title: Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.
abstract: BACKGROUND
  EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF.
  
  
  METHODS
  Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A.
  
  
  RESULTS
  Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases.
  
  
  CONCLUSIONS
  This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.
authors.affiliation: Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna, Italy. rossella.solmi@unibo.it
authors.forename: Rossella
authors.initials: R
authors.lastname: Solmi
authors.forename: Mattia
authors.initials: M
authors.lastname: Lauriola
authors.forename: Mirko
authors.initials: M
authors.lastname: Francesconi
authors.forename: Désirée
authors.initials: D
authors.lastname: Martini
authors.forename: Manuela
authors.initials: M
authors.lastname: Voltattorni
authors.forename: Claudio
authors.initials: C
authors.lastname: Ceccarelli
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Ugolini
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Rosati
authors.forename: Simone
authors.initials: S
authors.lastname: Zanotti
authors.forename: Isacco
authors.initials: I
authors.lastname: Montroni
authors.forename: Gabriella
authors.initials: G
authors.lastname: Mattei
authors.forename: Mario
authors.initials: M
authors.lastname: Taffurelli
authors.forename: Donatella
authors.initials: D
authors.lastname: Santini
authors.forename: Furio
authors.initials: F
authors.lastname: Pezzetti
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ruggeri
authors.forename: Gastone
authors.initials: G
authors.lastname: Castellani
authors.forename: Lia
authors.initials: L
authors.lastname: Guidotti
authors.forename: Domenico
authors.initials: D
authors.lastname: Coppola
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Strippoli
mesh_terms: D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D061067:Antibodies, Monoclonal, Humanized; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002470:Cell Survival; D000068818:Cetuximab; D016000:Cluster Analysis; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D004815:Epidermal Growth Factor / Q000008:administration & dosage*; D000077156:Gefitinib; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008871:Microvilli / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011799:Quinazolines / Q000008:administration & dosage*
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D011799:Quinazolines; D004815:Epidermal Growth Factor; D000068818:Cetuximab; D000077156:Gefitinib