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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.

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Upregulation of CYP2S1 protein expression increases oxaliplatin sensitivity in a p53-dependent manner in vivo.Identical (2 × 106) amounts of each cells were injected subcutaneously into the flanks of nude mice. When tumors reached approximately 100 mm3, the mice received either PBS or oxaliplatin (10 mg/kg) intraperitoneally (day 0). A second dose of either PBS or oxaliplatin was administered three days later. Tumor growth was analyzed by caliper measurements every 2 days. (A,B) Comparison of tumor volume between each group (means ± SD, n = 5). Oxaliplatin treatment markedly reduced tumor volume in p53+/+HCT116 tumor xenografts. *p < 0.05, **p < 0.01. (C) Tumors were harvested on day 12. Tumors from each group were analyzed for the presence of CYP2S1 and p53 by western blotting. GAPDH was used as an internal control. (D) Total proteins were extracted and the protein levels of the CYP2S1 were analyzed in Western blot analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., ** p < 0.01. (E,F) Weights of xenograft tumours derived from p53+/+ or p53−/−HCT116 cells expressing CYP2S1 knockdown or control (shRNA or shNT, respectively) after oxaliplatin treatment. n = 5 mice per group.
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f5: Upregulation of CYP2S1 protein expression increases oxaliplatin sensitivity in a p53-dependent manner in vivo.Identical (2 × 106) amounts of each cells were injected subcutaneously into the flanks of nude mice. When tumors reached approximately 100 mm3, the mice received either PBS or oxaliplatin (10 mg/kg) intraperitoneally (day 0). A second dose of either PBS or oxaliplatin was administered three days later. Tumor growth was analyzed by caliper measurements every 2 days. (A,B) Comparison of tumor volume between each group (means ± SD, n = 5). Oxaliplatin treatment markedly reduced tumor volume in p53+/+HCT116 tumor xenografts. *p < 0.05, **p < 0.01. (C) Tumors were harvested on day 12. Tumors from each group were analyzed for the presence of CYP2S1 and p53 by western blotting. GAPDH was used as an internal control. (D) Total proteins were extracted and the protein levels of the CYP2S1 were analyzed in Western blot analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., ** p < 0.01. (E,F) Weights of xenograft tumours derived from p53+/+ or p53−/−HCT116 cells expressing CYP2S1 knockdown or control (shRNA or shNT, respectively) after oxaliplatin treatment. n = 5 mice per group.

Mentions: As knockdown of CYP2S1 expression accelerates colorectal tumor growth in vivo25, we next investigated the effects of oxaliplatin on CYP2S1 protein expression in vivo. Isogenic p53+/+ and p53−/−HCT116 cell lines were injected into the flanks of immunocompromised nude mice, and (phosphate-buffered saline) PBS or oxaliplatin treatment was started when the tumors measured approximately 100 mm3. Tumor growth was monitored every 2 days until the mice were sacrificed on day 12, when tumors were excised and analyzed by western blotting. Oxaliplatin treatment had a striking antitumor effect on p53+/+HCT116 tumor xenografts (Fig. 5A), whereas the growth of p53−/−HCT116 xenografts was not significantly affected by oxaliplatin treatment (Fig. 5B). Oxaliplatin notably enhanced the induction of CYP2S1 expression in p53+/+HCT116 tumors but had virtually no effect on CYP2S1 expression in p53−/− HCT116 tumors (Fig. 5C,D).


Upregulation of CYP2S1 by oxaliplatin is associated with p53 status in colorectal cancer cell lines
Upregulation of CYP2S1 protein expression increases oxaliplatin sensitivity in a p53-dependent manner in vivo.Identical (2 × 106) amounts of each cells were injected subcutaneously into the flanks of nude mice. When tumors reached approximately 100 mm3, the mice received either PBS or oxaliplatin (10 mg/kg) intraperitoneally (day 0). A second dose of either PBS or oxaliplatin was administered three days later. Tumor growth was analyzed by caliper measurements every 2 days. (A,B) Comparison of tumor volume between each group (means ± SD, n = 5). Oxaliplatin treatment markedly reduced tumor volume in p53+/+HCT116 tumor xenografts. *p < 0.05, **p < 0.01. (C) Tumors were harvested on day 12. Tumors from each group were analyzed for the presence of CYP2S1 and p53 by western blotting. GAPDH was used as an internal control. (D) Total proteins were extracted and the protein levels of the CYP2S1 were analyzed in Western blot analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., ** p < 0.01. (E,F) Weights of xenograft tumours derived from p53+/+ or p53−/−HCT116 cells expressing CYP2S1 knockdown or control (shRNA or shNT, respectively) after oxaliplatin treatment. n = 5 mice per group.
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f5: Upregulation of CYP2S1 protein expression increases oxaliplatin sensitivity in a p53-dependent manner in vivo.Identical (2 × 106) amounts of each cells were injected subcutaneously into the flanks of nude mice. When tumors reached approximately 100 mm3, the mice received either PBS or oxaliplatin (10 mg/kg) intraperitoneally (day 0). A second dose of either PBS or oxaliplatin was administered three days later. Tumor growth was analyzed by caliper measurements every 2 days. (A,B) Comparison of tumor volume between each group (means ± SD, n = 5). Oxaliplatin treatment markedly reduced tumor volume in p53+/+HCT116 tumor xenografts. *p < 0.05, **p < 0.01. (C) Tumors were harvested on day 12. Tumors from each group were analyzed for the presence of CYP2S1 and p53 by western blotting. GAPDH was used as an internal control. (D) Total proteins were extracted and the protein levels of the CYP2S1 were analyzed in Western blot analysis. Results are of the average from three experiments. Data are represented as mean ± s.d., ** p < 0.01. (E,F) Weights of xenograft tumours derived from p53+/+ or p53−/−HCT116 cells expressing CYP2S1 knockdown or control (shRNA or shNT, respectively) after oxaliplatin treatment. n = 5 mice per group.
Mentions: As knockdown of CYP2S1 expression accelerates colorectal tumor growth in vivo25, we next investigated the effects of oxaliplatin on CYP2S1 protein expression in vivo. Isogenic p53+/+ and p53−/−HCT116 cell lines were injected into the flanks of immunocompromised nude mice, and (phosphate-buffered saline) PBS or oxaliplatin treatment was started when the tumors measured approximately 100 mm3. Tumor growth was monitored every 2 days until the mice were sacrificed on day 12, when tumors were excised and analyzed by western blotting. Oxaliplatin treatment had a striking antitumor effect on p53+/+HCT116 tumor xenografts (Fig. 5A), whereas the growth of p53−/−HCT116 xenografts was not significantly affected by oxaliplatin treatment (Fig. 5B). Oxaliplatin notably enhanced the induction of CYP2S1 expression in p53+/+HCT116 tumors but had virtually no effect on CYP2S1 expression in p53−/− HCT116 tumors (Fig. 5C,D).

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.


Related in: MedlinePlus