% in G₀/G₁, 14% in S, and 8% in G₂/M phase. Upon restimulation of the cells to enter the cell cycle by addition of complete medium, the cells started proliferating, which was indicated by the increase in S phase (26%) and G₂/M phase (19%) with a corresponding decrease in G₀/G₁ phase (53%). Synchronized MCF-7 cells when treated with Dtx and MonoD for 24 h in serum-containing medium showed an arrest in G₀/G₁ phase, with 25% increase (p < 0.05) in G₀/G₁ cell population with a significant (p < 0.05) decrease cells in S and G₂/M phases (Figure 2(a) and (b)). Similar cell-cycle arrest with a 25% increase G₀/G₁ phase (p < 0.05) in synchronized MDA-MB-468 cells came at the expense of G2/M phase alone (p < 0.05) with no significant change in S phase (Figure 2(c) and (d)).

Figure 2.

Dtx and MonoD induce cell-cycle arrest by increasing the fraction of cells in G₀/G₁ phase of MCF-7 and MDA-MB-468 breast cancer cells. Cells were synchronized before treating them with 25 nM of Dtx and MonoD in serum containing medium for 24 h. Cell-cycle distribution was determined by the DNA content as analyzed by propidium iodide staining. Increase in G₀/G₁ phase is shown by the histograms and bar graphs for (a and b) MCF-7 and (c and d) MDA-MB-468, respectively.

Digitoxin and MonoD inhibit migration of MCF-7 and MDA-MB-468 cells

The effect of Dtx and MonoD on cell migration was assessed using monolayer scratch assay.¹¹ The rate of migration was calculated by measuring the total area of the scratch at 0 h and 24 h. The treatment was done in the presence of 5% FBS in order to provide the cells a stimulant for migration and minimize the interference from cellular apoptosis. Dtx (p < 0.05) and MonoD (p < 0.05) reduced the migration of MCF-7 by over 75% (Figure 3(a), Supplementary Figure 1). While Dtx did not show a significant change in the rate of migration of MDA-MB-468 (Figure 3(b), Supplementary Figure 2), MonoD reduced the migration by over 60% (p < 0.05).

Figure 3.

Dtx and MonoD inhibit the migration of MCF-7 and MDA-MB-468 breast cancer cells. Cells were incubated with 50 nM of Dtx and MonoD in serum containing 5% FBS for 24 h. Monolayers of cells were scratched with a 1 mL sterile tip and photographed at 0 h as control and post 24 h to measure the effect of Dtx and MonoD on cell migration. Relative migration at 24 h is shown for (a) MCF7 and (b) MDA-MB-468, respectively.

Digitoxin and MonoD increase the production of superoxide ( $\overset{\cdot\cdot}{Y} O_{2}^{-}$ ) in MCF-7 and MDA-MB-468 cells

Reactive radicals including reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to play a crucial role in activating apoptosis.¹² A 4-electron reduction of oxygen to 1-electron form with the release of cytochrome c from the mitochondria during apoptosis is associated with production of superoxide.¹³ Therefore, we assessed for cellular ROS-RNS levels in Dtx- and MonoD-treated MCF-7 and MDA-MB-468 cells (Figure 4). While no significant change in the levels of nitric oxide and hydrogen peroxide was observed, both Dtx and MonoD induced higher levels of superoxide as assayed using DHE in both the breast cancer subtypes (p < 0.05).

Figure 4.

Dtx and MonoD increase the levels of superoxide ( $\overset{\cdot\cdot}{Y} O_{2}^{-}$ ) in (a) MCF-7 and (b) MDA-MB-468 breast cancer cells. Cells were incubated with 50 nM of Dtx or MonoD for 1 h. Cr (IV) was used as a positive control.

