Manisha Yadav1, Abhishek Kumar Singh1, Harish Kumar1, Geeta Rao1, Bandana Chakravarti2, Anagha Gurjar1, Shalini Dogra3, Sapana Kushwaha1, Achchhe Lal Vishwakarma4, Prem Narayan Yadav3, Dipak Datta1, Anil Kumar Tripathi5, Naibedya Chattopadhyay6, Arun Kumar Trivedi1, Sabyasachi Sanyal7. 1. Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow 226031, UP, India. 2. Department of Molecular Medicine, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, UP, India. 3. Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow 226031, UP, India. 4. Division of Sophisticated and Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow 226031, UP, India. 5. Department of Clinical Hematology and Medical Oncology, King George's Medical University, Lucknow 226003, Uttar Pradesh, India. 6. Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow 226031, UP, India. 7. Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow 226031, UP, India. Electronic address: sanyal@cdri.res.in.
Abstract
BACKGROUND: Epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) is used for treating non-small cell lung cancer. Gefitinib also induces differentiation in acute myeloid leukemia (AML) cell lines and patient samples lacking EGFR by an unknown mechanism. Here we dissected the mechanism of gefitinib action responsible for its EGFR-independent effects. METHODS: Signaling events were analyzed by homogenous time-resolved fluorescence and immunoblotting. Cellular proliferation and differentiation were assessed by ATP measurement, trypan blue exclusion, 5-bromo-2'-deoxyuridine incorporation and flow-cytometry. Gefitinib and G protein-coupled receptor (GPCR) interactions were assessed by β-arrestin recruitment, luciferase and radioligand competition assays. Role of histamine receptors (HR) in gefitinib actions were assessed by HR knockdown or pharmacological modulation. EGFR and HR interaction was assessed by co-immunoprecipitation. RESULTS: Gefitinib reduced cyclic AMP content in both AML and EGFR-expressing cells and induced ERK phosphorylation in AML cells. Dibutyryl-cAMP or PD98059 suppressed gefitinib-induced AML cell cytostasis and differentiation. Gefitinib bound to and modulated HRs with subtype selectivity. Pharmacological or genetic modulations of H2 and H4 HRs (H2R and H4R) not only suppressed gefitinib-induced cytostasis and differentiation of AML cells but also blocked EGFR and ERK1/2 inhibition in MDA-MB-231 cells. Moreover, in MDA-MB-231 cells gefitinib enhanced EGFR interaction with H4R that was blocked by H4R agonist 4-methyl histamine (4MH). CONCLUSION: HRs play critical roles in anti-cancer effects of gefitinib in both EGFR-deficient and EGFR-rich environments. GENERAL SIGNIFICANCE: We furnish fresh insights into gefitinib functions which may provide new molecular clues to its efficacy and safety issues.
BACKGROUND:Epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) is used for treating non-small cell lung cancer. Gefitinib also induces differentiation in acute myeloid leukemia (AML) cell lines and patient samples lacking EGFR by an unknown mechanism. Here we dissected the mechanism of gefitinib action responsible for its EGFR-independent effects. METHODS: Signaling events were analyzed by homogenous time-resolved fluorescence and immunoblotting. Cellular proliferation and differentiation were assessed by ATP measurement, trypan blue exclusion, 5-bromo-2'-deoxyuridine incorporation and flow-cytometry. Gefitinib and G protein-coupled receptor (GPCR) interactions were assessed by β-arrestin recruitment, luciferase and radioligand competition assays. Role of histamine receptors (HR) in gefitinib actions were assessed by HR knockdown or pharmacological modulation. EGFR and HR interaction was assessed by co-immunoprecipitation. RESULTS:Gefitinib reduced cyclic AMP content in both AML and EGFR-expressing cells and induced ERK phosphorylation in AML cells. Dibutyryl-cAMP or PD98059 suppressed gefitinib-induced AML cell cytostasis and differentiation. Gefitinib bound to and modulated HRs with subtype selectivity. Pharmacological or genetic modulations of H2 and H4 HRs (H2R and H4R) not only suppressed gefitinib-induced cytostasis and differentiation of AML cells but also blocked EGFR and ERK1/2 inhibition in MDA-MB-231 cells. Moreover, in MDA-MB-231 cells gefitinib enhanced EGFR interaction with H4R that was blocked by H4R agonist 4-methyl histamine (4MH). CONCLUSION: HRs play critical roles in anti-cancer effects of gefitinib in both EGFR-deficient and EGFR-rich environments. GENERAL SIGNIFICANCE: We furnish fresh insights into gefitinib functions which may provide new molecular clues to its efficacy and safety issues.
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