Cytotoxicity of glucoevatromonoside alone and in combination with chemotherapy drugs and their effects on Na+,K+-ATPase and ion channels on lung cancer cells.

Cardiac glycosides (CGs) are useful drugs to treat cardiac illnesses and have potent cytotoxic and anticancer effects in cultured cells and animal models. Their receptor is the Na+,K+ ATPase, but other plasma membrane proteins might bind CGs as well. Herein, we evaluated the short- and long-lasting cytotoxic effects of the natural cardenolide glucoevatromonoside (GEV) on non-small-cell lung cancer H460 cells. We also tested GEV effects on Na+,K+ -ATPase activity and membrane currents, alone or in combination with selected chemotherapy drugs. GEV reduced viability, migration, and invasion of H460 cells spheroids. It also induced cell cycle arrest and death and reduced the clonogenic survival and cumulative population doubling. GEV inhibited Na+,K+-ATPase activity on A549 and H460 cells and purified pig kidney cells membrane. However, it showed no activity on the human red blood cell plasma membrane. Additionally, GEV triggered a Cl-mediated conductance on H460 cells without affecting the transient voltage-gated sodium current. The administration of GEV in combination with the chemotherapeutic drugs paclitaxel (PAC), cisplatin (CIS), irinotecan (IRI), and etoposide (ETO) showed synergistic antiproliferative effects, especially when combined with GEV + CIS and GEV + PAC. Taken together, our results demonstrate that GEV is a potential drug for cancer therapy because it reduces lung cancer H460 cell viability, migration, and invasion. Our results also reveal a link between the Na+,K+-ATPase and Cl- ion channels.

Investigation of the cytotoxic activity of two novel digitoxigenin analogues on H460 lung cancer cells.

Cardiac glycosides (CGs) are natural compounds traditionally used for the treatment of heart disorders, and recently new therapeutic possibilities were proposed. Their antitumor reports and clinical trials have notably enhanced, including those targeted for lung cancer, the most lethal type that lacks of new treatment agents, instigating the research of these molecules. The CGs studied here, named C10 {3ß-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin} and C18 (3ß-(aminoacetyl)amino-3-deoxydigitoxigenin), are semisynthetic derivatives prepared from digitoxigenin scaffold. Both compounds demonstrated high cytotoxicity for different cancer cell lines, especially H460 lung cancer cells, and their cytotoxic effects were deeply investigated using different methodological approaches. C10 induced cell death at lower concentrations and during shorter periods of treatment than C18, and increased the number of small and irregular nuclei, which are characteristics of apoptosis. This type of cell death was confirmed by caspase-3/7 assay. Both compounds reduced H460 cells proliferative potential by long-term action, and C10 showed the strongest potential. Moreover, these compounds induced a significant decrease of the area and viability of H460 spheroids providing preclinical favorable profiles to develop new chemotherapeutic agents.

Elucidation of the mechanism of anti-herpes action of two novel semisynthetic cardenolide derivatives.

Human herpesviruses are among the most prevalent pathogens worldwide and have become an important public health issue. Recurrent infections and the emergence of resistant viral strains reinforce the need of searching new drugs to treat herpes virus infections. Cardiac glycosides are used clinically to treat cardiovascular disturbances, such as congestive heart failure and atrial arrhythmias. In recent years, they have sparked new interest in their potential anti-herpes action. It has been previously reported by our research group that two new semisynthetic cardenolides, namely C10 (3ß-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin) and C11 (3ß-(hydroxyacetyl)amino-3-deoxydigitoxigenin), exhibited potential anti-HSV-1 and anti-HSV-2 with selectivity index values > 1,000, comparable with those of acyclovir. This work reports the mechanism investigation of anti-herpes action of these derivatives. The results demonstrated that C10 and C11 interfere with the intermediate and final steps of HSV replication, but not with the early stages, since they completely abolished the expression of the UL42 (ß) and gD (γ) proteins and partially reduced that of ICP27 (α). Additionally, they were not virucidal and had no prophylactic effects. Both compounds inhibited HSV replication at nanomolar concentrations, but cardenolide C10 was more active than C11 and can be considered as an anti-herpes drug candidate including against acyclovir-resistant HSV-1 strains.

