Significance of p27 as potential biomarker for intracellular oxidative status

Abstract

Our previous proteomic study demonstrated that oxidative stress and antioxidant delphinidin regulated the cellular level of p27(kip1) (referred to as p27) as well as some heat shock proteins in human colon cancer HT 29 cells. Current study was conducted to validate and confirm the regulation of these proteins using both in vitro and in vivo systems. The level of p27 was decreased by hydrogen peroxide in a dose-dependent manner in human colon carcinoma HCT 116 (p53-positive) cells while it was increased upon exposure to hydrogen peroxide in HT 29 (p53-negative) cells. However, high concentration of hydrogen peroxide (100 µM) downregulated p27 in both cell lines, but delphindin, one of antioxidative anthocyanins, enhanced the level of p27 suppressed by 100 µM hydrogen peroxide. ICR mice were injected with varying concentrations of hydrogen peroxide, delphinidin and both. Western blot analysis for the mouse large intestinal tissue showed that the expression of p27 was upregulated by 25 mg/kg BW hydrogen peroxide. To investigate the association of p27 regulation with hypoxia-inducible factor 1-beta (HIF-1β), the level of p27 was analyzed in wild-type mouse hepatoma hepa1c1c7 and Aryl Hydrocarbon Nuclear Translocator (arnt, HIF-1β)-defective mutant BPRc1 cells in the absence and presence of hydrogen peroxide and delphinidin. While the level of p27 was responsive to hydrogen peroxide and delphinidin, it remained unchanged in BPRc1, suggesting that the regulation of p27 requires functional HIF-1β. We also found that hydrogen peroxide and delphinidin affected PI3K/Akt/mTOR signaling pathway which is one of upstream regulators of HIFs. In conclusion, hydrogen peroxide and antioxidant delphinidin seem to regulate intracellular level of p27 through regulating HIF-1 level which is, in turn, governed by its upstream regulators comprising of PI3K/Akt/mTOR signaling pathway. The results should also encourage further study for the potential of p27 as a biomarker for intracellular oxidative or antioxidant status.

Keywords: antioxidant; biomarker; delphinidin; oxidative status; p27kip1.

Fig. 1

Change in the expression of cell cycle inhibitor, p27^kip1 in HCT 116 cells treated with hydrogen peroxide and delphinidin for 12 h. Human colon carcinoma HCT 116 cells (wild type p53) were exposed to various doses of H₂O₂ and delphinidin for 12 h, followed by determination of p27^kip1 level by Western blot. Data are representative of three independent experiments as mean ± SD. Values on bars not sharing a common superscript significantly differ from each other (P < 0.05).

Fig. 2

Change in the expression of cell cycle inhibitor, p27^kip1 in HT 29 cells treated with hydrogen peroxide and delphinidin for 12 h. Human colon carcinoma HT 29 cells (nonfunctional p53) were exposed to various doses of H₂O₂ and delphinidin for 12 h, followed by determination of p27^kip1 level by Western blot. Data are representative of three independent experiments as mean ± SD. Values on bars not sharing a common superscript significantly differ from each other (P < 0.05).

Fig. 3

Modulation of p27^kip1 protein expression in large intestinal tissue of mice treated with delphinidin and hydrogen peroxide. ICR mice were injected with varying doses of H₂O₂ and delphinidin three times for a week, and epithelia of large intestine were collected and analyzed for p27^kip1 level. Data are representative of three independent experiments as mean ± SD. Values on bars not sharing a common superscript significantly differ from each other (P < 0.05).

Fig. 4

Effect of hydrogen peroxide and delphinidin on the level of p27^kip1 in wild type hepa1c1c7 (A) and HIF-1β-deficient BPRc1 (B) cells. Mouse hepatoma hepa1c1c7 (HIF-1β-positive) and its mutant BPRc1 (HIF-1β-negative) were treated with various doses of delphinidin in the presence of 100 µM H₂O₂, followed by the determination of p27^kip1 level. Data are representative of three independent experiments as mean ± SD. Values on bars not sharing a common superscript significantly differ from each other (P < 0.05).

Fig. 5

The levels of PI3K (A), Akt (B), mTOR (C) and their phosphorylated forms in HCT 116 cells treated with delphinidin and hydrogen peroxide. Human colon carcinoma HCT 116 cells were exposed to various doses of delphinidin in the presence of 100 µM H₂O₂, followed by the determination of PI3K, Akt, mTOR and their phosphorylation.