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P70S6 kinase phosphorylation: a new site to assess pharmacodynamy of sirolimus.

Wang JY, Fan H - Chin. Med. J. (2015)

Bottom Line: The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001).The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01).There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.

ABSTRACT

Background: The phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.

Methods: Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.

Results: Intra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

Conclusions: The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

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Assessing intra-assay variability by Western blotting (a and b).
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Figure 1: Assessing intra-assay variability by Western blotting (a and b).

Mentions: To compare and confirm the data generated in the phospho-flow cytometry assay, the phospho-flow technique was compared with Western blotting analysis of phosphorylation of p70S6K in healthy subject (n = 1) and liver transplant patients receiving sirolimus, tacrolimus or cyclosporine (n = 1 each) [Figure 1a]. The bar graphs showed the respective analysis of triplicate Western blotting densitometric measurements expressed in pixel values (HC: 0.79 ± 0.10; cyclosporine: 0.63 ± 0.07; tacrolimus: 0.55 ± 0.06; sirolimus: 0.18 ± 0.03). The mean CV value for intra-assay variability was 8.2% for healthy individuals, 18% for patients receiving tacrolimus, 15% for cyclosporine and 16% for sirolimus, respectively. A representative Western blotting for phosphorylation of p70S6K was assessed in PBMCs of a healthy subject and a patient receiving cyclosporine, tacrolimus or sirolimus, respectively [Figure 1b].


P70S6 kinase phosphorylation: a new site to assess pharmacodynamy of sirolimus.

Wang JY, Fan H - Chin. Med. J. (2015)

Assessing intra-assay variability by Western blotting (a and b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834780&req=5

Figure 1: Assessing intra-assay variability by Western blotting (a and b).
Mentions: To compare and confirm the data generated in the phospho-flow cytometry assay, the phospho-flow technique was compared with Western blotting analysis of phosphorylation of p70S6K in healthy subject (n = 1) and liver transplant patients receiving sirolimus, tacrolimus or cyclosporine (n = 1 each) [Figure 1a]. The bar graphs showed the respective analysis of triplicate Western blotting densitometric measurements expressed in pixel values (HC: 0.79 ± 0.10; cyclosporine: 0.63 ± 0.07; tacrolimus: 0.55 ± 0.06; sirolimus: 0.18 ± 0.03). The mean CV value for intra-assay variability was 8.2% for healthy individuals, 18% for patients receiving tacrolimus, 15% for cyclosporine and 16% for sirolimus, respectively. A representative Western blotting for phosphorylation of p70S6K was assessed in PBMCs of a healthy subject and a patient receiving cyclosporine, tacrolimus or sirolimus, respectively [Figure 1b].

Bottom Line: The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001).The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01).There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.

ABSTRACT

Background: The phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.

Methods: Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.

Results: Intra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

Conclusions: The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

Show MeSH
Related in: MedlinePlus