Pirarubicin reduces USP22 expression by inhibiting CREB-1 phosphorylation in HeLa cells

Abstract

The expression of ubiquitin specific peptidase 22 (USP22) is upregulated in several types of cancer, and has been implicated in tumorigenesis. Pirarubicin (THP), an anthracycline antineoplastic drug, can induce apoptosis of several types of cancer cells. However, the molecular mechanisms underlying the action of THP remain to be elucidated. In the current study, treatment with THP induced HeLa cell apoptosis and decreased USP22 expression in a dose- and time-dependent manner. THP reduced the USP22 promoter-regulated luciferase activity, regardless of the mutation of transcriptional activator MYB or E3 ubiquitin-protein ligase SP1 binding sequences; however, this effect was abrogated by the mutation of cyclic AMP-responsive element-binding protein (CREB) binding sequence in HeLa cells. Furthermore, the inhibition on the USP22 promoter activity by THP was not affected by overexpression of CREB-1 in HeLa cells. Additionally, treatment with THP significantly decreased the phosphorylation of CREB-1 at ser133 in HeLa cells. Quantitative chromatin immunoprecipitation assay revealed that THP significantly inhibited the binding of CREB-1 to the USP22 promoter in HeLa cells. The present study demonstrated that THP decreased USP22 expression and promoted HeLa cell apoptosis partially by inhibiting the phosphorylation of CREB-1. The current results may provide novel insights into the molecular mechanisms underlying the pharmacological effect of THP on cancer cell apoptosis.

Keywords: HeLa; cyclic AMP-responsive element-binding protein-1; phosphorylation; pirarubicin; ubiquitin specific peptidase 22.

Figure 1.

THP induces apoptosis in HeLa cells. HeLa cells were treated with the indicated doses of THP for 24 h, and stained with FITC-Annexin V and PI. The percentages of FITC⁺ early stage and FITC⁺PI⁺ late stage apoptotic cells were determined by flow cytometry. (A) Representative flow cytometry dot plots. (B) Quantitative data presented as the mean ± standard deviation of each group of cells from three separate experiments. *P<0.05 as indicated. THP, pirarubicin; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 2.

THP decreases USP22 expression in HeLa cells. HeLa cells were treated with different doses of THP for 12 h or with 500 ng/ml THP for 0–24 h. The relative levels of USP22 mRNA transcripts were determined by reverse transcription-quantitative polymerase chain reaction or western blotting. (A) THP decreases USP22 expression in a dose-dependent manner. (B) THP decreases USP22 expression compared with the vehicle group in what appears to be a time-dependent manner in. *P<0.05 vs. the vehicle group. (C) Western blot analysis of USP22 expression. Representative images or data presented as the mean ± standard deviation of each group from three separate experiments are included. *P<0.05 vs. 0 ng/ml THP. USP22, ubiquitin specific peptidase 22; THP, pirarubicin.

Figure 3.

THP inhibits the USP22 promoter activity partially dependent on the binding sequence of CREB in HeLa cells. (A) HeLa cells were transfected with the indicated plasmids for the USP22 promoter-controlled luciferase expression and treated with, or without, 500 ng/ml of THP. The luciferase activity in individual groups of cells was determined. (B) HeLa cells were transfected with P-210/MYB mut, P-210/CREB mut and P-210/SP1 mut, and treated with, or without, 500 ng/ml of THP. The luciferase activity in individual groups of cells was determined. *P<0.05 vs. the control group. (C) HeLa cells were transfected with CREB-1 overexpression plasmids and pCMV plasmids, which were used as the blank control. (D) CREB-1 overexpression did not rescue the THP-decreased USP22 promoter activity. HeLa and CREB-1 overexpressing HeLa cells were treated with, or without, THP and the luciferase activity of individual groups of cells was determined. Representative images or data presented as the mean ± standard deviation of each group from three separate experiments are included. USP22, ubiquitin specific peptidase 22; CREB, cyclic AMP-responsive element-binding protein-1; THP, pirarubicin; MYB, transcriptional activator MYB; SP1, E3 ubiquitin-protein ligase SP1; mut, mutant.

Figure 4.

THP decreases CREB phosphorylation in HeLa cells. HeLa cells were treated with the indicated doses of THP for 24 h and the relative levels of CREB expression and phosphorylation at ser133 were determined by western blotting. (A) Western blot analysis. (B) Quantitative analysis of the ratios of p-CREB to t-CREB in individual groups of cells. Representative images or data presented as the mean ± standard deviation of each group from three separate experiments are included. *P<0.05 vs. 0 ng/ml THP. CREB, cyclic AMP-responsive element-binding protein-1; THP, pirarubicin; p, phosphorylated; t, total.

Figure 5.

THP inhibits the binding of CREB to the USP22 promoter in HeLa cells. HeLa cells were treated with vehicle or 500 ng/ml THP for 3 h, lysed, sonicated and cross-linked. The DNA/protein complexes were immunoprecipitated with control IgG or anti-CREB, followed by polymerase chain reaction amplification of the targeted DNA fragment using specific primers for the USP22 promoter region. Whole cell lysates served as the input control. (A) A representative image. (B) Quantitative analysis. Representative images or data presented as the mean % ± standard deviation of each group from three separate experiments are included. *P<0.05 as indicated. USP22, ubiquitin specific peptidase 22; CREB, cyclic AMP-responsive element-binding protein-1; THP, pirarubicin; Ig, immunoglobulin.