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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 siS100A4 transfection of HT29 cells. A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 2: Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 cells. A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p < 0.05, ** p < 0.01, *** p < 0.001.

Mentions: We used iRNA technology to study the role of S100A4 in MTX resistance. Treatment of sensitive HT29 cells with increasing concentrations (10-100 nM) of the siRNA against S100A4 (siS100A4) showed a progressive decrease in its mRNA levels (Figure 2A), causing an 80% reduction at 100 nM. This treatment also caused a vast decrease in S100A4 protein levels (Figure 2B) and increased the sensitivity of HT29 cells toward MTX by about 50% (Figure 2C). To further assess siRNA specificity for S100A4, we determined the mRNA levels of other cellular genes upon incubation with siS100A4 (off-target effects). These experiments did not show any significant variation of the mRNA levels for Enolase 2, Topoisomerase II, Clusterin and UGT1A7 (Table 3)


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 siS100A4 transfection of HT29 cells. A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p < 0.05, ** p < 0.01, *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 cells. A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p < 0.05, ** p < 0.01, *** p < 0.001.
Mentions: We used iRNA technology to study the role of S100A4 in MTX resistance. Treatment of sensitive HT29 cells with increasing concentrations (10-100 nM) of the siRNA against S100A4 (siS100A4) showed a progressive decrease in its mRNA levels (Figure 2A), causing an 80% reduction at 100 nM. This treatment also caused a vast decrease in S100A4 protein levels (Figure 2B) and increased the sensitivity of HT29 cells toward MTX by about 50% (Figure 2C). To further assess siRNA specificity for S100A4, we determined the mRNA levels of other cellular genes upon incubation with siS100A4 (off-target effects). These experiments did not show any significant variation of the mRNA levels for Enolase 2, Topoisomerase II, Clusterin and UGT1A7 (Table 3)

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