title: The curcuminoid CLEFMA selectively induces cell death in H441 lung adenocarcinoma cells via oxidative stress.
abstract: CLEFMA or 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] is a curcuminoid being developed as an anticancer drug. We recently reported that it potently inhibits proliferation of various cancer cells. In this project, we investigated the effect of CLEFMA on gene expression profile in H441 lung adenocarcinoma cells, and studied its mechanism of action. In microarray data, we observed a deregulation of genes involved in redox and glutamate metabolism. Based on the affected ontologies, we hypothesized that antiproliferative activity of CLEFMA could be a result of the induction of reactive oxygen species (ROS). We tested this hypothesis by determining the levels of glutathione (GSH) and ROS in H441 cells treated with CLEFMA. We observed a rapid depletion of intracellular GSH/GSSG ratio. Using a cell-permeable fluorogenic substrate, we found that CLEFMA significantly induced ROS in a time- and dose-dependent manner (p < 0.05). Flow-cytometry with a mitochondria-selective fluorescent reporter of ROS indicated that the CLEFMA-induced ROS was of mitochondrial origin. In contrast to the cancer cells, the normal lung fibroblasts (CCL-151) did not show any increase in ROS and were resistant to CLEFMA-induced cell death. Furthermore, the addition of antioxidants, such as catalase, superoxide dismutase and N-acetylcysteine, rescued cancer cells from CLEFMA-induced cell death. Gene expression pathway analysis suggested that a transcription factor regulator Nrf2 is a pivotal molecule in the CLEFMA-induced deregulation of redox pathways. The immunoblotting of Nrf2 showed that CLEFMA treatment resulted in phosphorylation and nuclear translocation of Nrf2 in a time-dependent fashion. Based on these results, we conclude that induction of ROS is critical for the antiproliferative activity of CLEFMA and the Nrf2-mediated oxidative stress response fails to salvage H441 cells.
authors.affiliation: Department of Pharmaceutical Sciences, University of Oklahoma Health Science Center, 1110 North Stonewall Avenue, Oklahoma, OK 73117, USA.
authors.forename: Kaustuv
authors.initials: K
authors.lastname: Sahoo
authors.forename: Mikhail G
authors.initials: MG
authors.lastname: Dozmorov
authors.forename: Shrikant
authors.initials: S
authors.lastname: Anant
authors.forename: Vibhudutta
authors.initials: V
authors.lastname: Awasthi
mesh_terms: D021581:Active Transport, Cell Nucleus; D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D001597:Benzylidene Compounds / Q000494:pharmacology*; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003474:Curcumin; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects* / Q000235:genetics; D010766:Phosphorylation; D010881:Piperidones / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors
chemical_list: C572622:4-(3,5-bis(2-chlorobenzylidene)-4-oxopiperidine-1-yl)-4-oxo-2-butenoic acid; D000972:Antineoplastic Agents, Phytogenic; D000975:Antioxidants; D001597:Benzylidene Compounds; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D010881:Piperidones; D017382:Reactive Oxygen Species; D005978:Glutathione; D003474:Curcumin