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The role of MMP7 and its cross-talk with the FAS/FASL system during the acquisition of chemoresistance to oxaliplatin.

Almendro V, Ametller E, García-Recio S, Collazo O, Casas I, Augé JM, Maurel J, Gascón P - PLoS ONE (2009)

Bottom Line: Some of these effects are caused by alterations in Fas receptor.Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor.Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals.

View Article: PubMed Central - PubMed

Affiliation: Facultat de Medicina, Hospital Clínic, Institut d'Investigacions Biomèdiques Agustí Pi y Sunyer, Universitat de Barcelona, Barcelona, Spain. almendro@clinic.ub.es

ABSTRACT

Background: The efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells.

Principal findings: For this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53(+/+), RHCT116 p53(-/-)) from the parental HT29, HCT116 p53(+/+) and HCT116 p53(-/-) colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53(-/-) but not in the RHCT116 p53(+/+). Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53(-/-) but not in the RHCT116 p53(+/+). Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells.

Conclusions: Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals.

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Modulation of FasL expression in HT29 and RHT29 cells lines.FasL expression was detected in HT29 and RHT29 cell lines treated or not with oxaliplatin 10 µM by A) FACS analysis of its surface expression or B) qPCR of its mRNA levels. C) NF-κB p65 was detected by Western Blot by the use of an antibody that recognizes the nuclear localization signal (NLS) epitope. Tubulin expression was used as endogenous control. D) Sensitivity of both cell lines to the inhibition of the NF-κB pathway was measured by an MTS assay in which cells were treated with different doses of BAY117085, a known inhibitor of IKK activation. Values that are significantly different between control and treated group by ANOVA analysis are indicated by ***p<0.0001. OXA: oxaliplatin.
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pone-0004728-g006: Modulation of FasL expression in HT29 and RHT29 cells lines.FasL expression was detected in HT29 and RHT29 cell lines treated or not with oxaliplatin 10 µM by A) FACS analysis of its surface expression or B) qPCR of its mRNA levels. C) NF-κB p65 was detected by Western Blot by the use of an antibody that recognizes the nuclear localization signal (NLS) epitope. Tubulin expression was used as endogenous control. D) Sensitivity of both cell lines to the inhibition of the NF-κB pathway was measured by an MTS assay in which cells were treated with different doses of BAY117085, a known inhibitor of IKK activation. Values that are significantly different between control and treated group by ANOVA analysis are indicated by ***p<0.0001. OXA: oxaliplatin.

Mentions: We next determined the expression of FasL, a molecule known to be cleaved and released from the cell surface by MMP7 [4]. The analysis of FasL expression under oxaliplatin treatment shows that the drug does not increase significantly the expression of FasL at the cell surface of both cell lines (Figure 6A). However, the mRNA content of FasL was strongly upregulated in both cells lines by oxaliplatin treatment, being these effects more pronounced in the resistant cell line (20-fold) than in the normal one (4-fold) (Figure 6B). It has been observed in other cell lines that cytotoxic treatment could induce FasL expression by upregulation of the NF-κB system. To determine if this system was altered in our model, we decided to analyze the expression of the p65 subunit by using an antibody that recognizes the nuclear localization signal (NLS), an epitope that could be detected when p65 is activated [22]. The expression of NLS-p65 was similar in both cell lines, but under oxaliplatin treatment its activation was higher in the resistant cell line (Figure 6C). Moreover, RHT29 cells showed higher sensitivity to the inhibition of the NF-κB pathway by the IKK inhibitor Bay117085 (Figure 6D) than the non-resistant cells, indicating a metabolic dependence on this pathway by the resistant cell line. Therefore, the increased expression of FasL is correlated with the increased activation of the NF-κB system. These results suggest that, although oxaliplatin treatment induces the synthesis of FasL in both cell lines, its detection at the cell surface is not increased by the drug treatment probably by the shedding of FasL by MMP7.


