ABSTRACT
Title
Characterization of the effects of celecoxib and 2,5 di-methylcelecoxib on human Chronic Myeloid Leukemia
Authors
B. Riva, F. Condorelli and P.L. Canonico
Dept. of Chemical, Food, Pharmaceutical and Pharmacological Sciences, University of Piemonte Orientale A. Avogadro, Novara, Italy
Dept. of Chemical, Food, Pharmaceutical and Pharmacological Sciences, University of Piemonte Orientale A. Avogadro, Novara, Italy
Abstract
Celecoxib (CEL), widely known as selective cyclo-oxygenase 2 (COX2) inhibitor, has been ascribed of anti-cancer effects, at least in solid tumor models (e.g. colon carcinoma), both through COX2-dependent and independent mechanisms. Conversely, the influence of CEL on leukaemia progression and its mechanism of action have not been fully elucidated so far. Thus we endeavoured the characterization of the possible anti-proliferative, COX2-independent, effects of CEL in a cellular model Chronic Myelogenous Leukemia (CML). To this purpose we used the COX2-negative human cell line, named K562 (known to express the bcr/abl chimeric oncogene), which was isolated from a CML patient in the “blast crisis” phase of the disease.
As revealed by MTT assays, CEL and its di-methyl analogue DMC, which is devoid of COX-2 inhibitory activity, both inhibited K562 cell proliferation unlike Rofecoxib (ROFE), another selective COX-2 inhibitor. The absorbance values measured in these experiments demonstrated that CEL impairs cell proliferation in a dose- and time-dependent manner, peaking at 72 h from treatments with the 50 mM concentration (78±7,3% reduction as compared to untreated controls), without significantly affecting cell viability. Conversely, MTT experiments, “trypan blue exclusion” tests, FACS analysis of propidium iodide fluorescence, and DRAQ5-staining of nuclei demonstrated that DMC is more potent and efficient in reducing K562 cell proliferation since it causes apoptotic cell death (more evident after 48 h treatments with the 25 µM concentration). Accordingly, only DMC was able to alter mitochondrial membrane potential, as from FACS analysis of TMRM-loaded cells, an event that suggests the involvement of these organelles in the execution of the apoptotic program (the so-called “mitochondrial pathway”). On the other hand, CEL-induced (25-50 µM) cytostatic effects were associated to the induction of autophagy, at least after 48-72 h from drug administration. This was documented in western blot and immunofluorescence experiments set to respectively detect the processing and re-localization of LC3 protein, a typical marker of autophagy.Experiments with lentivirally delivered miRNAs targeting the up-stream activator of autophagy, ATG7, demonstrated that the engagement of this pathway is required by K562 cells in order to express the cytostatic response to CEL treatments. In line with these observations, K562 cells cultured in limiting culture conditions (serum starvation) respond to CEL with massive cell death (MTT and FACS assays). Collectively, these data demonstrated that CEL strikes CML cells metabolism (as verified also in other cellular models), causing the induction of the autophagy “salvage-response”, and that serum starvation adds up with this mechanism, thus inducing cell death. In other words, CEL is able to restore, in a COX2-independent manner, the dependency of leukaemia cells from the growth factors present in the culture media, as it is also confirmed by its ability to synergize with the tyrosine-kinase inhibitor imatinib (affecting Bcr/Abl funtion), the front-line drug in the management of CML patients.
As revealed by MTT assays, CEL and its di-methyl analogue DMC, which is devoid of COX-2 inhibitory activity, both inhibited K562 cell proliferation unlike Rofecoxib (ROFE), another selective COX-2 inhibitor. The absorbance values measured in these experiments demonstrated that CEL impairs cell proliferation in a dose- and time-dependent manner, peaking at 72 h from treatments with the 50 mM concentration (78±7,3% reduction as compared to untreated controls), without significantly affecting cell viability. Conversely, MTT experiments, “trypan blue exclusion” tests, FACS analysis of propidium iodide fluorescence, and DRAQ5-staining of nuclei demonstrated that DMC is more potent and efficient in reducing K562 cell proliferation since it causes apoptotic cell death (more evident after 48 h treatments with the 25 µM concentration). Accordingly, only DMC was able to alter mitochondrial membrane potential, as from FACS analysis of TMRM-loaded cells, an event that suggests the involvement of these organelles in the execution of the apoptotic program (the so-called “mitochondrial pathway”). On the other hand, CEL-induced (25-50 µM) cytostatic effects were associated to the induction of autophagy, at least after 48-72 h from drug administration. This was documented in western blot and immunofluorescence experiments set to respectively detect the processing and re-localization of LC3 protein, a typical marker of autophagy.Experiments with lentivirally delivered miRNAs targeting the up-stream activator of autophagy, ATG7, demonstrated that the engagement of this pathway is required by K562 cells in order to express the cytostatic response to CEL treatments. In line with these observations, K562 cells cultured in limiting culture conditions (serum starvation) respond to CEL with massive cell death (MTT and FACS assays). Collectively, these data demonstrated that CEL strikes CML cells metabolism (as verified also in other cellular models), causing the induction of the autophagy “salvage-response”, and that serum starvation adds up with this mechanism, thus inducing cell death. In other words, CEL is able to restore, in a COX2-independent manner, the dependency of leukaemia cells from the growth factors present in the culture media, as it is also confirmed by its ability to synergize with the tyrosine-kinase inhibitor imatinib (affecting Bcr/Abl funtion), the front-line drug in the management of CML patients.