Inhibition of cell proliferation, invasion and migration by the cardenolides digitoxigenin monodigitoxoside and convallatoxin in human lung cancer cell line

<div><p>Cardiac glycosides consist of a large family of naturally derived compounds that are clinically used to treat congestive heart failure, and also present anticancer properties. In this study, the cytotoxic effects of two cardenolides, digitoxigenin monodigitoxoside (DGX) and convallatoxin (CON) were screened in four human tumour cell lines. Both compounds showed anti-proliferative effects in all tumour cells, at nanomolar concentrations. Since the human lung cancer cell line A549 was the most sensitive, we investigated the anti-proliferative, anti-migratory and anti-invasive effects of these cardenolides. DGX and CON reduced A549 cell migration, being able to reduce more than 90% of cell invasion. Their effects on the expression of key regulators of metastatic mechanism showed decreased levels of MMP-2, MMP-9 and p-FAK. Both compounds also presented low toxicity for healthy cells. Finally, this work provides the first insights into the effects of these cardenolides on key steps of lung cancer metastasis.</p></div

Effects of DACE on TNFα-mediated activation of signaling pathways.

<p>(A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.</p

Scheme for preparation of a novel semisynthetic derivative of cucurbitacin B (DACE).

<p>Scheme for preparation of a novel semisynthetic derivative of cucurbitacin B (DACE).</p

Effects of DACE on c-RAF-1-induced lung tumor growth in mice.

<p>(A and B) c-RAF-1-BxB transgenic mice were injected daily with either DMSO (n = 4, A) or 1 mg/kg of DACE (n = 4, B). On day 21, the lungs were fixed, embedded into paraffin and stained for H&E (left images) or for human c-RAF-1 protein (right images). Bars, 1000 μm for upper images and 100 μm for lower images. Arrow heads on upper images indicate lung tumor areas shown on lower images. (C) The total amount of tumor tissue in the lungs of untreated control mice (<i>n =</i> 4) and DACE-treated mice (<i>n =</i> 4) measured after immunohistochemistry. The lungs of treated mice exhibited 58% (*<i>p</i><0.05, <i>t</i> test) less tumor tissue in comparison to untreated control animals. (D) Western blotting of tumor lysates of untreated control mice (<i>n</i> = 4) and DACE-treated mice (<i>n</i> = 4) for c-RAF-1-BxB expression. Beta-actin was used as a loading control. (E) Densitometric quantitation of the human c-RAF-1-BxB protein expressed in the lungs of untreated and DACE-treated mice. The lungs of treated mice exhibited 66% (*<i>p</i><0.05, <i>t</i> test) less c-RAF-1-BxB expressed protein in comparison to untreated control animals. (G) Total RNA was isolated from the lungs of untreated mice (<i>n =</i> 4) and DACE-treated mice (<i>n</i> = 4), reverse transcribed, and the expression of c-RAF-1-BxB mRNA was determined by quantitative real-time PCR. The expression of c-RAF-1-BxB mRNA was reduced by 37% after systemic treatment with DACE, albeit the means are not statistically significant when compared by <i>t</i> test. (<i>p</i>>0.05). Relationship between tumor tissue amount and c-RAF-1-BxB protein (F) and c-RAF-1-BxB mRNA (H) in lungs of all mice analyzed. The relative amounts of tumors, and c-RAF-1-BxB protein and mRNA, were measured by immunohistochemistry, Western blotting, and qRT-PCR as shown in (A/B, D and G).</p

Effects of DACE on cell cycle arrest and apoptosis.

<p>(A) A549 cells (5x10⁵) were treated with DACE and analyzed 24h later by flow cytometry. The values indicate the percentage of A549 cells in the indicated phases of the cell cycle (sub-G0/G1, G0/G1, S and G2/M). *<i>p</i><0.001 and **<i>p</i><0.0001 as compared with control. (B) The A549 cells were treated for 12h with DACE, stained with Annexin V/PI, and submitted to flow cytometry for analysis of the apoptotic cell proportion. *<i>p</i><0.05 as compared with control. (C) The A549 cells were either untreated or treated with 0.5μM and 1.0μM DACE for 12h, fixed, stained with Hoechst and TRITC-labeled-phalloidin and analyzed by confocal microscopy. Overlay images are shown. (D) The A549 cells were treated for 12h with DACE and their cytosolic fraction was analyzed for changes in the activity of caspase-3. *<i>p</i><0.05 as compared with control. (E) A549 cells were treated with DACE for 12h and then subjected to Western blotting using antibodies as indicated. Equal protein loading was confirmed by probing for beta-actin. Representative images of three independently repeated experiments are shown. The values represent means of three independent experiments and SD.</p

Cell and clonogenic growth inhibition by DACE.

<p>(A) Human non-small cell lung cancer cells (A549 cells) were treated with different concentrations of the DACE for 48h and 72h. The growth inhibition effects were determined by MTT assay and the IC₅₀ was calculated by GraphPad Prism 5 software through a nonlinear fit-curve (log of compound concentration <i>versus</i> normalized response—variable slope). (B) A549 cells were treated with 0.5 and 1 μM of the DACE for 48h, followed by two washes for compound removal. Then, cells were plated for clonogenic assay; left—representative images of colonies formed from A549 cells under the different treatment conditions; right—number of colonies after 12 days. ***<i>p</i><0.001 as compared with control.</p

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<p>Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na⁺/K⁺-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.</p

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<p>Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na⁺/K⁺-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.</p

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<p>Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na⁺/K⁺-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.</p

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<p>Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na⁺/K⁺-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.</p