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Upregulation of CYP2S1 by oxaliplatin is associated with p53 status in colorectal cancer cell lines

View Article: PubMed Central - PubMed

ABSTRACT

Oxaliplatin displays a wide spectrum of antitumor activities and is widely used in the treatment of metastatic colorectal cancer (CRC). However, tumor responses to this agent are variable, and the underlying mechanisms are poorly understood. In the present study, oxaliplatin was found to strongly inhibit the growth of HCT116 cells harboring wild-type p53 but to only weakly inhibit SW480 cells, HT29 cells or p53−/− HCT116 cells, which all lack p53 expression. Administration of oxaliplatin significantly induced p53 accumulation and enhanced expression of CYP2S1 in HCT116 cells with wild-type p53. CYP2S1 knockdown conferred a cell survival advantage after oxaliplatin treatment to cells harboring wild-type p53 in vitro and in vivo. Interestingly, enzyme immunoassays, TOPFlash/FOPFlash reporter activity assays and western blotting analysis demonstrated oxaliplatin-mediated downregulation of PGE2 and Wnt/β-catenin signaling in a manner dependent on p53. Moreover, oxaliplatin treatment of mice with subcutaneous tumor xenografts drastically reduced the volume of wild-type p53 HCT116 tumors but had no effect on isogenic p53−/− HCT116 tumors. These results suggest that oxaliplatin exerts its inhibitory effects in human CRC cells via upregulation of CYP2S1 expression in a p53-dependent manner.

No MeSH data available.


Effect of oxaliplatin on PGE2 production and Wnt/β-catenin signaling and correlation with p53 status.(A) Enzyme-linked immunosorbent assay of PGE2 levels. PGE2 levels in cell culture supernatants were examined after oxaliplatin (OXA) treatment, and the results are expressed in terms of pg/mg protein. The error bars indicate the variability among wells, n = 3. *p < 0.05, **p < 0.01. Data analysis was performed using Student’s t-test. (B) Treatment of cells with or without exogenous PGE2 influence oxaliplatin treatment in p53+/+ and p53−/− HCT116 cells. The data on cell growth was performed using CCK8 assay from three experiments, data analysis was performed using student’s t-test. *p < 0.05. (C) Luciferase activity assay shows that oxaliplatin treatment decreases the transcriptional activity of TCF/LEF in p53+/+ HCT116 cells. TOPFlash and FOPFlash activities were evaluated in p53+/+ HCT116 and p53−/− HCT116 cells 24 h after oxaliplatin treatment. The data are expressed as the TOP/FOP ratio relative to the control. The results represent the mean ± SD from three independent experiments. **p < 0.01. (D) p53+/+ HCT116 and p53−/− HCT116 cells were treated with or without oxaliplatin for 24 h, and the expression levels of β-catenin or GAPDH (internal control) were analyzed by western blotting. One of three representative experiments is shown. (E) Total proteins were extracted and the protein levels of the β-catenin were analyzed in Western blot analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., **p < 0.01. (F, G) The overexpression of β-catenin conferred cell survival advantage after oxaliplatin treatment in p53+/+ HCT116 cells. Cell viability after oxaliplatin treatment (24 h or 48 h) was determined using the CCK8 assay. Increased β-catenin protein expression in cells transfected with β-catenin was confirmed by Western blotting. One of three representative experiments is shown.
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f4: Effect of oxaliplatin on PGE2 production and Wnt/β-catenin signaling and correlation with p53 status.(A) Enzyme-linked immunosorbent assay of PGE2 levels. PGE2 levels in cell culture supernatants were examined after oxaliplatin (OXA) treatment, and the results are expressed in terms of pg/mg protein. The error bars indicate the variability among wells, n = 3. *p < 0.05, **p < 0.01. Data analysis was performed using Student’s t-test. (B) Treatment of cells with or without exogenous PGE2 influence oxaliplatin treatment in p53+/+ and p53−/− HCT116 cells. The data on cell growth was performed using CCK8 assay from three experiments, data analysis was performed using student’s t-test. *p < 0.05. (C) Luciferase activity assay shows that oxaliplatin treatment decreases the transcriptional activity of TCF/LEF in p53+/+ HCT116 cells. TOPFlash and FOPFlash activities were evaluated in p53+/+ HCT116 and p53−/− HCT116 cells 24 h after oxaliplatin treatment. The data are expressed as the TOP/FOP ratio relative to the control. The results represent the mean ± SD from three independent experiments. **p < 0.01. (D) p53+/+ HCT116 and p53−/− HCT116 cells were treated with or without oxaliplatin for 24 h, and the expression levels of β-catenin or GAPDH (internal control) were analyzed by western blotting. One of three representative experiments is shown. (E) Total proteins were extracted and the protein levels of the β-catenin were analyzed in Western blot analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., **p < 0.01. (F, G) The overexpression of β-catenin conferred cell survival advantage after oxaliplatin treatment in p53+/+ HCT116 cells. Cell viability after oxaliplatin treatment (24 h or 48 h) was determined using the CCK8 assay. Increased β-catenin protein expression in cells transfected with β-catenin was confirmed by Western blotting. One of three representative experiments is shown.