Digitoxin and MonoD induce apoptosis in MCF-7 and MDA-MB-468 cells

Given that Dtx and MonoD reduce the viability/proliferation in both these breast cancer cells, we wanted to investigate the ability of these drugs to induce apoptotic cell death. Cells were treated with Dtx and MonoD (50 nM) in serum-free media for 24 h. Apoptotic cells were detected by Hoechst 33342 staining. Figure 5 shows that Dtx and MonoD induce apoptosis as characterized by nuclear condensation and fragmentation in MCF-7 and MDA-MB-468 cells. Moreover, as observed with the MTT assay, the effect of MonoD on MCF-7 cells was significantly higher (p < 0.05) as compared with Dtx. Both MCF-7 as well MDA-MB-468 showed same level of sensitivity to apoptotic stimuli by MonoD.

Figure 5.

Dtx and MonoD induce apoptosis in (a and b) MCF-7 and (c and d) MDA-MB-468 breast cancer cells. Cells were incubated with 50 nM of Dtx, MonoD, and 300 µM Cisplatin (positive control) for 24 h and stained with Hoechst 33342 as indicated in section “Materials and Methods.” Bar graphs show the numbers of apoptotic nuclei. Data (mean ± SD) are representative of three independent experiments performed in triplicates (*p < 0.05 for each drug treated data point as compared to un-treated control).

Validation of cell-cycle arrest and apoptosis by immunoblotting for protein markers

Apoptosis was confirmed by western blot analysis that showed a marked increase in cleaved PARP, a hallmark of apoptosis and caspase activation.^14,15 Figure 6 shows that Dtx treatment showed a marked increase in cleaved PARP at 24 h in MCF-7 as compared to 12 h. However, MonoD had comparable levels of cleaved PARP in MCF-7 at 12 h as well as 24 h, suggesting that MonoD potentiated apoptosis faster as compared to Dtx. Bcl-2 is a pro-survival factor which prevents the release of cytochrome c from mitochondria, thereby inhibiting the activation of caspases and inhibiting apoptosis.¹⁶ We observed decreased expression of Bcl-2 with a 24 h treatment in both cell lines. Both Dtx and MonoD treatment reduced the levels of pro-caspase-9 and showed an increase in cleaved caspase-9 fragments. The activated caspase-9 activates caspase-3 and caspase-7 downstream along the apoptotic pathway.¹⁷ Akt activation has been shown to positively regulate cellular proliferation and negatively regulate apoptosis.¹⁸ Dtx and MonoD treatment decreased Akt activation in MCF-7 cells, thereby potentiating apoptosis. Interestingly, both these drugs, and MonoD to a larger extent, caused an induction of Akt in MDA-MB-468.

Figure 6.

Dtx and MonoD induced modulation in the expression levels of markers of apoptosis, cell cycle, and the NF-κB pathway. Cells treated for 12 and 24 h with 50 nM Dtx and MonoD were lysed and assayed for detection of markers of (a and b) apoptotic, (c) cell cycle, and (d and e) NF-κB pathway. Blots were re-probed with β-actin antibody to confirm equal loading of the samples.

NF-κB is known to inhibit programmed cell death through the activation of target genes, which block the induction of apoptosis.¹⁹ IKK-β upon activation phosphorylates IκB and liberates NF-κB bound to it, which is translocated to the nucleus to activate the target genes involved in the inhibition of apoptosis.²⁰ Both CGs showed downregulation of IKK-β and NF-κB over a period of 24 h.

p27 is a key regulator of G1–S phase transition and its activity is often impaired in breast and other cancers, with loss of p27 being a strong indicator of poor prognosis particularly in breast cancer.²¹ Both these drugs induced expression of p27 over 24 h. Cyclin D3 is another protein that regulates the cell-cycle progression from G1 to S phase by forming a complex with CDK4/6.²² MCF-7 and MDA-MB-468 showed a downregulation of cyclin D3 with 24 h treatment, indicating impairment in the activity of cyclin D3–dependent kinases, thereby causing cell-cycle arrest. CDK2 is a key regulator of G1–S phase progression by binding to cyclin E or cyclin A.²³ Our test drugs reduced the expression of CDK2, thereby blocking the cell-cycle progression from G1 to S phase. CDK inhibitor p21, in addition to its function as an inhibitor of proliferation, also acts as an inhibitor of apoptosis, thereby counteracting its function as a tumor suppressor.^24,25 We observed a downregulation of p21, suggesting that p21 acts as an inhibitor of apoptosis in our system.

