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Curcumin mediates oxaliplatin-acquired resistance reversion in colorectal cancer cell lines through modulation of CXC-Chemokine/NF-κB signalling pathway.

Ruiz de Porras V, Bystrup S, Martínez-Cardús A, Pluvinet R, Sumoy L, Howells L, James MI, Iwuji C, Manzano JL, Layos L, Bugés C, Abad A, Martínez-Balibrea E - Sci Rep (2016)

Bottom Line: Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells.High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin.In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients.

View Article: PubMed Central - PubMed

Affiliation: Health Sciences Research Institute of the Germans Trias i Pujol Foundation (IGTP), Can Ruti Campus, Ctra. Can Ruti- Camí de les escoles s/n, 08916, Badalona, Spain.

ABSTRACT
Resistance to oxaliplatin (OXA) is a complex process affecting the outcomes of metastatic colorectal cancer (CRC) patients treated with this drug. De-regulation of the NF-κB signalling pathway has been proposed as an important mechanism involved in this phenomenon. Here, we show that NF-κB was hyperactivated in in vitro models of OXA-acquired resistance but was attenuated by the addition of Curcumin, a non-toxic NF-κB inhibitor. The concomitant combination of Curcumin + OXA was more effective and synergistic in cell lines with acquired resistance to OXA, leading to the reversion of their resistant phenotype, through the inhibition of the NF-κB signalling cascade. Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells. Moreover, CXCL8 and CXCL1 gene silencing made resistant cells more sensitive to OXA through the inhibition of the Akt/NF-κB pathway. High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin. In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients.

No MeSH data available.


Related in: MedlinePlus

Effect of OXA treatment on NF-κB activation in HT29 and HTOXAR3 cells.Representative western blot images showing protein expression changes of phosphorylated p65 (p-p65 Ser536), phosphorylated IκBα (p-IκBα Ser32/36), Survivin, Bcl-2 and Cyclin D1 in HT29 and HTOXAR3 cells after treatment with 10 μM or 30 μM of OXA respectively, for 0 to 120 minutes (a) and for 24 hours (b,c) Bar graphs representing mean ± SEM expression changes of the indicated proteins after a 24-hour OXA treatment with the corresponding IC50 dose for HT29 (10 μM) and HTOXAR3 (30 μM). Alpha-tubulin was used as endogenous control. (d) Western blots showing p65 protein expression in cytoplasmic and nuclear extracts of HT29 cells after OXA treatment at short exposure times (0 to 60 min) and at 24 hours (e). TNFα was used as positive control at 50 ng/mL. Msh2 and α-tubulin were used as nuclear- and cytoplasmic-specific protein controls, respectively. NT: Non-treated cells. Results shown were obtained from at least 3 independent experiments. *p-value < 0.05 and **p-value < 0.01; as compared to NT. #p-value < 0.05 relative to protein expression in the HT29 cell line.
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f2: Effect of OXA treatment on NF-κB activation in HT29 and HTOXAR3 cells.Representative western blot images showing protein expression changes of phosphorylated p65 (p-p65 Ser536), phosphorylated IκBα (p-IκBα Ser32/36), Survivin, Bcl-2 and Cyclin D1 in HT29 and HTOXAR3 cells after treatment with 10 μM or 30 μM of OXA respectively, for 0 to 120 minutes (a) and for 24 hours (b,c) Bar graphs representing mean ± SEM expression changes of the indicated proteins after a 24-hour OXA treatment with the corresponding IC50 dose for HT29 (10 μM) and HTOXAR3 (30 μM). Alpha-tubulin was used as endogenous control. (d) Western blots showing p65 protein expression in cytoplasmic and nuclear extracts of HT29 cells after OXA treatment at short exposure times (0 to 60 min) and at 24 hours (e). TNFα was used as positive control at 50 ng/mL. Msh2 and α-tubulin were used as nuclear- and cytoplasmic-specific protein controls, respectively. NT: Non-treated cells. Results shown were obtained from at least 3 independent experiments. *p-value < 0.05 and **p-value < 0.01; as compared to NT. #p-value < 0.05 relative to protein expression in the HT29 cell line.

