Our results open new possibilities for the treatment of drug-resistant tumors through combination strategies based on the use of well-tolerated pH modulators such as PPIs.
The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H 2 O 2 and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H 2 O 2 . Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells.
The search for innovative therapeutic approaches based on the use of new substances is gaining more interest in clinical oncology. In this in vitro study the potential anti-tumoral activity of tea tree oil, distilled from Melaleuca alternifolia, was analyzed against human melanoma M14 WT cells and their drug-resistant counterparts, M14 adriamicin-resistant cells. Both sensitive and resistant cells were grown in the presence of tea tree oil at concentrations ranging from 0.005 to 0.03%. Both the complex oil (tea tree oil) and its main active component terpinen-4-ol were able to induce caspase-dependent apoptosis of melanoma cells and this effect was more evident in the resistant variant cell population. Freeze-fracturing and scanning electron microscopy analyses suggested that the effect of the crude oil and of the terpinen-4-ol was mediated by their interaction with plasma membrane and subsequent reorganization of membrane lipids. In conclusion, tea tree oil and terpinen-4-ol are able to impair the growth of human M14 melanoma cells and appear to be more effective on their resistant variants, which express high levels of P-glycoprotein in the plasma membrane, overcoming resistance to caspase-dependent apoptosis exerted by P-glycoprotein-positive tumor cells.
The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H2O2 and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H2O2 generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.
© F e r r a t a S t o r t i F o u n d a t i o nIn this study we showed that human PBMC-derived CD14 + Methods Cell sortingCD3, CD19, CD14 and CD34 MicroBeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were used for magnetic-activated cell sorting (MACS) from PBMC (manufacturer's protocol). Prior to sorting, monocytes/macrophages were purified from PBMC by counterflow centrifugal elutriation (JE-6B Beckman-Coulter centrifuge, Beckman Instruments Inc.; Palo Alto, CA, USA). Monocyte/macrophage subsets and hematopoietic precursors were sorted on a FACS-Aria II/III (BD Biosciences; Oxford, UK). Cell cultureHuman cells were cultured in StemSpan (Stem Cell Technologies; Grenoble, France) supplemented with stem cell factor (SCF; supernatant equivalent to 100 ng/mL), erythropoietin (2 U/mL, ProSpec; East Brunswick, NJ, USA), dexamethasone (1 μM, Sigma; St. Louis, MO, USA) and cholesterol-rich lipids (40 μg/mL, Sigma) as described elsewhere.14,15 Informed consent was given in accordance with the Declaration of Helsinki and Dutch national and Sanquin internal ethic boards. Conditioned media were collected from CD14 + cells cultured for 2 days at 5-10x10 6 cells/8 mL, filtered (0.22 μm) and stored at 4°C. Isolated CD34 + cells were cultured with conditioned media diluted 1:2 with fresh culture medium. The media were replenished every 2 days. Co-culture experiments CD34+ cells were co-cultured with purified hematopoietic effector cells using ratios found in PBMC (1:100 CD14 Transwell assays CD14+ and CD34 + cells were seeded into transwells (0.4 µm polyester membrane, Corning; NY, USA) with CD34 + cells inside the transwell and CD14 + cells in the well (at a ratio of 1:100). Cells were analyzed after 2-8 days on the flow cytometer. Colony assaysColony assays were started with freshly purified, sorted, or cultured cells mixed with methacult (Medium ColonyGel ™ Cell Systems; Troisdorf, Germany). After 14 days, total colony numbers and colony-forming units were manually scored twice using a wide-field microscope (Axiovert 200M, Carl Zeiss Inc.; Thornwood, NY, USA). Proliferation assays CD34+ cells were washed in phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA, Sigma; PSA). Cells were labeled with 0.5 μM carboxyfluoroscein succinimidyl ester (CFSE, Molecular Probes; Leiden, The Netherlands) for 8 min with gentle shaking at 37°C. Cells were washed in 1% PSA and cultured with or without CD14+ cells (at a ratio of 1:100) for 2 days. CFSE dilution was measured by flow cytometry. Flow cytometryCells were washed in PBS and re-suspended in 1% PSA. Cells were incubated with primary antibodies for 30 min at 4°C, washed in PBS and measured on a FACS Canto II or LSR Fortessa (both BD Biosciences) and analyzed using FlowJo software (FlowJo v7.6.4/v10; Ashland, OR, USA). The antibodies are detailed in the Online Supplementary Methods. For apoptosis experiments, cells were stained in 100 μL binding buffer with annexin V-APC (BD Biosciences; 1:200) at 4°C. After 30 min, 100 μL binding buffer and...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.