Discussion

Of the several subtypes of breast cancer, the hormone-refractory “triple-negative breast cancer” (TNBC; indicating lack of estrogen, progesterone, and HER2neu receptor expression) is less responsive to treatment and has a much worse prognosis as compared to receptor-positive breast cancer.^26,27 The 5-year survival rate for TNBC is 77% versus 93% of women with other types of breast cancer,²⁸ emphasizing an unmet pressing need to identify novel compounds that can target this particular subtype of breast cancer. The chemotherapeutic potential of CGs and particularly Dtx has been surprisingly underinvestigated in TNBCs, given its ability to induce cytotoxicity in other cancer types. The therapeutic plasma levels of Dtx are considered to be in the range of 13–33 nM^4,29 and up to 46 nM.⁶ An epidemiologic study showed that control patients were 10 times more likely to have recurrence of breast cancer as compared to patients who were administered digitalis.³⁰ Thus, digitoxin at therapeutic, non-toxic concentrations of CGs seems to be a promising anticancer drug. We have synthesized its monosaccharide analog MonoD that has higher cytotoxicity compared to the parent compound at the same concentration and can therefore exert potent anti-tumor effects at sub-physiologic doses as compared to Dtx in lung cancer.^8,10 Here, we have demonstrated the anti-tumorigenic potential of Dtx as well as MonoD in TNBC.

In this study, we first demonstrated a concentration-dependent effect on the antiproliferative activity of Dtx and its synthetic analog MonoD on MCF-7 (triple positive) and MDA-MB-468 (triple negative) cell lines at clinically tolerated concentrations (Figure 1). While both CGs showed cytotoxic potential against breast cancers independent of receptor status, MonoD showed higher efficacy in MCF-7 as compared to Dtx. Several in vitro models of apoptotic cell death share the common features of cell-cycle arrest and inhibition of migration, eventually leading to apoptosis.^31,32 Both CGs significantly increased the percentage of cells in the G₀/G₁ phase of the cell cycle (Figure 2) and inhibited the migration of cancer cells (Figure 3) as measured by the scratch assay. This assay is a straight forward method to study cell migration in vitro that mimics migration of cells in vivo.¹¹ Reactive radicals, and ROS in particular, are considered to be pivotal mediators of apoptosis and is a feature of many effective chemotherapeutic agents.^33,34 Both CGs were found to induce oxidative stress by increasing the production of superoxide ( $\overset{\cdot\cdot}{Y} O_{2}^{-}$ ) without altering the production of H₂O₂ or NO (Figure 4). Increased production of $\overset{\cdot\cdot}{Y} O_{2}^{-}$ was reported previously for the cardenolide oleandrin in human melanoma cells.³⁵ Increased superoxide that causes tumor cell injury through mitochondrial disruption³⁶ partially explains the decreased cellular viability observed in our system. This notion is supported by the fact that both Dtx and MonoD decreased the levels of Bcl-2, a key pro-survival protein associated with mitochondrial integrity.³⁷ Induction of apoptosis by Dtx and MonoD was assessed by Hoechst staining (Figure 5) and confirmed by western blot, which showed increased levels of protein markers associated with apoptosis such as cleaved PARP and cleaved caspase-9 (Figure 6). The decrease in the levels of Bcl-2 indicates that apoptosis occurred via activation of the intrinsic pathway that is known to be activated by stress stimuli.³⁸

