ABSTRACT
Dysregulation of the epidermal growth factor receptor (EGFR) signaling network has been frequently reported in pancreatic cancer. Inhibition of EGFR was associated with antitumor effects in both in vitro and in vivo studies of pancreatic cancer. We have previously reported the isolation and characterization of an EGFR-related protein (ERRP), which seems to be a negative regulator of EGFR. In the present investigation, we tested our hypothesis whether recombinant ERRP could be an effective inhibitor of growth of BxPC3 pancreatic cancer cells. Cell growth and apoptosis were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis ELISA assay, respectively, in the presence and absence of recombinant ERRP in BxPC3 cells. To evaluate activation of EGFR and its downstream signaling events, levels of phospho-EGFR, phospho-AKT, and phospho-extracellular signal-regulated kinase (phospho-ERK) were determined by Western blot analysis. NF-kappaB activity was measured by electrophoretic mobility shift assay. Our data show, for the first time, that ERRP inhibits the growth of BxPC3 cells in a dose- and time-dependent manner. The EGF or transforming growth factor (TGF)-alpha-induced stimulation of cell growth and activation of EGFR was also inhibited by ERRP. These changes were accompanied by a concomitant attenuation of activation of mitogen-activated protein (MAP) kinases, AKT, and NF-kappaB. ERRP also induced apoptosis as evidenced by increased poly(ADP-ribose) polymerase cleavage and reduction in procaspase3. From these results, we conclude that ERRP is a potent inhibitor of growth of BxPC-3 pancreatic cancer cells, which could be due to attenuation of EGFR cellular signaling processes. We also suggest that ERRP could be a potential therapeutic agent for pancreatic cancer.
Subject(s)
Apoptosis/drug effects , Glycoproteins/pharmacology , Pancreatic Neoplasms/drug therapy , Caspase 3 , Caspases/metabolism , Cell Growth Processes/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Administration Schedule , Epidermal Growth Factor/antagonists & inhibitors , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Humans , NF-kappa B/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Phosphorylation , Poly(ADP-ribose) Polymerases/metabolism , Recombinant Proteins/pharmacology , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/pharmacologyABSTRACT
Interference with the activation of growth factor receptors, specifically epidermal growth factor receptor (EGFR), represents a promising strategy for the development of novel and selective anticancer therapies. We reported that EGFR-related peptide (ERRP), a recently isolated negative regulator of EGFR, could be a potential therapeutic agent for colorectal cancer. To determine whether ERRP could potentially be a therapeutic agent for prostate carcinoma, we examined the effect of recombinant ERRP on the growth of the prostate cancer cell line PC-3 in vitro. Events of the EGFR signal transduction pathways were also examined. ERRP caused a marked inhibition of cell growth in a dose- and time-dependent manner and also induced apoptosis. The latter was evidenced by increased number of apoptotic cells, activation of caspase-3, and cleavage of poly(ADP-ribose)polymerase. The transforming growth factor-alpha-induced stimulation of cell growth and activation of EGFR was also inhibited by ERRP. These changes were accompanied by a concomitant attenuation of activation of Akt and mitogen-activated protein kinases as well as basal and transforming growth factor-alpha-induced activation of nuclear factor-kappaB. Inhibition of EGFR activation by ERRP could be partly attributed to increased sequestration of EGFR ligands. In summary, our data show that ERRP inhibits the growth of prostate cancer cells by attenuating EGFR signaling processes. ERRP could potentially be an effective therapeutic agent for prostate cancer.
Subject(s)
Apoptosis/drug effects , ErbB Receptors/metabolism , Glycoproteins/pharmacology , Prostatic Neoplasms/pathology , Humans , Male , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Prostatic Neoplasms/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Signal Transduction , Transforming Growth Factor alpha/pharmacology , Tumor Cells, CulturedABSTRACT
BACKGROUND & AIMS: Epidermal growth factor receptor is frequently implicated in epithelial cancers and is, therefore, being considered as a potential target for therapy. Recently, we reported the isolation and characterization of epidermal growth factor receptor-related protein, a negative regulator of epidermal growth factor receptor. To discern whether epidermal growth factor receptor-related protein could be an effective therapeutic agent for colorectal cancer, we generated epidermal growth factor receptor-related protein fusion protein and studied its effect on the growth of colon cancer cells in vivo and in vitro. We also studied whether epidermal growth factor receptor-related protein expression is altered in colorectal cancer. METHODS: A 55-kilodalton epidermal growth factor receptor-related protein fusion protein with V5 and His tags was generated in a drosophila expression system and subsequently purified by a His antibody affinity column. Rabbit polyclonal antibodies against epidermal growth factor receptor-related protein were used to examine the expression of epidermal growth factor receptor-related protein. RESULTS: Epidermal growth factor receptor-related protein expression was found to be high in benign human colonic epithelium but low in adenocarcinoma. Exposure of the colon cancer cell lines HCT-116 and Caco-2 to purified recombinant epidermal growth factor receptor-related protein caused a marked inhibition of proliferation, as well as attenuation of basal and ligand-induced stimulation of epidermal growth factor receptor phosphorylation. Epidermal growth factor receptor-related protein-induced inhibition of proliferation of colon cancer cells was prevented by epidermal growth factor receptor-related protein antibodies. Reduced epidermal growth factor receptor phosphorylation was partly due to sequestration of epidermal growth factor receptor ligands by epidermal growth factor receptor-related protein, resulting in the formation of inactive heterodimers with epidermal growth factor receptor. Intratumoral or subcutaneous (away from the tumor site) injections of purified epidermal growth factor receptor-related protein caused regression of palpable colon cancer xenograft tumors in some severely compromised immunodeficient mice and arrested tumor growth in others. CONCLUSIONS: We propose that epidermal growth factor receptor-related protein inhibits cellular growth by attenuating epidermal growth factor receptor signaling processes and is an effective therapeutic agent for colorectal cancer.
