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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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Analysis of HT29 and RHT29 growth rate.A) HT29 and RHT29 cells were subcutaneously inoculated into nude mice. Two weeks later, animals were treated with oxaliplatin 10 mg/kg once a week for 27 days. Results illustrate tumor volume and are represented as mean±SEM for 10 animals per group. The lower panel is a representative image of tumors obtained from animals treated or not with oxaliplatin. B) Growth kinetics was studied by colony formation assay. This representative image shows that HT29 cells are not able to grow under oxaliplatin treatment compared to RHT29 cell line. However, HT29 cells have a higher clonogenic capacity than RHT29 after 21 days of growth. OXA: oxaliplatin.
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pone-0004728-g002: Analysis of HT29 and RHT29 growth rate.A) HT29 and RHT29 cells were subcutaneously inoculated into nude mice. Two weeks later, animals were treated with oxaliplatin 10 mg/kg once a week for 27 days. Results illustrate tumor volume and are represented as mean±SEM for 10 animals per group. The lower panel is a representative image of tumors obtained from animals treated or not with oxaliplatin. B) Growth kinetics was studied by colony formation assay. This representative image shows that HT29 cells are not able to grow under oxaliplatin treatment compared to RHT29 cell line. However, HT29 cells have a higher clonogenic capacity than RHT29 after 21 days of growth. OXA: oxaliplatin.

Mentions: Some authors have observed that addition of MMP7 to cell cultures protects cells from drug cytotoxicity [4] and from cytotoxic T-cell killing [8], but whether MMP7 exerts these effects physiologically or if its expression is related to the acquisition of drug resistance remains unresolved. To analyze the role of MMP7 in these processes, we first generated cell lines resistant to oxaliplatin treatment by exposure of the parental cells to the drug over 5 months. These oxaliplatin-resistant cells show an IC50 several fold higher (RHT29 80 µM, RHCT116 p53+/+ ∼8 µM and ∼15 µM RHCT116 p53−/−) than the parental (HT29 ∼5–7 µM, HCT116 p53+/+ ∼1.75 µM and ∼2 µM HCT116 p53−/−),as determined by MTS assay (Figure 1A), and they are more resistant to oxaliplatin induced apoptosis (Figure 1B), as analyzed by annexin/IP staining (5-fold double staining for HT29 compared to 2-fold double staining for RHT29). We chose the HT29 normal and resistant cells for the in vivo experiments since they shown the most resistant phenotype. In vivo, RHT29 also shows resistance when the drug is administered as a single dose of 10 mg/kg once a week for 4 weeks (Figure 2A). Moreover, characterization of their growth dynamics and their colony formation properties show that, although resistant cells can grow under drug treatment, its growing rate is much lower compared to non-resistant cells (Figure 2B), in agreement with other studies [17]. This lower growth rate was also observed in vivo (Figure 2A). On the other hand, the RHT29 cell line shows phenotypic changes consistent with a more mesenchymal phenotype, as it was observed by increased intercellular separation, spindle-shape morphology and formation of pseudopodia (not shown), as previously described for this model of chemoresistance [17].


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)

Analysis of HT29 and RHT29 growth rate.A) HT29 and RHT29 cells were subcutaneously inoculated into nude mice. Two weeks later, animals were treated with oxaliplatin 10 mg/kg once a week for 27 days. Results illustrate tumor volume and are represented as mean±SEM for 10 animals per group. The lower panel is a representative image of tumors obtained from animals treated or not with oxaliplatin. B) Growth kinetics was studied by colony formation assay. This representative image shows that HT29 cells are not able to grow under oxaliplatin treatment compared to RHT29 cell line. However, HT29 cells have a higher clonogenic capacity than RHT29 after 21 days of growth. OXA: oxaliplatin.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004728-g002: Analysis of HT29 and RHT29 growth rate.A) HT29 and RHT29 cells were subcutaneously inoculated into nude mice. Two weeks later, animals were treated with oxaliplatin 10 mg/kg once a week for 27 days. Results illustrate tumor volume and are represented as mean±SEM for 10 animals per group. The lower panel is a representative image of tumors obtained from animals treated or not with oxaliplatin. B) Growth kinetics was studied by colony formation assay. This representative image shows that HT29 cells are not able to grow under oxaliplatin treatment compared to RHT29 cell line. However, HT29 cells have a higher clonogenic capacity than RHT29 after 21 days of growth. OXA: oxaliplatin.
Mentions: Some authors have observed that addition of MMP7 to cell cultures protects cells from drug cytotoxicity [4] and from cytotoxic T-cell killing [8], but whether MMP7 exerts these effects physiologically or if its expression is related to the acquisition of drug resistance remains unresolved. To analyze the role of MMP7 in these processes, we first generated cell lines resistant to oxaliplatin treatment by exposure of the parental cells to the drug over 5 months. These oxaliplatin-resistant cells show an IC50 several fold higher (RHT29 80 µM, RHCT116 p53+/+ ∼8 µM and ∼15 µM RHCT116 p53−/−) than the parental (HT29 ∼5–7 µM, HCT116 p53+/+ ∼1.75 µM and ∼2 µM HCT116 p53−/−),as determined by MTS assay (Figure 1A), and they are more resistant to oxaliplatin induced apoptosis (Figure 1B), as analyzed by annexin/IP staining (5-fold double staining for HT29 compared to 2-fold double staining for RHT29). We chose the HT29 normal and resistant cells for the in vivo experiments since they shown the most resistant phenotype. In vivo, RHT29 also shows resistance when the drug is administered as a single dose of 10 mg/kg once a week for 4 weeks (Figure 2A). Moreover, characterization of their growth dynamics and their colony formation properties show that, although resistant cells can grow under drug treatment, its growing rate is much lower compared to non-resistant cells (Figure 2B), in agreement with other studies [17]. This lower growth rate was also observed in vivo (Figure 2A). On the other hand, the RHT29 cell line shows phenotypic changes consistent with a more mesenchymal phenotype, as it was observed by increased intercellular separation, spindle-shape morphology and formation of pseudopodia (not shown), as previously described for this model of chemoresistance [17].

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