Conclusion

In summary, we have demonstrated that Dtx and MonoD, previously shown to be effective cytotoxic agents against lung cancer, have great potential as a chemotherapeutic agent in breast cancer. Chemotherapeutic treatment options for cancer still remain limited, particularly for breast cancers that have no receptors that can be targeted. Therefore, there is an urgent need to synthesize novel anticancer drugs such as MonoD that can be effective against cancers independent of their receptor status. Furthermore, the effectiveness of MonoD against TNBCs from African origin is particularly exciting, given the dearth of effective therapeutic strategies that can combat this particular type of breast cancer. This study will form the basis for future investigation into the pathways associated with MonoD action and will allow for further characterization of CGs as a viable class of drugs to treat cancer.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research,authorship,and/or publication of this article: This work was supported by National Institutes of Health grants HL112630 (NA) and CA173069 (AKVI).

References

Peddi

Ellis

Molecular basis of triple negative breast cancer and implications for therapy. Int J Breast Cancer 2012; 2012: 217185.

Palma

Frasci

Chirico

. Triple negative breast cancer: looking for the missing link between biology and treatments. Oncotarget 2015; 6: 26560–26574.

Simpson

Mawji

Anyiwe

. Inhibition of the sodium potassium adenosine triphosphatase pump sensitizes cancer cells to anoikis and prevents distant tumor formation. Cancer Res 2009; 69: 2739–2747.

Kometiani

Liu

Askari

Digitalis-induced signaling by Na⁺/K⁺-ATPase in human breast cancer cells. Mol Pharmacol 2005; 67: 929–936.

Einbond

Shimizu

. Actein inhibits the Na⁺-K⁺-ATPase and enhances the growth inhibitory effect of digitoxin on human breast cancer cells. Biochem Biophys Res Commun 2008; 375: 608–613.

Einbond

. Digitoxin activates EGR1 and synergizes with paclitaxel on human breast cancer cells. J Carcinog 2010; 9: 10.

Lehmann

Bauer

Chen

. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 2011; 121: 2750–2767.

Iyer

Zhou

Azad

. A direct comparison of the anticancer activities of digitoxin MeON-neoglycosides and O-glycosides: oligosaccharide chain length-dependent induction of caspase-9-mediated apoptosis. ACS Med Chem Lett 2010; 1: 326–330.

Azad

Iyer

Wang

. Reactive oxygen species-mediated p38 MAPK regulates carbon nanotube-induced fibrogenic and angiogenic responses. Nanotoxicology 2013; 7: 157–168.

10.

Kulkarni

Kaushik

Azad

. Autophagy-induced apoptosis in lung cancer cells by a novel digitoxin analog. J Cell Physiol 2015; 231: 817–828.

11.

Liang

Park

Guan

JL.

In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2: 329–333.

12.

Simon

Haj-Yehia

Levi-Schaffer

Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 2000; 5: 415–418.

13.

Cai

Jones

DP.

Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem. 1998; 273: 11401–11404.

14.

Yang

Zhao

Song

Caspase-dependent apoptosis and -independent poly(ADP-ribose) polymerase cleavage induced by transforming growth factor beta1. Int J Biochem Cell Biol 2004; 36: 223–234.

15.

Boulares

Yakovlev

Ivanova

. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J Biol Chem 1999; 274: 22932–22940.

16.

Yang

Liu

Bhalla

. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275: 1129–1132.

17.

Nijhawan

Budihardjo

. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479–489.

18.

Kim

Khursigara

Sun

. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol 2001; 21: 893–901.

19.

Wang

Mayo

Baldwin

ASJ

. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science 1996; 274: 784–787.

20.

Luo

Kamata

Karin

IKK/NF-kappaB signaling: balancing life and death—a new approach to cancer therapy. J Clin Invest 2005; 115: 2625–2632.

21.

Alkarain

Jordan

Slingerland

p27 deregulation in breast cancer: prognostic significance and implications for therapy. J Mammary Gland Biol Neoplasia 2004; 9: 67–80.

22.

Sherr

CJ.

