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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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Effects on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells. A) mRNA levels of S100A4 determined by RT-Real-Time PCR 48 h after treatment of HT29 cells (30,000) with 250 ng of the expression vector for S100A4 (pCMV-S100A4). B) A representative image of the intracellular protein levels of S100A4 determined by Western Blotting 72 h after ectopic transfection with its expression vector is shown in the upper panel, and the quantification of the blots is shown in the lower panel. Purified S100A4 protein was used as a reference marker (Abnova; first lane). An additional panel showing endogenous S100A4 protein levels in HT29 sensitive (S) and resistant (R) cells is also provided. C) Effects of S100A4 overexpression on cell viability. HT29 cells (100,000) were treated with 1 μg of pCMV-S100A4 and 5 × 10-8M MTX was added 48 h later. Cell viability was assessed by the MTT assay six days after MTX treatment. D) Extracellular S100A4 protein levels quantified by ELISA 72 h after S100A4 overexpression upon pCMV-S100A4 transfection. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p < 0.05, **p < 0.01, *** p < 0.001.
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Figure 1: Effects on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells. A) mRNA levels of S100A4 determined by RT-Real-Time PCR 48 h after treatment of HT29 cells (30,000) with 250 ng of the expression vector for S100A4 (pCMV-S100A4). B) A representative image of the intracellular protein levels of S100A4 determined by Western Blotting 72 h after ectopic transfection with its expression vector is shown in the upper panel, and the quantification of the blots is shown in the lower panel. Purified S100A4 protein was used as a reference marker (Abnova; first lane). An additional panel showing endogenous S100A4 protein levels in HT29 sensitive (S) and resistant (R) cells is also provided. C) Effects of S100A4 overexpression on cell viability. HT29 cells (100,000) were treated with 1 μg of pCMV-S100A4 and 5 × 10-8M MTX was added 48 h later. Cell viability was assessed by the MTT assay six days after MTX treatment. D) Extracellular S100A4 protein levels quantified by ELISA 72 h after S100A4 overexpression upon pCMV-S100A4 transfection. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p < 0.05, **p < 0.01, *** p < 0.001.

Mentions: HT29 MTX-resistant cells displayed the highest S100A4 expression values, considering both the mRNA and protein levels (Table 2). Thus, HT29 cells were selected for further studies using an expression vector for S100A4 (pCMV-S100A4). Transfection with 250 ng of this vector produced a 8-fold increase in the levels of S100A4 RNA after 48 hours (Figure 1A). The intracellular levels of S100A4 protein were also increased, reaching almost 5 fold when transfecting 1 μg of the expression plasmid (Figure 1B). The overexpression of S100A4 was associated to a decrease in the sensitivity toward MTX (Figure 1C).


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 on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells. A) mRNA levels of S100A4 determined by RT-Real-Time PCR 48 h after treatment of HT29 cells (30,000) with 250 ng of the expression vector for S100A4 (pCMV-S100A4). B) A representative image of the intracellular protein levels of S100A4 determined by Western Blotting 72 h after ectopic transfection with its expression vector is shown in the upper panel, and the quantification of the blots is shown in the lower panel. Purified S100A4 protein was used as a reference marker (Abnova; first lane). An additional panel showing endogenous S100A4 protein levels in HT29 sensitive (S) and resistant (R) cells is also provided. C) Effects of S100A4 overexpression on cell viability. HT29 cells (100,000) were treated with 1 μg of pCMV-S100A4 and 5 × 10-8M MTX was added 48 h later. Cell viability was assessed by the MTT assay six days after MTX treatment. D) Extracellular S100A4 protein levels quantified by ELISA 72 h after S100A4 overexpression upon pCMV-S100A4 transfection. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p < 0.05, **p < 0.01, *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effects on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells. A) mRNA levels of S100A4 determined by RT-Real-Time PCR 48 h after treatment of HT29 cells (30,000) with 250 ng of the expression vector for S100A4 (pCMV-S100A4). B) A representative image of the intracellular protein levels of S100A4 determined by Western Blotting 72 h after ectopic transfection with its expression vector is shown in the upper panel, and the quantification of the blots is shown in the lower panel. Purified S100A4 protein was used as a reference marker (Abnova; first lane). An additional panel showing endogenous S100A4 protein levels in HT29 sensitive (S) and resistant (R) cells is also provided. C) Effects of S100A4 overexpression on cell viability. HT29 cells (100,000) were treated with 1 μg of pCMV-S100A4 and 5 × 10-8M MTX was added 48 h later. Cell viability was assessed by the MTT assay six days after MTX treatment. D) Extracellular S100A4 protein levels quantified by ELISA 72 h after S100A4 overexpression upon pCMV-S100A4 transfection. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p < 0.05, **p < 0.01, *** p < 0.001.
Mentions: HT29 MTX-resistant cells displayed the highest S100A4 expression values, considering both the mRNA and protein levels (Table 2). Thus, HT29 cells were selected for further studies using an expression vector for S100A4 (pCMV-S100A4). Transfection with 250 ng of this vector produced a 8-fold increase in the levels of S100A4 RNA after 48 hours (Figure 1A). The intracellular levels of S100A4 protein were also increased, reaching almost 5 fold when transfecting 1 μg of the expression plasmid (Figure 1B). The overexpression of S100A4 was associated to a decrease in the sensitivity toward MTX (Figure 1C).

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