Mentions: NF-κB pathway could be activated through two mechanisms: signals that originate at cell receptors and signals that originate in the nucleus in response to DNA damage. Treatment with OXA potentiated NF-κB activation in different cell types, including CRC cell lines1030. Accordingly, we assessed the phosphorylation status of p65 and IκBα, and the expression of three NF-κB target genes in HT29 and HTOXAR3 cell lines after short OXA-exposure times (0 to 120 min) and after 24 h of OXA treatment at 10 and 30 μM (IC50 values), respectively. We observed that OXA induced a fast phosphorylation of p65 and IκBα after the first 15 min of treatment and potentiated the expression of Bcl-2, Survivin and Cyclin D1 in both cell lines. Despite a slightly decrease in the expression of these proteins was observed after 30’ post-treatment, higher levels as compared to non-treated cells were maintained until at least 120’ after OXA treatment. Interestingly, we observed a trend to a more evident activation in HT29 as compared with HTOXAR3 cell line (Fig. 2a and Supplementary Fig. S1). In fact, these differences were more evident after a 24 h exposure since p65 and IκBα phosphorylation and Bcl-2 and Survivin expression increased in HT29 but not in HTOXAR3 cell line in a statistically significant way. In contrast, Cyclin D1 protein levels decreased significantly in both cell lines, this effect being more evident in the parental cell line. As previously described in HT29 cells31 we did not observe a significant IκBα protein degradation (Fig. 2b,c). When NF-κB is activated, the p65 subunit translocates to the cell nucleus. To confirm that OXA treatment promotes p65 nuclear translocation, HT29 cells were treated with OXA (10 μM) for varying lengths of times, and p65 expression in nuclear and cytoplasmic extracts was analysed by western blot. Nuclear p65 increased as early as 15 min after OXA exposure (Fig. 2d) and was sustained at least after 24 h of OXA treatment (Fig. 2e). TNFα was used as a positive control due to its ability to activate the NF-κB pathway.


Curcumin mediates oxaliplatin-acquired resistance reversion in colorectal cancer cell lines through modulation of CXC-Chemokine/NF-κB signalling pathway.

Ruiz de Porras V, Bystrup S, Martínez-Cardús A, Pluvinet R, Sumoy L, Howells L, James MI, Iwuji C, Manzano JL, Layos L, Bugés C, Abad A, Martínez-Balibrea E - Sci Rep (2016)

Effect of OXA treatment on NF-κB activation in HT29 and HTOXAR3 cells.Representative western blot images showing protein expression changes of phosphorylated p65 (p-p65 Ser536), phosphorylated IκBα (p-IκBα Ser32/36), Survivin, Bcl-2 and Cyclin D1 in HT29 and HTOXAR3 cells after treatment with 10 μM or 30 μM of OXA respectively, for 0 to 120 minutes (a) and for 24 hours (b,c) Bar graphs representing mean ± SEM expression changes of the indicated proteins after a 24-hour OXA treatment with the corresponding IC50 dose for HT29 (10 μM) and HTOXAR3 (30 μM). Alpha-tubulin was used as endogenous control. (d) Western blots showing p65 protein expression in cytoplasmic and nuclear extracts of HT29 cells after OXA treatment at short exposure times (0 to 60 min) and at 24 hours (e). TNFα was used as positive control at 50 ng/mL. Msh2 and α-tubulin were used as nuclear- and cytoplasmic-specific protein controls, respectively. NT: Non-treated cells. Results shown were obtained from at least 3 independent experiments. *p-value < 0.05 and **p-value < 0.01; as compared to NT. #p-value < 0.05 relative to protein expression in the HT29 cell line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835769&req=5

