Silencing of PYGB suppresses growth and promotes the apoptosis of prostate cancer cells via the NF‑κB/Nrf2 signaling pathway

Abstract

Brain‑type glycogen phosphorylase (PYGB) is an enzyme that metabolizes glycogen, whose function is to provide energy for an organism in an emergency state. The present study purposed to investigate the role and mechanism of PYGB silencing on the growth and apoptosis of prostate cancer cells. A cell counting kit‑8 assay and flow cytometry were performed to determine the cell viability, apoptosis and reactive oxygen species (ROS) content, respectively. Colorimetry was performed to analyze the activity of caspase‑3. Western blotting and reverse transcription‑quantitative polymerase chain reaction were used to evaluate the associated mRNA and protein expression levels. The results revealed that PYGB was upregulated in prostate cancer tissues and was associated with disease progression. In addition, PYGB silencing suppressed the cell viability of PC3 cells. PYGB silencing promoted apoptosis of PC3 cells via the regulation of the expression levels of cleaved‑poly (adenosine diphosphate‑ribose) polymerase, cleaved‑caspase‑3, B‑cell lymphoma‑2 (Bcl‑2) and Bcl‑2‑associated X protein. PYGB silencing increased the ROS content in PC3 cells, and affected nuclear factor (NF)‑κB/nuclear factor‑erythroid 2‑related factor 2 (Nrf2) signaling pathways in PC3 cells. In conclusion, PYGB silencing suppressed the growth and promoted the apoptosis of prostate cancer cells by affecting the NF‑κB/Nrf2 signaling pathway. The present study provided evidence that may lead to the development of a potential therapeutic strategy for prostate cancer.

Figure 1.

PYGB is upregulated in prostate cancer tissues. (A) Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression levels of PYGB in prostate cancer tissues and matched adjacent normal prostate tissues from 50 prostate cancer patients. (B) Western blot assay was performed to measure the expression levels of PYGB in prostate cancer tissues and matched adjacent normal prostate tissues from prostate cancer patients. **P<0.01 and ***P<0.001 vs. normal tissue. PYGB, brain-type glycogen phosphorylase.

Figure 2.

PYGB is associated with the disease progression of prostate cancer and PYGB silencing suppresses the cell viability of PC3 cells. (A) The overall survival rates of prostate cancer patients based on the expression levels of PYGB measured by Kaplan-Meier survival analysis, and the P-values were calculated by log-rank test. (B) RT-qPCR was performed to determine the expression levels of PYGB in several prostate cancer cell lines, including LNCap, PC3 and DU145, together with the normal prostate cell line PrEC. (C) RT-qPCR and (D) western blot analysis was performed to measure the expression levels of PYGB in PC3 cells transfected with the siRNA negative control (mock group) and PYGB siRNA (si-PYGB group); the control group was untreated. (E) Cell counting kit-8 assay was performed to evaluate the cell viability of PC3 cells. *P<0.05, **P<0.01 vs. mock group. No significant differences were detected between the control and mock groups. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; PYGB, brain-type glycogen phosphorylase; si-/siRNA, small interfering RNA.

Figure 3.

PYGB silencing promotes the apoptosis of PC3 cells. PC3 cells were transfected with siRNA negative control (mock group) and PYGB siRNA (si-PYGB group). The cells in control group received no treatment. (A) Flow cytometric analysis was performed to determine the levels of cell apoptosis in PC3 cells. (B) Colorimetry was performed to measure the activity of caspase-3 in PC3 cells. (C) Western blotting was performed to measure the expression levels of PARP, cleaved-PARP and cleaved-caspase-3 in PC3 cells transfected with the siRNA negative control (mock group) and PYGB siRNA (si-PYGB group); the control group was untreated. **P<0.01 vs. mock group. No significant differences were detected between the control and mock groups. PYGB, brain-type glycogen phosphorylase; PARP, poly (adenosine diphosphate-ribose) polymerase; siRNA, small interfering RNA; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 4.

PYGB silencing modulates the expression levels of Bax and Bcl-2 in PC3 cells. PC3 cells were transfected with the siRNA negative control (mock group) and PYGB siRNA (si-PYGB group). The cells in control group received no treatment. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blotting were carried out to detect the mRNA and protein expression levels of Bax and Bcl-2, respectively, in PC3 cells transfected with the siRNA negative control (mock group) and PYGB siRNA (si-PYGB group); the control group was untreated. **P<0.01 vs. mock group. No significant differences were detected between the control and mock groups. PYGB, brain-type glycogen phosphorylase; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; si-/siRNA, small interfering RNA.

Figure 5.

PYGB silencing enhances the ROS content in PC3 cells transfected with the siRNA negative control (mock group) and PYGB siRNA (si-PYGB group); the control group was untreated. Flow cytometric analysis was carried out to evaluate the ROS content in PC3 cells. ROS, reactive oxygen species; PYGB, brain-type glycogen phosphorylase; si-/siRNA, small interfering RNA.

Figure 6.

PYGB silencing affects the NF-κB/Nrf2 signaling pathway in PC3 cells. (A) Reverse transcription-quantitative polymerase chain reaction was performed to measure the expression levels of NF-κB and Nrf2 in PC3 cells. (B) Western blotting was performed to measure the expression levels of NF-κB and Nrf2 in PC3 cells. **P<0.01, ***P<0.001 vs. mock group. No significant differences were detected between the control and mock groups. PYGB, brain-type glycogen phosphorylase; NF-κB, nuclear factor-κB; Nrf2, nuclear factor-erythroid 2-related factor 2; si-/siRNA, small interfering RNA.