Synthesis and biological evaluation of isoxazolyl-sulfonamides: A non-cytotoxic scaffold active against Trypanosoma cruzi, Leishmania amazonensis and Herpes Simplex Virus.

In this study we report the synthesis, characterization, biological evaluation, and druglikeness assessment of a series of 20 novel isoxazolyl-sulfonamides, obtained by a four-step synthetic route. The compounds had their activity against Trypanosoma cruzi, Leishmania amazonensis, Herpes Simplex Virus type 1 and cytotoxicity evaluated in phenotypic assays. All compounds have drug-like properties, showed low cytotoxicity and were promising regarding all other biological activities reported herein, especially the inhibitory activity against T. cruzi. The compounds 8 and 16 showed significant potency and selectivity against T. cruzi (GI50 = 14.3 µM, SI > 34.8 and GI50 = 11.6 µM, SI = 29.1, respectively). These values, close to the values of the reference drug benznidazole (GI50 = 10.2 µM), suggest that compounds 8 and 16 represent promising candidates for further pre-clinical development targeting Chagas disease.

Cytotoxic effects of the cardenolide convallatoxin and its Na,K-ATPase regulation.

Cardenolides are cardiac glycosides, mostly obtained from natural sources. They are well known for their inhibitory action on the Na,K-ATPase, an effect that regulates cardiovascular alterations such as congestive heart failure and atrial arrhythmias. In recent years, they have also sparked new interest in their anticancer potential. In the present study, the cytotoxic effects of the natural cardenolide convallatoxin (CON) were evaluated on non-small cell lung cancer (A549 cells). It was found that CON induced cytostatic and cytotoxic effects in A549 cells, showing essentially apoptotic cell death, as detected by annexin V-propidium iodide double-staining, as well as changes in cell form. In addition, it prompted cell cycle arrest in G2/M and reduced cyclin B1 expression. This compound also increased the number of cells in subG1 in a concentration- and time-dependent manner. At a long term, the reduction of cumulative population doubling was shown along with an increase of ß-galactosidase positive cells and larger nucleus, indicative of senescence. Subsequently, CON inhibited the Na,K-ATPase in A549 cells at nM concentrations. Interestingly, at the same concentrations, CON was unable to directly inhibit the Na,K-ATPase, either in pig kidney or in red blood cells. Additionally, results of docking calculations showed that CON binds with high efficiency to the Na,K-ATPase. Taken together, our data highlight the potent anticancer effects of CON in A549 cells, and their possible link with non-classical inhibition of Na,K-ATPase.

Anticancer and Immunogenic Properties of Cardiac Glycosides.

Cardiac glycosides (CGs) are natural compounds widely used in the treatment of several cardiac conditions and more recently have been recognized as potential antitumor compounds. They are known to be ligands for Na/K-ATPase, which is a promising drug target in cancer. More recently, in addition to their antitumor effects, it has been suggested that CGs activate tumor-specific immune responses. This review summarizes the anticancer aspects of CGs as new strategies for immunotherapy and drug repositioning (new horizons for old players), and the possible new targets for CGs in cancer cells.

Cytotoxic effects of a triterpene-enriched fraction of Cecropia pachystachya on the human hormone-refractory prostate cancer PC3 cell line.