The role of MMP7 and its cross-talk with the FAS/FASL system during the acquisition of chemoresistance to oxaliplatin.

Almendro V, Ametller E, García-Recio S, Collazo O, Casas I, Augé JM, Maurel J, Gascón P - PLoS ONE (2009)

Modulation of FasL expression in HT29 and RHT29 cells lines.FasL expression was detected in HT29 and RHT29 cell lines treated or not with oxaliplatin 10 µM by A) FACS analysis of its surface expression or B) qPCR of its mRNA levels. C) NF-κB p65 was detected by Western Blot by the use of an antibody that recognizes the nuclear localization signal (NLS) epitope. Tubulin expression was used as endogenous control. D) Sensitivity of both cell lines to the inhibition of the NF-κB pathway was measured by an MTS assay in which cells were treated with different doses of BAY117085, a known inhibitor of IKK activation. Values that are significantly different between control and treated group by ANOVA analysis are indicated by ***p<0.0001. OXA: oxaliplatin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2648894&req=5

pone-0004728-g006: Modulation of FasL expression in HT29 and RHT29 cells lines.FasL expression was detected in HT29 and RHT29 cell lines treated or not with oxaliplatin 10 µM by A) FACS analysis of its surface expression or B) qPCR of its mRNA levels. C) NF-κB p65 was detected by Western Blot by the use of an antibody that recognizes the nuclear localization signal (NLS) epitope. Tubulin expression was used as endogenous control. D) Sensitivity of both cell lines to the inhibition of the NF-κB pathway was measured by an MTS assay in which cells were treated with different doses of BAY117085, a known inhibitor of IKK activation. Values that are significantly different between control and treated group by ANOVA analysis are indicated by ***p<0.0001. OXA: oxaliplatin.
Mentions: We next determined the expression of FasL, a molecule known to be cleaved and released from the cell surface by MMP7 [4]. The analysis of FasL expression under oxaliplatin treatment shows that the drug does not increase significantly the expression of FasL at the cell surface of both cell lines (Figure 6A). However, the mRNA content of FasL was strongly upregulated in both cells lines by oxaliplatin treatment, being these effects more pronounced in the resistant cell line (20-fold) than in the normal one (4-fold) (Figure 6B). It has been observed in other cell lines that cytotoxic treatment could induce FasL expression by upregulation of the NF-κB system. To determine if this system was altered in our model, we decided to analyze the expression of the p65 subunit by using an antibody that recognizes the nuclear localization signal (NLS), an epitope that could be detected when p65 is activated [22]. The expression of NLS-p65 was similar in both cell lines, but under oxaliplatin treatment its activation was higher in the resistant cell line (Figure 6C). Moreover, RHT29 cells showed higher sensitivity to the inhibition of the NF-κB pathway by the IKK inhibitor Bay117085 (Figure 6D) than the non-resistant cells, indicating a metabolic dependence on this pathway by the resistant cell line. Therefore, the increased expression of FasL is correlated with the increased activation of the NF-κB system. These results suggest that, although oxaliplatin treatment induces the synthesis of FasL in both cell lines, its detection at the cell surface is not increased by the drug treatment probably by the shedding of FasL by MMP7.

Bottom Line: Some of these effects are caused by alterations in Fas receptor.Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor.Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals.

View Article: PubMed Central - PubMed

Affiliation: Facultat de Medicina, Hospital Clínic, Institut d'Investigacions Biomèdiques Agustí Pi y Sunyer, Universitat de Barcelona, Barcelona, Spain. almendro@clinic.ub.es

ABSTRACT

Background: The efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells.

Principal findings: For this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53(+/+), RHCT116 p53(-/-)) from the parental HT29, HCT116 p53(+/+) and HCT116 p53(-/-) colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53(-/-) but not in the RHCT116 p53(+/+). Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53(-/-) but not in the RHCT116 p53(+/+). Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells.

Conclusions: Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals.

Show MeSH
Related in: MedlinePlus