D-type cyclins. Trends Biochem Sci 1995; 20: 187–190.

23.

Rosenblatt

Morgan

DO.

Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A. Proc Natl Acad Sci U S A 1992; 89: 2824–2828.

24.

Gartel

Tyner

AL.

The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther 2002; 1: 639–649.

25.

Bunz

Hwang

Torrance

. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest 1999; 104: 263–269.

26.

Carey

Perou

Livasy

. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 2006; 295: 2492–2502.

27.

Perou

Sorlie

Eisen

. Molecular portraits of human breast tumours. Nature 2000; 406: 747–752.

28.

Pal

Childs

Pegram

Triple negative breast cancer: unmet medical needs. Breast Cancer Res Treat 2011; 125: 627–636.

29.

López-Lázaro

Pastor

Azrak

. Digitoxin inhibits the growth of cancer cell lines at concentrations commonly found in cardiac patients. J Nat Prod 2005; 68: 1642–1645.

30.

Stenkvist

Bengtsson

Eklund

. Evidence of a modifying influence of heart glucosides on the development of breast cancer. Anal Quant Cytol 1980; 2: 49–54.

31.

Xiong

Zhang

Tian

. Inhibition of JAK1, 2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells. Neoplasia 2008; 10: 287–297.

32.

Johnson

Saigal

Talpaz

. Dasatinib (BMS-354825) tyrosine kinase inhibitor suppresses invasion and induces cell cycle arrest and apoptosis of head and neck squamous cell carcinoma and non-small cell lung cancer cells. Clin Cancer Res 2005; 11: 6924–6932.

33.

Kannan

Jain

SK.

Oxidative stress and apoptosis. Pathophysiology 2000; 7: 153–163.

34.

Conklin

KA.

Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr Cancer Ther 2004; 3: 294–300.

35.

Newman

Yang

Hittelman

. Oleandrin-mediated oxidative stress in human melanoma cells. J Exp Ther Oncol 2006; 5: 167–181.

36.

Kumar

Mishra

. Oleandrin: a cardiac glycosides with potent cytotoxicity. Pharmacogn Rev 2013; 7: 131–139.

37.

Dewson

Mitochondria and apoptosis: emerging concepts. F1000Prime Rep 2015; 7: 42.

38.

Plati

Bucur

Khosravi-Far

Apoptotic cell signaling in cancer progression and therapy. Integr Biol 2011; 3: 279–296.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.27 MB

0.58 MB

Anti-tumorigenic effects of a novel digitoxin derivative on both estrogen receptor–positive and triple-negative breast cancer cells

Abstract

Keywords

Introduction

Materials and methods

Synthesis of MonoD

Chemicals and reagents

Preparation of anticancer drugs

Cell culture

MTT assay

Cell-cycle synchronization and analysis

Apoptosis assay

Detection of reactive oxygen species and nitric oxide

Western blotting

Wound healing assay (scratch assay)

Statistic analysis

Results

Digitoxin and MonoD inhibit viability of MCF-7 and MDA-MB-468 cells

Digitoxin and MonoD arrest MCF-7 and MDA-MB-468 cells in G0/G1

Digitoxin and MonoD inhibit migration of MCF-7 and MDA-MB-468 cells

Digitoxin and MonoD increase the production of superoxide ( Y ·· O 2 − ) in MCF-7 and MDA-MB-468 cells

Digitoxin and MonoD induce apoptosis in MCF-7 and MDA-MB-468 cells

Validation of cell-cycle arrest and apoptosis by immunoblotting for protein markers

Discussion

Conclusion

Footnotes

Declaration of conflicting interests

Funding

References

Supplementary Material

Digitoxin and MonoD arrest MCF-7 and MDA-MB-468 cells in G₀/G₁

Digitoxin and MonoD increase the production of superoxide ( $\overset{\cdot\cdot}{Y} O_{2}^{-}$ ) in MCF-7 and MDA-MB-468 cells