Overexpression of S100A4 in human cancer cell lines resistant to methotrexate.
BACKGROUND
Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance.


METHODS
The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for beta-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway.


RESULTS
S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. beta-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells.


CONCLUSIONS
S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistance.
Mencía
Nuria
N
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.
Selga
Elisabet
E
Rico
Isabel
I
de Almagro
M Cristina
MC
Villalobos
Xenia
X
Ramirez
Sara
S
Adan
Jaume
J
Hernández
Jose L
JL
Noé
Véronique
V
Ciudad
Carlos J
CJ
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D019073:HT29 Cells; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D051176:beta Catenin; C077591:S100A4 protein, human; D008727:Methotrexate