Original Papers A Hydroalcoholic Extract from the Leaves of Nerium oleander Inhibits Glycolysis and Induces Selective Killing of Lung Cancer Cells Authors José Manuel Calderón-Montaño1, Estefanía Burgos-Morón1, Manuel Luis Orta2, Santiago Mateos2, Miguel López-Lázaro1 Affiliations 1 Department of Pharmacology, Faculty of Pharmacy, University of Seville, Seville, Spain 2 Department of Cell Biology, Faculty of Biology, University of Seville, Seville, Spain Key words Abstract consumption and lactate production) in A549 l" Nerium oleander ! cells, comparable to that of the glycolysis inhibitor l" Apocynaceae Recent evidence suggests that cardiac glycosides dichloroacetate (currently in clinical develop- l" cardiac glycosides might be used for the treatment of cancer. The or- ment for cancer therapy). Because platinum com- l" cardiotonic steroids namental shrub Nerium oleander has been used in pounds are widely used in the treatment of lung l" anticancer traditional medicine for treating several disorders cancer, we tested the cytotoxicity of several com- including cancer, and extracts from the leaves of binations of cisplatin with the extract and found a this plant have already entered phase I clinical tri- moderate synergism when Nerium oleander ex- als. In this communication, we have prepared a tract was administered after cisplatin but a mod- hydroalcoholic extract from the leaves of Nerium erate antagonism when it was added before cis- oleander (containing 4.75 ± 0.32% of cardenolides) platin. Our results suggest that extracts from Ne- and have assessed its cytotoxic activity in A549 rium oleander might induce anticancer effects in lung cancer cells vs. MRC5 nonmalignant lung fi- patients with lung cancer and support their possi- broblasts. The results showed that the cytotoxic- ble advancement into phase II clinical trials for ity of the Nerium oleander extract against the can- the treatment of this type of cancer. cer cell line was significantly higher than that against the nonmalignant cell line, with a potency and selectivity similar to those of the anticancer Abbreviations drug cisplatin. Pretreatment of A549 cells with ! the antioxidants N-acetylcysteine and catalase BRCA2: breast cancer type 2 slightly prevented the cytotoxicity of the extract, CI: combination index Downloaded by: Universidad de Sevilla. Copyrighted material. therefore suggesting that the formation of reac- DAPI: 4′,6-diamidino-2-phenylindole tive oxygen species participates in its cytotoxic dihydrochloride activity but does not play a major role. Nerium DCA: dichloroacetate received April 25, 2013 oleander extract-induced cytotoxicity and DNA DSB: double strand break revised May 24, 2013 damage (gamma-H2AX focus formation) were HR: homologous recombination accepted May 31, 2013 slightly higher in cells lacking BRCA2 (deficient NAC: N-acetylcysteine Bibliography in homologous recombination repair) than in par- NOE: Nerium oleander extract DOI http://dx.doi.org/ ental cells; this indicates that the induction of PFK: phosphofructokinase 10.1055/s-0032-1328715 DNA damage may also play a role in the cytotoxic- SGLTs: sodium glucose transporters Published online ity of the extract. Nerium oleander extract in- ROS: reactive oxygen species Planta Med © Georg Thieme Verlag KG Stuttgart · New York · duced a marked inhibition of glycolysis (glucose SEM: standard error of the means ISSN 0032‑0943 Correspondence Dr. M. López-Lázaro, Associate Introduction ranone) and bufadienolides (with the lactone 2- Professor ! pyrone). Numerous cardiac glycosides (e.g., digi- Department of Pharmacology Cardiac glycosides are a group of natural products toxin, digoxin, ouabain, oleandrin, and proscillar- Faculty of Pharmacy C/Profesor Garcia Gonzalez 2 that share a steroid-like structure with an un- idin) have been isolated from plants (e.g., Digitalis 41012 Sevilla saturated lactone ring and the ability to inhibit purpurea, Digitalis lannata, Strophantus gratus, Spain the Na+/K+-ATPase pump. The lactone moiety at Nerium oleander,andUrginea martima). Several Phone: + 34954556348 Fax: + 349 54556074 position 17 defines the two classes of cardiac gly- cardiac glycosides have also been found in am- [email protected] coside: cardenolides (with the lactone 2-fu- phibians and mammals, including digoxin, oua- Calderón-Montaño JM et al. A Hydroalcoholic Extract… Planta Med Original Papers bain, bufalin, marinobufagenin, and telecinobufagin. Some car- water solution was lyophilized, with an extraction yield of 2.3%. diac glycosides are used in cardiology for the treatment of cardiac The cardiac glycoside content of N. oleander was determined with congestion and some types of cardiac arrhythmias. The mecha- the Kedde reaction, a colorimetric technique that allows the de- nism by which these drugs affect cardiac contractility is known termination of unsaturated pentacyclic lactones (present in car- to be mediated by a highly specific inhibition of Na+/K+-ATPase diac glycosides from N. oleander) by using 3,5-dinitrobenzoic acid [1–3]. [20]. Briefly, a 3% solution of 3,5-dinitrobenzoic acid in ethanol Over the years, there have been a variety of periodic reports sug- was mixed in the ratio 1:1 with a solution of 2 M NaOH in dis- gesting that cardiac glycosides may have an anticancer utilization tilled water. 