f2: Effect of OXA treatment on NF-κB activation in HT29 and HTOXAR3 cells.Representative western blot images showing protein expression changes of phosphorylated p65 (p-p65 Ser536), phosphorylated IκBα (p-IκBα Ser32/36), Survivin, Bcl-2 and Cyclin D1 in HT29 and HTOXAR3 cells after treatment with 10 μM or 30 μM of OXA respectively, for 0 to 120 minutes (a) and for 24 hours (b,c) Bar graphs representing mean ± SEM expression changes of the indicated proteins after a 24-hour OXA treatment with the corresponding IC50 dose for HT29 (10 μM) and HTOXAR3 (30 μM). Alpha-tubulin was used as endogenous control. (d) Western blots showing p65 protein expression in cytoplasmic and nuclear extracts of HT29 cells after OXA treatment at short exposure times (0 to 60 min) and at 24 hours (e). TNFα was used as positive control at 50 ng/mL. Msh2 and α-tubulin were used as nuclear- and cytoplasmic-specific protein controls, respectively. NT: Non-treated cells. Results shown were obtained from at least 3 independent experiments. *p-value < 0.05 and **p-value < 0.01; as compared to NT. #p-value < 0.05 relative to protein expression in the HT29 cell line.
Mentions: NF-κB pathway could be activated through two mechanisms: signals that originate at cell receptors and signals that originate in the nucleus in response to DNA damage. Treatment with OXA potentiated NF-κB activation in different cell types, including CRC cell lines1030. Accordingly, we assessed the phosphorylation status of p65 and IκBα, and the expression of three NF-κB target genes in HT29 and HTOXAR3 cell lines after short OXA-exposure times (0 to 120 min) and after 24 h of OXA treatment at 10 and 30 μM (IC50 values), respectively. We observed that OXA induced a fast phosphorylation of p65 and IκBα after the first 15 min of treatment and potentiated the expression of Bcl-2, Survivin and Cyclin D1 in both cell lines. Despite a slightly decrease in the expression of these proteins was observed after 30’ post-treatment, higher levels as compared to non-treated cells were maintained until at least 120’ after OXA treatment. Interestingly, we observed a trend to a more evident activation in HT29 as compared with HTOXAR3 cell line (Fig. 2a and Supplementary Fig. S1). In fact, these differences were more evident after a 24 h exposure since p65 and IκBα phosphorylation and Bcl-2 and Survivin expression increased in HT29 but not in HTOXAR3 cell line in a statistically significant way. In contrast, Cyclin D1 protein levels decreased significantly in both cell lines, this effect being more evident in the parental cell line. As previously described in HT29 cells31 we did not observe a significant IκBα protein degradation (Fig. 2b,c). When NF-κB is activated, the p65 subunit translocates to the cell nucleus. To confirm that OXA treatment promotes p65 nuclear translocation, HT29 cells were treated with OXA (10 μM) for varying lengths of times, and p65 expression in nuclear and cytoplasmic extracts was analysed by western blot. Nuclear p65 increased as early as 15 min after OXA exposure (Fig. 2d) and was sustained at least after 24 h of OXA treatment (Fig. 2e). TNFα was used as a positive control due to its ability to activate the NF-κB pathway.

Bottom Line: Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells.High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin.In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients.

View Article: PubMed Central - PubMed

Affiliation: Health Sciences Research Institute of the Germans Trias i Pujol Foundation (IGTP), Can Ruti Campus, Ctra. Can Ruti- Camí de les escoles s/n, 08916, Badalona, Spain.

ABSTRACT
Resistance to oxaliplatin (OXA) is a complex process affecting the outcomes of metastatic colorectal cancer (CRC) patients treated with this drug. De-regulation of the NF-κB signalling pathway has been proposed as an important mechanism involved in this phenomenon. Here, we show that NF-κB was hyperactivated in in vitro models of OXA-acquired resistance but was attenuated by the addition of Curcumin, a non-toxic NF-κB inhibitor. The concomitant combination of Curcumin + OXA was more effective and synergistic in cell lines with acquired resistance to OXA, leading to the reversion of their resistant phenotype, through the inhibition of the NF-κB signalling cascade. Transcriptomic profiling revealed the up-regulation of three NF-κB-regulated CXC-chemokines, CXCL8, CXCL1 and CXCL2, in the resistant cells that were more efficiently down-regulated after OXA + Curcumin treatment as compared to the sensitive cells. Moreover, CXCL8 and CXCL1 gene silencing made resistant cells more sensitive to OXA through the inhibition of the Akt/NF-κB pathway. High expression of CXCL1 in FFPE samples from explant cultures of CRC patients-derived liver metastases was associated with response to OXA + Curcumin. In conclusion, we suggest that combination of OXA + Curcumin could be an effective treatment, for which CXCL1 could be used as a predictive marker, in CRC patients.

No MeSH data available.


Related in: MedlinePlus