Table 1.
Oligonucleotides used in this study.
Figure 1.
CSTP1 mRNA is down-regulated in bladder cancer tissues and cell lines.
(A) Real-time PCR analysis of CSTP1 mRNA levels in six kinds of human cancer tissues. CSTP1 mRNA was selectively down-regulated in bladder cancer tissues compared to adjacent non-cancerous tissues. (B) Northern blot analysis of CSTP1 mRNA in bladder cancer cell lines. CSTP1 mRNA was expressed at a moderate level in SV-HUC1 cells, but could be hardly detected in RT4, EJ and T24 bladder cancer cell lines, GAPDH was used as internal control. (C) CSTP1 mRNA expression analysis on Oncomine. Decreased CSTP1 mRNA expression in bladder cancers in two sets of dataset, with p-values of 0.005(up) and 2.62E-4(down) respectively.(1, bladder mucosa; 2, infiltrating bladder urothelial carcinoma; 3, superficial bladder cancer).
Figure 2.
(A) the nucleotide sequence of CSTP1 open reading frame and the deduced amino acid sequence. The nucleotide sequence is shown in the 5' to 3' direction. The predicted amino acid sequence is shown below the nucleotide sequence. The amino acid residues corresponding to the phosphorase 2A catalytic unit domain (PP2Ac) are in red letters. (B) Bioinformatics analysis indicated that CSTP1 protein contained a conserved PP2Ac domain between 50 to 250 amino acids. (C) Phylogenetic analysis showed that CSTP1 protein is conserved in chimpanzee, dog, cow, mouse, rat, chicken, zebrafish, and P.falciparum. (D) Western blot analysis of CSTP1 expression in HeLa cells. HeLa cells were transfected with pCDNA3.1 Myc/His C-CSTP1 plasmid, 48 h later, the level of CSTP1-Myc fusion protein was tested by anti-Myc antibody. Actin was used as internal control. (E) CSTP1 displayed a PP2B-like protein phosphatase activity in vitro. 293T cells were transfected with pcDNA3.1Myc/His C-CSTP1, 48 h later, the CSTP1-His fusion protein was purified for phosphatase assay. The release of Pi was determined in the presence of 1 mM EGTA, 50 mM MgCl2, 5 mM NiCl2 and 250 µg/ml calmodulin. 200 nM trifluoroperazine or 0.5 µmol of Okadaic Acid was also added to abrogate the phosphatase activity of PP2B or PP2A.
Figure 3.
Cellular localization of CSTP1 protein.
Hela cells were transfected with pEGFPN1 and pEGFPN1-CSTP1 plasmid respectively, 48 hours later, cells were fixed with 4% paraformaldehyde and visualized by fluorescence confocal microscopy. 4, 6-Diamidino-2-phenylindole dihydrochloride staining was used to indicate the cell nucleus. Results were representative of three independent experiments.
Figure 4.
Over-expression of CSTP1 suppresses bladder cancer cells proliferation.
(A) EJ cells were transduced with lentiviruses overexpressing CSTP1 (Lv-CSTP1), the PP2Ac domian deleted CSTP1 (Lv-CSTP1 ΔPP2Ac), or lenti-vector control (Lv-ctr). Cell proliferation was detected by MTT assay. Data at each time point represents the mean ± SD of 8 samples. Results were representative of three independent experiments. (B) The transduced EJ cells were cultured for 14 days, and colonies were stained using crystal violet and counted within the field of a ×40 objective lens. Results were representative of three independent experiments.(C) The xenograft tumor growth in nude mice were remarkably suppressed following CSTP1 overexpression. Data at each time point represents the mean ± SD, n = 6. (D) Chamber assays were performed with transduced EJ cells. The results showed that CSTP1 has no effect on cell invasion . Data shown were mean number of invading cells (×100 field) ± SD from three independent experiments. **, p<0.01.
Figure 5.
CSTP1 inhibits cell cycle progression.
(A) EJ cells overexpressing CSTP1 (Lv-CSTP1), CSTP1 ΔPP2Ac (Lv-CSTP1 ΔPP2Ac) and control cells were treated with two rounds of 2 mM thymidine. Cell cycles were analyzed by FACS after releasing from G0/G1 phase at indicated time point. Results were representative of three independent experiments. (B) Immunoblotting of CSTP1 in extracts of SV-HUC1cells 48 h after transfection with a control siRNA (Mock) or a siRNA for CSTP1 (CSTP1 siRNA). (C) Cell cycle analysis of SV-HUC1 cells after transfection with control siRNA or siRNA for CSTP1. *,p<0.05. Results were representative of three independent experiments.
Figure 6.
CSTP1 promotes cell apoptosis.
EJ cells stably over-expressing CSTP1 or CSTP1 ΔPP2Ac and control cells were cultured in complete medium with or without gemcitabine(10−8 mol/L) and cisplatin(2 µg/mL), 48 hours later, cells were double stained with annexin V-FITC and PI, cell apoptosis was analyzed by FACS. Results were representative of three independent experiments. **, p<0.01.
Figure 7.
CSTP1 interacts with and dephosphorylates Akt at Ser473 site.
(A) EJ cells overexpressing CSTP1 and control cells were transfected with a series of cignal reported plasmid, 48 h later, luciferase activity was measured using the luciferase assay system with a luminometer. luciferase-based reporter signal is normalized to the expression of a cotransfected Renilla luciferase control plasmid. (B) Western blot analysis of the phosphorylation levels of FOXO3A, Akt,GSK3α, GSK3β, p70S6K and TSC2 in EJ cells overexpressing CSTP1. Unphosphorylated FOXO3A, Akt, GSK3α, GSK3β, p70S6K and TSC2 proteins were detected as internal controls. (C) Western blot analysis of the phosphorylation levels of FOXO3A and Akt after knocking down of CSTP1 in SV-HUC1 cells. (D) CSTP1 interacts with Akt in 293T cells. 293T cells were cotransfected with pcDNA3.1-CSTP1 and Flag-Akt expressing plasmids, interaction between CSTP1 and Akt was analyzed by co-ip experiment with anti-Flag (up panel) or anti-CSTP1 antibody(low panel). (E) Dephosphorylation of Akt by purified CSTP1 in vitro. Pure His-Akt was incubated with GST-tagged CSTP1 or GST-tagged CSTP1ΔPP2Ac in the phosphatase buffer. For a negative control, CSTP1 protein was omitted from the reaction mixture(Ctr). (F) EJ cells were overexpressed CSTP1 (Lv-CSTP1) or CSTP1(Lv-CSTP1) plus phosphor-mimetic S473D construct of Akt (ca-Akt) by lentivirus, cell cycle was analyzed by FACS. All experiments were performed at least three times.
Figure 8.
Decreased CSTP1 protein expression is correlated with tumor recurrence in patients with non-muscular invasive bladder cancer.
(A) Representative immunohistochemistry staining of CSTP1 protein in formalin-fixed paraffin-embedded tumor or normal urothelia tissues. (B) Recurrence-free survival was analyzed by Kaplan-Meier method. The expression level of CSTP1 in bladder cancer tissues positively correlated with the recurrence-free survival in patients suffered from non-invasive bladder cancers. p = 0.039, Log-rank test.