Mentions: CYP2S1 depletion enhances CRC cell proliferation and is associated with PGE2-mediated activation of β-catenin signaling, and p53 downregulates Wnt signaling2537. We therefore examined whether p53 plays a role in oxaliplatin mediated regulation of CYP2S1-PGE2-Wnt signaling. CRC cells were treated with or without oxaliplatin for 24 h, and the level of PGE2 in the culture supernatants was examined by competitive ELISA. Treatment with oxaliplatin decreased the production of PGE2 in all cell lines (Fig. 4A). A significant decrease in PGE2 synthesis was observed in p53+/+ HCT116 cells, whereas only a slight decrease in PGE2 production was observed in p53-deficient cells (SW480, HT29 and p53−/− HCT116 cells) (Fig. 4A). Next, we examined whether exogenous PGE2 could affect cell growth in the processing of oxaliplatin treatment. For this purpose, Cells were treated with or without 50 nM PGE2 and 20 μM oxaliplatin for 24 h and then cell growth were determined. We found that the treatment of cells with exogenous PGE2 resulted in a significant decrease effect of inhibiting cell growth after oxaliplatin treatment in p53+/+ HCT116 (p < 0.05) (Fig. 4B).


Upregulation of CYP2S1 by oxaliplatin is associated with p53 status in colorectal cancer cell lines
Effect of oxaliplatin on PGE2 production and Wnt/β-catenin signaling and correlation with p53 status.(A) Enzyme-linked immunosorbent assay of PGE2 levels. PGE2 levels in cell culture supernatants were examined after oxaliplatin (OXA) treatment, and the results are expressed in terms of pg/mg protein. The error bars indicate the variability among wells, n = 3. *p < 0.05, **p < 0.01. Data analysis was performed using Student’s t-test. (B) Treatment of cells with or without exogenous PGE2 influence oxaliplatin treatment in p53+/+ and p53−/− HCT116 cells. The data on cell growth was performed using CCK8 assay from three experiments, data analysis was performed using student’s t-test. *p < 0.05. (C) Luciferase activity assay shows that oxaliplatin treatment decreases the transcriptional activity of TCF/LEF in p53+/+ HCT116 cells. TOPFlash and FOPFlash activities were evaluated in p53+/+ HCT116 and p53−/− HCT116 cells 24 h after oxaliplatin treatment. The data are expressed as the TOP/FOP ratio relative to the control. The results represent the mean ± SD from three independent experiments. **p < 0.01. (D) p53+/+ HCT116 and p53−/− HCT116 cells were treated with or without oxaliplatin for 24 h, and the expression levels of β-catenin or GAPDH (internal control) were analyzed by western blotting. One of three representative experiments is shown. (E) Total proteins were extracted and the protein levels of the β-catenin were analyzed in Western blot analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., **p < 0.01. (F, G) The overexpression of β-catenin conferred cell survival advantage after oxaliplatin treatment in p53+/+ HCT116 cells. Cell viability after oxaliplatin treatment (24 h or 48 h) was determined using the CCK8 assay. Increased β-catenin protein expression in cells transfected with β-catenin was confirmed by Western blotting. One of three representative experiments is shown.
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f4: Effect of oxaliplatin on PGE2 production and Wnt/β-catenin signaling and correlation with p53 status.(A) Enzyme-linked immunosorbent assay of PGE2 levels. PGE2 levels in cell culture supernatants were examined after oxaliplatin (OXA) treatment, and the results are expressed in terms of pg/mg protein. The error bars indicate the variability among wells, n = 3. *p < 0.05, **p < 0.01. Data analysis was performed using Student’s t-test. (B) Treatment of cells with or without exogenous PGE2 influence oxaliplatin treatment in p53+/+ and p53−/− HCT116 cells. The data on cell growth was performed using CCK8 assay from three experiments, data analysis was performed using student’s t-test. *p < 0.05. (C) Luciferase activity assay shows that oxaliplatin treatment decreases the transcriptional activity of TCF/LEF in p53+/+ HCT116 cells. TOPFlash and FOPFlash activities were evaluated in p53+/+ HCT116 and p53−/− HCT116 cells 24 h after oxaliplatin treatment. The data are expressed as the TOP/FOP ratio relative to the control. The results represent the mean ± SD from three independent experiments. **p < 0.01. (D) p53+/+ HCT116 and p53−/− HCT116 cells were treated with or without oxaliplatin for 24 h, and the expression levels of β-catenin or GAPDH (internal control) were analyzed by western blotting. One of three representative experiments is shown. (E) Total proteins were extracted and the protein levels of the β-catenin were analyzed in Western blot analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., **p < 0.01. (F, G) The overexpression of β-catenin conferred cell survival advantage after oxaliplatin treatment in p53+/+ HCT116 cells. Cell viability after oxaliplatin treatment (24 h or 48 h) was determined using the CCK8 assay. Increased β-catenin protein expression in cells transfected with β-catenin was confirmed by Western blotting. One of three representative experiments is shown.
Mentions: CYP2S1 depletion enhances CRC cell proliferation and is associated with PGE2-mediated activation of β-catenin signaling, and p53 downregulates Wnt signaling2537. We therefore examined whether p53 plays a role in oxaliplatin mediated regulation of CYP2S1-PGE2-Wnt signaling. CRC cells were treated with or without oxaliplatin for 24 h, and the level of PGE2 in the culture supernatants was examined by competitive ELISA. Treatment with oxaliplatin decreased the production of PGE2 in all cell lines (Fig. 4A). A significant decrease in PGE2 synthesis was observed in p53+/+ HCT116 cells, whereas only a slight decrease in PGE2 production was observed in p53-deficient cells (SW480, HT29 and p53−/− HCT116 cells) (Fig. 4A). Next, we examined whether exogenous PGE2 could affect cell growth in the processing of oxaliplatin treatment. For this purpose, Cells were treated with or without 50 nM PGE2 and 20 μM oxaliplatin for 24 h and then cell growth were determined. We found that the treatment of cells with exogenous PGE2 resulted in a significant decrease effect of inhibiting cell growth after oxaliplatin treatment in p53+/+ HCT116 (p < 0.05) (Fig. 4B).

View Article: PubMed Central - PubMed

ABSTRACT

Oxaliplatin displays a wide spectrum of antitumor activities and is widely used in the treatment of metastatic colorectal cancer (CRC). However, tumor responses to this agent are variable, and the underlying mechanisms are poorly understood. In the present study, oxaliplatin was found to strongly inhibit the growth of HCT116 cells harboring wild-type p53 but to only weakly inhibit SW480 cells, HT29 cells or p53&minus;/&minus; HCT116 cells, which all lack p53 expression. Administration of oxaliplatin significantly induced p53 accumulation and enhanced expression of CYP2S1 in HCT116 cells with wild-type p53. CYP2S1 knockdown conferred a cell survival advantage after oxaliplatin treatment to cells harboring wild-type p53 in vitro and in vivo. Interestingly, enzyme immunoassays, TOPFlash/FOPFlash reporter activity assays and western blotting analysis demonstrated oxaliplatin-mediated downregulation of PGE2 and Wnt/&beta;-catenin signaling in a manner dependent on p53. Moreover, oxaliplatin treatment of mice with subcutaneous tumor xenografts drastically reduced the volume of wild-type p53 HCT116 tumors but had no effect on isogenic p53&minus;/&minus; HCT116 tumors. These results suggest that oxaliplatin exerts its inhibitory effects in human CRC cells via upregulation of CYP2S1 expression in a p53-dependent manner.

No MeSH data available.