Protein kinase C-α (PKCα) modulates cell apoptosis by stimulating nuclear translocation of NF-kappa-B p65 in urothelial cell carcinoma of the bladder

Abstract

Background: The protein kinase C (PKC) family comprises central regulators of multiple signal transduction processes and is involved in the progression of many cancers. Nuclear factor Kappa-B (NF-κB) is constitutively expressed in cancer tissues and stimulates the transcription of various tumor-related genes. The present study aims to investigate the clinical significance of PKCα and NF-κB p65 in bladder cancer tissues and the mechanism underlying PKCα induction of bladder cancer cell apoptotic resistance through stimulation of p65 nuclear translocation.

Methods: Expression of PKCα and NF-κB subunit p65 was detected in seven bladder cancer cell lines by western blot and in 30 bladder cancer tissue specimens by immunostaining. Immunofluorescence was performed to evaluate p65 nuclear translocation induced by Phorbol 12-myristate 13-acetate (PMA). PKCα/β selective inhibitor Gö6976, PKC pan-inhibitor sotrastaurin, and the PKC siRNA were employed to conduct PKC inhibition/knockdown in bladder cancer cells. Luciferase reporter assays were performed to measure the activity of NF-κB. Flow cytometry and TUNEL analysis were used to assess cell apoptosis.

Results: Expression of PKCα and NF-κB was found to positively correlate with tumor progression in 30 tumor tissue specimens. Furthermore, a Pearson's correlation coefficient analysis revealed a positive correlation between PKCα and NF-κB expression. Among the PKC inhibitors, the PKCα/β selective inhibitor Gö6976 yielded the most significant block of PKCα and NF-κB activation by PMA. Knockdown of NF-κB p65 remarkably induced cell apoptosis, but PMA restored p65 expression and significantly suppressed cell apoptosis that was otherwise induced by the p65 knockdown alone.

Conclusion: Our study showed that PKCα modulated cell resistance to apoptosis by stimulating NF-κB activation and thus promoted the tumorigenesis of bladder cancer.

Keywords: Apoptosis; NF-κB; PKCα; Urothelial cell cancer.

Figures

Fig. 1

Expression profile of the PKC isotypes and NF-κB p65 subunit in bladder cancer cell lines and tissue specimens. a The expression profile of nine PKC isotypes in four bladder urothelial cancer cell lines was measured by real-time PCR. The expression levels were normalized to β-actin. The statistical analysis results are as follows. RT4 cell line (left upper panel): PKCδ, PKCι, PKCβ, PKCη vs PKCα: p < 0.01**; PKCζ vs PKCα: not significant. 5637 cell line (right upper panel): PKCδ, PKCι, PKCζ vs PKCα: p < 0.01**. T24 cell line (left lower panel): PKCδ vs PKCα: p < 0.01**; PKCα vs PKCι, PKCζ: p < 0.05*. TCC-SUP cell line (right lower panel): PKCα vs PKCι: not significant; PKCα vs PKCδ: p < 0.01**. b Protein expression of PKCα in seven bladder cancer cell lines was detected by western blot. The gels were run under the same experimental conditions. The band intensities were calculated using the ImageJ 1.46r software. β-Tubulin was used as an internal control for total protein measurements, and Histone was used as a nucleoprotein reference. The ratio of the target gene to β-Tubulin/Histone was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student’s T-test. c PKCα and NF-κB p65 expression were associated with tumor progression in 30 clinical bladder cancer specimens. Two representative cases are shown. The gene expression level was evaluated in three random visual fields. Original magnifications: 200× and 400×. The gene expression of PKCα and NF-κB p65 between tumor tissue samples staged as pT1 and pT4 was compared d and a Pearson’s correlation coefficient analysis was performed to analyze the expression correlation between the two genes

Fig. 2

PMA significantly induces overexpression of PKCα, p-PKCα and NF-κB p65 nuclear translocation in bladder cancer cell lines. a 5637 and T24 cells were treated with PMA (10 ng/ml) for 0, 15, 30, 60, and 240 min, and the total, nuclear and cytoplasmic proteins were extracted at the indicated time point; p-PKCα and nuclear/cytoplasmic p65 were measured by western blot. b Normalized protein expression levels were calculated and analyzed. The gels were run under the same experimental conditions. The band intensities were calculated using the ImageJ 1.46r software. β-Tubulin was used as an internal control for the total protein measurement, and Histone was used as a nucleoprotein reference. The ratio of the target gene to β-Tubulin/Histone was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student’s T-test. c Cells were treated with DMSO or PMA (10 ng/ml) for 1 h, and p65 localization was detected by immunofluorescence. The cells with nuclear translocation of p65 are indicated with red arrows for the 5637 and T24 cell lines. For the BIU-87 cell line, nuclear translocation of p65 is evident in almost all cells within the visual field after the PMA treatment, and p65 expression can be observed in both the cytosol and nucleus. Original magnification: 400×. Comparisons between the control and PMA groups were made based on the statistical analysis of the cells with nuclear localization of p65 counted in three random fields

Fig. 3

PKCα is the key player in PMA-induced NF-κB activation. Three pairs of small interfering RNA against PKCα were designed, and the knockdown efficiencies were analyzed by real-time PCR (b) and western blot (a). c Cells were treated/transfected with DMSO/negative control (NC), PMA/NC or PMA/siPKCα for 12 h, and protein expression of PKCα and nuclear/cytoplasmic p65 were detected by western blot. The experiment was repeated three times with each pair of siRNAs against PKCα, and similar results were obtained. A dual luciferasy reporter assay was performed in parallel to confirm the result (g). d Protein expression levels were normalized to Tubulin/Histone, and the band intensities were calculated and analyzed. (e) Cells were pretreated with DMSO, Gö6976 (100 nM) or Sotrastaurin (100 nM) for 1 h and then challenged with PMA (10 ng/ml) for 12 h. Cells without any treatment were used as the blank control. Protein expression of PKCα, p-PKCα and nuclear/cytoplasmic p65 were detected by western blot, normalized and analyzed against the internal control (f). Also a dual-luciferasy reporter assay was performed in parallel to confirm the reslut (h). The gels were run under the same experimental conditions. The band intensities were calculated using the ImageJ 1.46r software. β-Tubulin was used as an internal control for the total protein measurements, and Histone was used as a nucleoprotein reference. The ratio of the target gene to β-Tubulin/Histone was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student’s t-test

Fig. 4

PKCα suppressed cell apoptosis by activating NF-κB signaling. Transfection efficiency of siRNA against p65 was measured by western blot a and real-time PCR b. c, d PMA restored the NF-κB expression that was effectively knocked down by the siRelA transfection. e Cells were transfected with the negative control (NC), p65 siRNA, or p65 siRNA plus the PMA (10 ng/ml) treatment for 24 h and then collected, stained with Annexin-V/PI and subjected to FACS for apoptotic analysis. The percentage of apoptotic cells (early and late apoptosis) were analyzed. f TUNEL staining was also performed to determine the cell apoptosis level. TUNEL-positive cells were counted and analyzed. Original magnification: 200×. *P < 0.05 and **P < 0.01, as determined by Student’s T-test. The experiment was repeated three times with each pair of siRNAs against p65, and similar results were obtained