BACKGROUND: Prostate cancer (PCa) is the most diagnosed invasive cancer and a leading cause of death in men in western countries. Most patients initially respond to androgen deprivation but finally develop hormone-refractory disease, which results in advanced clinical failure and death. Since hormone-refractory disease is highly radiotherapy and chemotherapy resistant, increasing interest has been placed on finding novel therapies for this advanced type of Pca. PURPOSE: The potential cytotoxic effects of the crude extract and fractions obtained from the leaves of Cecropia pachystachya Trécul on different human cancer cell lines were investigated. Additionally, the mechanism of cell death induction of the most active sample (triterpene-enriched fraction, TEF) on the human hormone-refractory prostate PC3 cell line was examined. METHODS: Sulforhodamine B assay was used to measure the viability of human tumor and non-tumor cell lines. To elucidate the mechanism of PC3 cells death induced by TEF, different methodological approaches were used: cell cycle analysis and annexin V/PI staining, nuclear morphological analysis, and senescence-associated-ß-galactosidase assay. Moreover, the mitochondrial membrane potential was measured, and the long-term effects of TEF on PC3 cells were evaluated. RESULTS: TEF exerted cytotoxic effects on PC3 cells but not on human non-tumor cells. The analysis of nuclear morphology of PC3 cells treated with TEF increased the number of cells with large and regular nuclei suggesting senescence induction, which was supported by ß-galactosidase overexpression. Regarding PC3 cells cycle, TEF reduced the number of cells in G1 phase and increased that in sub G0/G1. Apoptosis was not involved in PC3 cell death. However, there was a decrease in mitochondrial membrane potential without the participation of reactive oxygen species (ROS) in the cytotoxic effects detected. Furthermore, there was a decrease in the number of viable cells able to duplicate after long-term TEF treatment. CONCLUSIONS: The results showed the in vitro cytotoxic potential of the triterpene-enriched fraction obtained from the leaves of C. pachystachya on human prostate cancer PC3 cell line.

Cytotoxic and cytostatic effects of digitoxigenin monodigitoxoside (DGX) in human lung cancer cells and its link to Na,K-ATPase.

Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells). It was found that DGX induced cytotoxic effects in both cells and the apoptotic effects were more pronounced on H460 cells. In long-term analysis, using the clonogenic and the cumulative population doubling (CPD) assays, DGX showed a reduction of cell survival, after 15days without re-treatment. To better understand DGX effects in A549 cells, several assays were conducted. In cell cycle analysis, DGX caused an arrest in S and G2/M phases. This compound also increased the number of cells in subG1 phase in a concentration- and time-dependent manner. The presence of ß-galactosidase positive cells, large nucleus and flattened cells indicated senescence. Additionally, DGX inhibited Na,K-ATPase activity in A549 cells, as well as in purified pig kidney and in human red blood cell membrane preparations, at nanomolar range. Moreover, results of molecular docking showed that DGX binds with high efficiency (-11.4Kcal/mol) to the Na,K-ATPase (PDB:4HYT). Taken together, our results highlight the potent effects of DGX both in A549 and H460 cells, and disclose its link with Na,K-ATPase inhibition.

Cytotoxicity of AMANTADIG - a semisynthetic digitoxigenin derivative - alone and in combination with docetaxel in human hormone-refractory prostate cancer cells and its effect on Na+/K+-ATPase inhibition.

Cardiac glycosides (CGs) are natural compounds used to treat congestive heart failure. They have garnered attention as a potential cancer treatment option, especially because they bind to Na+/K+-ATPase as a target and activate intracellular signaling pathways leading to a variety of cellular responses. In this study we evaluated AMANTADIG, a semisynthetic cardenolide derivative, for its cytotoxic activity in two human androgen-insensitive prostate carcinoma cell lines, and the potential synergistic effects with docetaxel. AMANTADIG induced cytotoxic effects in both cell lines, and a combination with docetaxel showed a moderate and strong synergism in DU145 and PC-3 cells, respectively, at concentrations considerably lower than their IC50 values. Cell cycle analyses showed that AMANTADIG and its synergistic combination induced G2/M arrest of DU145 and PC-3 cells by modulating Cyclin B1, CDK1, p21 and, mainly, survivin expression, a promising target in cancer therapy. Furthermore, AMANTADIG presented reduced toxicity toward non-cancerous cell type (PBMC), and computational docking studies disclosed high-affinity binding to the Na+/K+-ATPase α subunit, a result that was experimentally confirmed by Na+/K+-ATPase inhibition assays. Hence, AMANTADIG inhibited Na+/K+-ATPase activity in PC-3 cells, as well as in purified pig kidney at nanomolar range. Altogether, these data highlight the potent effects of AMANTADIG in combination with docetaxel and offer important insights for the development of more effective and selective therapies against prostate cancer.