digitoxin and Carcinoma--Non-Small-Cell-Lung

Cardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes.. We treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration.. Digitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling.. Our study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity.. Discovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cytoskeleton; Digitoxin; Humans; Lung Neoplasms

Non-small-cell lung cancer (NSCLC) dominates over 85% of all lung cancer cases. Epidermal growth factor receptor (EGFR) activating mutation is a common situation in NSCLC. In the clinic, molecular-targeting with Gefitinib as a tyrosine kinase inhibitor (TKI) for EGFR downstream signaling is initially effective. However, drug resistance frequently happens due to additional mutation on EGFR, such as substitution from threonine to methionine at amino acid position 790 (T790M). In this study, we screened a traditional Chinese medicine (TCM) compound library consisting of 800 single compounds in TKI-resistance NSCLC H1975 cells, which contains substitutions from leucine to arginine at amino acid 858 (L858R) and T790M mutation on EGFR. Attractively, among these compounds there are 24 compounds CC50 of which was less than 2.5 μM were identified. We have further investigated the mechanism of the most effective one, Digitoxin. It showed a significantly cytotoxic effect in H1975 cells by causing G2 phase arrest, also remarkably activated 5' adenosine monophosphate-activated protein kinase (AMPK). Moreover, we first proved that Digitoxin suppressed microtubule formation through decreasing α-tubulin. Therefore, it confirmed that Digitoxin effectively depressed the growth of TKI-resistance NSCLC H1975 cells by inhibiting microtubule polymerization and inducing cell cycle arrest.

Topics: AMP-Activated Protein Kinases; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cardiac Glycosides; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Digitoxin; Drug Resistance, Neoplasm; ErbB Receptors; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Medicine, Chinese Traditional; Microtubules; Mutation; Quinazolines; Tubulin

Advanced stage cancers acquire anoikis resistance which provides metastatic potential to invade and form tumors at distant sites. Suppression of anoikis resistance by novel molecular therapies would greatly benefit treatment strategies for metastatic cancers. Recently, digitoxin and several of its novel synthetic derivatives, such as α-l-rhamnose monosaccharide derivative (D6-MA), have been synthesized and studied for their profound anticancer activity in various cancer cell lines. In this study, we investigated the anoikis sensitizing effect of D6-MA compared with digitoxin to identify their anti-metastatic mechanism of action. D6-MA sensitized NSCLC H460 cells to detachment-induced apoptosis with significantly greater cytotoxicity (IC50=11.9 nM) than digitoxin (IC50=90.7 nM) by activating caspase-9. Screening of the Bcl-2 protein family revealed that degradation of anti-apoptotic Mcl-1 protein is a favorable target. Mcl-1 over-expression and knockdown studies in D6-MA and digitoxin exposed cells resulted in rescue and enhancement, respectively, indicating a facilitative role for decreased Mcl-1 expression in NSCLC anoikis. Transfection with mutant Mcl-1S159 attenuated detachment-induced cell death and correlated with a remaining of Mcl-1 level. Furthermore, D6-MA suppressed Mcl-1 expression via ubiquitin proteasomal degradation that is dependent on activation of glycogen synthase kinase (GSK)-3β signaling. In addition, D6-MA also targeted Mcl-1 degradation causing an increased anoikis in A549 lung cancer cells. Anoikis sensitizing effect on normal small airway epithelial cells was not observed indicating the specificity of D6-MA and digitoxin for NSCLC. These results identify a novel cardiac glycoside (CG) sensitizing anoikis mechanism and provide a promising anti-metastatic target for lung cancer therapy.

Topics: Anoikis; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Culture Techniques; Cell Line, Tumor; Digitoxin; Humans; Lung Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein

Digitoxin belongs to a naturally occurring class of cardiac glycosides (CG); digitoxin is clinically approved for heart failure and known for its anti-cancer effects against non-small lung cancer cells (NSCLC). However, concerns associated with its narrow therapeutic index and its concentration-dependent mechanism of action are rising. Thus, before digitoxin implementation in designing and developing safer and more effective CG-based anti-cancer therapies, its pharmacological and safety profiles need to be fully elucidated. In this research we used a combinatorial approach to evaluate the anti-cancer mechanisms of digitoxin in real-time. Our approach employed a non-invasive electric cell impedance sensing technique as a proxy to monitor NSCLC behavior post-exposure to toxic, therapeutic and sub-therapeutic concentrations of the drug. By developing structure-function combinatorial relations we showed that digitoxin targets cancer cells in a time and dose-dependant manner by activating pro-apoptotic and anti-proliferative signaling cascades that results in strengthening cellular adhesion and sequestration of key regulatory proliferation protein from the nucleus.

Topics: Antineoplastic Agents; Apoptosis; Biosensing Techniques; Carcinoma, Non-Small-Cell Lung; Cardiotonic Agents; Cell Adhesion; Cell Line, Tumor; Digitoxin; Humans; Lung; Lung Neoplasms; Signal Transduction