Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.
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.
Solmi
Rossella
R
Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna, Italy. rossella.solmi@unibo.it
Lauriola
Mattia
M
Francesconi
Mirko
M
Martini
Désirée
D
Voltattorni
Manuela
M
Ceccarelli
Claudio
C
Ugolini
Giampaolo
G
Rosati
Giancarlo
G
Zanotti
Simone
S
Montroni
Isacco
I
Mattei
Gabriella
G
Taffurelli
Mario
M
Santini
Donatella
D
Pezzetti
Furio
F
Ruggeri
Alessandro
A
Castellani
Gastone
G
Guidotti
Lia
L
Coppola
Domenico
D
Strippoli
Pierluigi
P
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*
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D011799:Quinazolines; D004815:Epidermal Growth Factor; D000068818:Cetuximab; D000077156:Gefitinib