100 µL of this mixture was mixed with 150 µL of (reviewed in [4–8]). In vitro and ex vivo experiments have re- NOE or 2-furanone in ethanol at different concentrations. Optical vealed that some cardiac glycosides (e.g., digitoxin) induce selec- densities were measured at 540 nm on a multiwell plate spectro- tive anticancer effects [4,9,10], which may occur at concentra- photometer reader. Based on the standard 2-furanone (lactone of tions commonly found in the plasma of patients treated with cardenolides), the percentage of cardenolides in the extract was these drugs [11]. Some cardiac glycosides have also shown potent determined, and was expressed as the mean ± SEM. and selective anticancer effects in mice harboring human malig- nant cells [12,13]; however, these results should be interpreted Chemicals and cell lines cautiously, as mouse cells are much more resistant than human Cisplatin (99.9%), dichloroacetate (98%), NAC (99%), 2-furanone cells to the cytotoxic effects of cardiac glycosides, and it is not (97%), and catalase were purchased from Sigma. The human clear whether such selectivity is due to selective inhibition of tu- A549 lung cancer cell line, the human embryo lung fibroblastic mor cells or to interspecies differences in sensitivity [14]. The MRC-5 cell line and the human HT29 colon adenocarcinoma cell cardiac drugs digitoxin and digoxin, the semisynthetic cardiac line were purchased from European Collection of Cell Cultures. glycoside UNBS1450, and two extracts from Nerium oleander The human UACC-62 melanoma cell line was purchased from Apocynaceae have entered clinical trials for the treatment of can- American Type Culture Collection. The HR-deficient VC8 cell line cer (see http://clinicaltrials.gov/ and ref. [6,7,15,16]). Recently, (V79 Chinese hamster lung cells mutated in BRCA2) and the however, Perne et al. reported results suggesting that cardiac gly- VC8B2 cell line (VC8 cells complemented with human BRCA2) coside-induced cytotoxicity was mediated by general protein were kindly provided by Dr. Helleday. All cell lines were main- synthesis inhibition and was not selective for cancer cells, raising tained in DMEM supplemented with 2 mM glutamine, 50 µg/mL concerns about ongoing clinical trials testing cardiac glycosides penicillin, 50 µg/mL streptomycin, and 10% fetal bovine serum, as anticancer agents [17]. Later, Hallbook et al. observed that and were cultured at 37°C in a humidified atmosphere contain- some cardiac glycosides, particularly digitoxin, induced ex vivo ing 5% CO2. Cell culture reagents were obtained from Life Tech- selective anticancer effects in leukemia cells and found that pro- nologies. tein synthesis inhibition by cardiac glycosides at concentrations corresponding to IC50 values did not occur in all types of cancer Assay for cytotoxic activity cells [10]. These data suggest that cardiac glycosides may induce The MTT assay is a colorimetric technique that allows the quanti- selective anticancer effects only in some types of cancer. tative determination of cell viability. It is based on the capability Several mechanisms of action have been proposed to participate of viable cells to transform the MTT salt (3-(4,5-dimethylthiazol- in the cytotoxic activity of cardiac glycosides (reviewed in [5–8, 2-yl)-2,5-diphenyltetrazolium bromide) into a formazan dye. Ex- 16]). However, it is unclear why cancer cells are generally more ponentially growing cells were seeded into 96-well plates and susceptible than nonmalignant cells to the cytotoxic activity of drugs were added 24 h later. Following the incubation period these compounds. Recent data have revealed that cancer cells specified in the figures and table legends, the medium was re- have a higher reliance on glycolysis for their survival than normal moved, and 125 µL MTT (1 mg/mL in medium) was added to each cells, and that the inhibition of glycolysis may cause selective well for 4 hours. Then, 80 µL 20% SDS in 0.02 M HCl were added, Downloaded by: Universidad de Sevilla. Copyrighted material. anticancer effects [5,18, 19]. In this communication, we have as- plates were incubated for 10 hours at 37°C, and optical densities sessed the selective cytotoxic activity of a hydroalcoholic extract