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Overexpression of S100A4 in human cancer cell lines resistant to methotrexate.

Mencía N, Selga E, Rico I, de Almagro MC, Villalobos X, Ramirez S, Adan J, Hernández JL, Noé V, Ciudad CJ - BMC Cancer (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.

ABSTRACT

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.

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Related in: MedlinePlus

Effects of transfecting an expression vector encoding for β-Catenin on S100A4 mRNA levels. Transfection with β-Catenin expression vector (pcDNA3-β-Catenin) was performed in HT29 cells, both sensitive (Figure 4A) and resistant (Figure 4B) as described in Methods. S100A4 mRNA levels were determined by RT-Real-Time PCR 48 h after transfection. All results are expressed as percentages referred to untreated cells. Values are the mean of three independent experiments ± SE. * p < 0.05.
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Figure 4: Effects of transfecting an expression vector encoding for β-Catenin on S100A4 mRNA levels. Transfection with β-Catenin expression vector (pcDNA3-β-Catenin) was performed in HT29 cells, both sensitive (Figure 4A) and resistant (Figure 4B) as described in Methods. S100A4 mRNA levels were determined by RT-Real-Time PCR 48 h after transfection. All results are expressed as percentages referred to untreated cells. Values are the mean of three independent experiments ± SE. * p < 0.05.

Mentions: It had been previously described that S100A4 was a target of the Wnt signaling pathway and a functional TCF binding site has been identified in its promoter sequence [18]. To investigate whether S100A4 expression was regulated through this pathway in HT29 cells, we overexpressed β-catenin in HT29 sensitive or resistant cells and quantified S100A4 mRNA levels 48 h after transfection. A 2-fold increase in S100A4 mRNA levels was observed upon transfection of 1 μg of pcDNA3-β-catenin in HT29 sensitive cells (Figure 4A) whereas no changes were obtained when transfection was performed in HT29 resistant cells (Figure 4B).


Overexpression of S100A4 in human cancer cell lines resistant to methotrexate.

Mencía N, Selga E, Rico I, de Almagro MC, Villalobos X, Ramirez S, Adan J, Hernández JL, Noé V, Ciudad CJ - BMC Cancer (2010)

Effects of transfecting an expression vector encoding for β-Catenin on S100A4 mRNA levels. Transfection with β-Catenin expression vector (pcDNA3-β-Catenin) was performed in HT29 cells, both sensitive (Figure 4A) and resistant (Figure 4B) as described in Methods. S100A4 mRNA levels were determined by RT-Real-Time PCR 48 h after transfection. All results are expressed as percentages referred to untreated cells. Values are the mean of three independent experiments ± SE. * p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903526&req=5

Figure 4: Effects of transfecting an expression vector encoding for β-Catenin on S100A4 mRNA levels. Transfection with β-Catenin expression vector (pcDNA3-β-Catenin) was performed in HT29 cells, both sensitive (Figure 4A) and resistant (Figure 4B) as described in Methods. S100A4 mRNA levels were determined by RT-Real-Time PCR 48 h after transfection. All results are expressed as percentages referred to untreated cells. Values are the mean of three independent experiments ± SE. * p < 0.05.
Mentions: It had been previously described that S100A4 was a target of the Wnt signaling pathway and a functional TCF binding site has been identified in its promoter sequence [18]. To investigate whether S100A4 expression was regulated through this pathway in HT29 cells, we overexpressed β-catenin in HT29 sensitive or resistant cells and quantified S100A4 mRNA levels 48 h after transfection. A 2-fold increase in S100A4 mRNA levels was observed upon transfection of 1 μg of pcDNA3-β-catenin in HT29 sensitive cells (Figure 4A) whereas no changes were obtained when transfection was performed in HT29 resistant cells (Figure 4B).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus