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

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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.


CYP2S1 knockdown elevate resistance of p53+/+HCT116 cells to oxaliplatin treatment in vitro.CYP2S1 knockdown conferred cell survival advantage after oxaliplatin treatment to cells harboring wild-type p53 but not to isogenic cells lacking p53. Cells were treated with the shNT (denotes non-targeting control shRNA) or CYP2S1 shRNA, respectively. Cell viability after Oxaliplatin (20 μM) treatment was determined by the CCK8 assay. (A,B) p53+/+ HCT116 CYP2S1 knockdown cells are highly resistant to oxaliplatin treatment. Reduced CYP2S1 protein expression in cells was confirmed by Western blot, one of three representative experiments is shown. (C,D) Total RNA was extracted and the mRNA levels of the CYP2S1 gene were analyzed in qRT–PCR analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., *p < 0.05, **p < 0.01.
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f3: CYP2S1 knockdown elevate resistance of p53+/+HCT116 cells to oxaliplatin treatment in vitro.CYP2S1 knockdown conferred cell survival advantage after oxaliplatin treatment to cells harboring wild-type p53 but not to isogenic cells lacking p53. Cells were treated with the shNT (denotes non-targeting control shRNA) or CYP2S1 shRNA, respectively. Cell viability after Oxaliplatin (20 μM) treatment was determined by the CCK8 assay. (A,B) p53+/+ HCT116 CYP2S1 knockdown cells are highly resistant to oxaliplatin treatment. Reduced CYP2S1 protein expression in cells was confirmed by Western blot, one of three representative experiments is shown. (C,D) Total RNA was extracted and the mRNA levels of the CYP2S1 gene were analyzed in qRT–PCR analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., *p < 0.05, **p < 0.01.

Mentions: Considering that CYP2S1 expression is associated with pharmacological drug metabolism in CRC cancer. ShRNA plasmids (genechem NM_030622) was used to knockdown CYP2S1 mRNA and protein and examined whether CYP2S1 expression alters cell viability upon oxaliplatin treatment. To validate the knockdown efficiency, CYP2S1 protein and mRNA levels were measured by Western blot analysis and by quantitative real-time PCR, respectively. The results indicated that CYP2S1 mRNA and protein were clearly reduced (Fig. 3A–D). CYP2S1 knockdown led to a significant cell survival advantage after oxaliplatin treatment (20 μM, 24–72 h) in p53+/+ HCT116 cells but not in isogenic p53−/− HCT116 cells (Fig. 3A,B). The findings indicate that p53 may be potentially important for the CYP2S1effect to inhibit cell survival after oxaliplatin treatment.


Upregulation of CYP2S1 by oxaliplatin is associated with p53 status in colorectal cancer cell lines
CYP2S1 knockdown elevate resistance of p53+/+HCT116 cells to oxaliplatin treatment in vitro.CYP2S1 knockdown conferred cell survival advantage after oxaliplatin treatment to cells harboring wild-type p53 but not to isogenic cells lacking p53. Cells were treated with the shNT (denotes non-targeting control shRNA) or CYP2S1 shRNA, respectively. Cell viability after Oxaliplatin (20 μM) treatment was determined by the CCK8 assay. (A,B) p53+/+ HCT116 CYP2S1 knockdown cells are highly resistant to oxaliplatin treatment. Reduced CYP2S1 protein expression in cells was confirmed by Western blot, one of three representative experiments is shown. (C,D) Total RNA was extracted and the mRNA levels of the CYP2S1 gene were analyzed in qRT–PCR analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., *p < 0.05, **p < 0.01.
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Related In: Results  -  Collection

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f3: CYP2S1 knockdown elevate resistance of p53+/+HCT116 cells to oxaliplatin treatment in vitro.CYP2S1 knockdown conferred cell survival advantage after oxaliplatin treatment to cells harboring wild-type p53 but not to isogenic cells lacking p53. Cells were treated with the shNT (denotes non-targeting control shRNA) or CYP2S1 shRNA, respectively. Cell viability after Oxaliplatin (20 μM) treatment was determined by the CCK8 assay. (A,B) p53+/+ HCT116 CYP2S1 knockdown cells are highly resistant to oxaliplatin treatment. Reduced CYP2S1 protein expression in cells was confirmed by Western blot, one of three representative experiments is shown. (C,D) Total RNA was extracted and the mRNA levels of the CYP2S1 gene were analyzed in qRT–PCR analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., *p < 0.05, **p < 0.01.
Mentions: Considering that CYP2S1 expression is associated with pharmacological drug metabolism in CRC cancer. ShRNA plasmids (genechem NM_030622) was used to knockdown CYP2S1 mRNA and protein and examined whether CYP2S1 expression alters cell viability upon oxaliplatin treatment. To validate the knockdown efficiency, CYP2S1 protein and mRNA levels were measured by Western blot analysis and by quantitative real-time PCR, respectively. The results indicated that CYP2S1 mRNA and protein were clearly reduced (Fig. 3A–D). CYP2S1 knockdown led to a significant cell survival advantage after oxaliplatin treatment (20 μM, 24–72 h) in p53+/+ HCT116 cells but not in isogenic p53−/− HCT116 cells (Fig. 3A,B). The findings indicate that p53 may be potentially important for the CYP2S1effect to inhibit cell survival after oxaliplatin treatment.

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.