Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration‑resistant prostate cancer cells

Abstract

Cancer cells are characterized by increased glycolysis, known as the Warburg effect, which leads to increased production of cytotoxic methylglyoxal (MGO) and apoptotic cell death. Cancer cells often activate the protective nuclear factor erythroid 2‑related factor2 (Nrf2)/glyoxalase1 (Glo1) system to detoxify MGO. The effects of sodium butyrate (NaB), a product of gut microbiota, on Nrf2/Glos/MGO pathway and the underlying mechanisms in prostate cancer (PCa) cells were investigated in the present study. Treatment with NaB induced the cell death and reduced the proliferation of PCa cells (DU145 and LNCap). Moreover, the protein kinase RNA-like endoplasmic reticulum kinase/Nrf2/Glo1 pathway was greatly inhibited by NaB, thereby accumulating MGO-derived adduct hydroimidazolone (MG-H1). In response to a high amount of MGO, the expression of Nrf2 and Glo1 was attenuated, coinciding with an increased cellular death. NaB also markedly inhibited the Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (Stat3) pathway. Conversely, co‑treatment with Colivelin, a Stat3 activator, significantly reversed the effects of NaB on Glo1 expression, MG-H1 production, and the cell migration and viability. As expected, overexpression of Stat3 or Glo1 reduced NaB‑induced cell death. The activation of calcium/calmodulin dependent protein kinase II gamma and reactive oxygen species production also contributed to the anticancer effect of NaB. The present study, for the first time, demonstrated that NaB greatly increases MGO production through suppression of the JAK2/Stat3/Nrf2/Glo1 pathway in DU145 cells, a cell line mimicking castration‑resistant PCa (CRPC), suggesting that NaB may be a potential agent for PCa therapy.

Keywords: butyrate; glyoxalases; methylglyoxal; nuclear factor erythroid 2‑related factor2; prostate cancer; signal transducer and activator of transcription 3.

Figure 1.

Effects of NaB on the cell viability, proliferation, Nrf2/Glos pathway in PCa cells. (A) The DU145 cells or LNCaP cell were treated with NaB (2.5 or 5 mM) for 48 h, the cell viability and proliferation, (B) the expression of target genes and (C) the production of D-lactate and MG-H1 were determined. Results are expressed as the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the Control group. NaB, sodium butyrate; Nrf2, Nuclear factor erythroid 2-related factor 2; Glos, glyoxalases; PCa, prostate cancer; MG-H1, hydroimidazolone; HO-1, heme oxygenase-1.

Figure 2.

The effects of NaB on the activation of PERK and Nrf2. (A) DU145 cells were treated with NaB (2.5 or 5 mM) for 48 h, the PERK expression was determined. (B) The changes of Nrf2 expression and the cell viability were examined in cct020312 and NaB-treated cells. (C) After the cells treated with NaB for 24 h, the levels of cytoplasmic and nuclear Nrf2 and Bach1 were determined. (D) The distribution of Nrf2 in cytoplasm and nuclei in DU145 cells was evaluated by immunofluorescence assay. (E) The DU145 cells were incubated with AI-1 (50 µM) for 2 h followed by treatment with NaB for another 48 h, then the expression of Nrf2 and Glo1 was determined. Results are expressed as the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the Control group; ^#P<0.05 vs. respective NaB-treated alone cells. NaB, sodium butyrate; PERK, eukaryotic translation initiation factor 2 alpha kinase 3; Nrf2, nuclear factor erythroid 2-related factor 2; Bach1, BTB domain and CNC homolog 1; Glo1, glyoxalase1.

Figure 3.

The effects of NaB on Stat1, JAK2 and Stat3 expression. (A) DU145 cells were treated with NaB (2.5 or 5 mM) for 48 h and the expression of these genes was determined. (B) The DU145 cells were incubated with ruxolitinib (0.5 µM) for 2 h followed by treatment with NaB for 48 h, the expression of p-Stat3 and Glo1 was determined. (C) The relative ROS was measured in cells treated with NaB (2.5 or 5 mM) for 48 h. (D) After the cells were incubated with NAC (5 mM) or medium for 2 h followed by treatment with NaB for another 48 h, the protein level of p-Stat3 was determined in various groups. (E and F) Cells were pretreated with Colivelin (10 µM) or medium for 2 h followed by treatment with NaB for another 48 h. Then, the expression of the (E) target genes and (F) MG-H1 production were determined. Results are expressed as the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the Control group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. respective NaB-treated alone cells. NaB, sodium butyrate; Stat, signal transducer and activator of transcription; JAK2, Janus kinase 2; Glo1, glyoxalase1; ROS, reactive oxygen species; NAC, N-acetyl-cysteine; MG-H1, hydroimidazolone; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; p-, phosphorylated.

Figure 4.

The effects of MGO, Glo1, Stat3 and CaMKII phosphorylation on NaB-mediated target gene expression and cell viability. (A) After treatment with MGO (0.2-1.6 mM) for 48 h, the expression of Nrf2, Glo1 and HO-1, as well as the cell viability were determined. (B) The cell viability of the DU145 cells with overexpression of Glo1 or Stat3 was examined. (C) Cells were pretreated with KN-93 (10 µM) for 2 h followed by treatment with NaB for 48 h. Then, the expression of MAPKs and pro-apoptotic proteins were determined. Results are expressed as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the Vector or Control group; ^#P<0.05, ^###P<0.001 vs. respective NaB-treated alone cells. MGO, methylglyoxal; Glo1, glyoxalase1; Stat3, signal transducer and activator of transcription 3; CaMKII, calcium/calmodulin dependent protein kinase II gamma; NaB, sodium butyrate; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; PARP, Poly (ADP-Ribose) polymerase.

Figure 5.

Effects of NaB on cell apoptosis and migration. After treatment with NaB (2.5 or 5 mM) for 48 h or indicated time, (A) the apoptotic cells (%), (B) the cell migration and (C) the cell viability in the presence or absence of Colivelin (10 µM), KN-93 (10 µM) or NAC (5 mM) for 48 h were examined in various groups. (D) The production of MG-H1 was measured. The cells were treated with KN-93 (10 µM) for 2 h followed by treatment with NaB for 48 h. Results are expressed as the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs. the Control group; ^#P<0.05 and ^##P<0.01 vs. respective NaB-treated alone cells; NaB, sodium butyrate; NAC, N-acetyl-cysteine; MG-H1, hydroimidazolone; HO-1, heme oxygenase-1.

Figure 6.

Effects of KN-93 on cell proliferation the effects of NaB on normal RWPE-1 cells. (A) The relative cell proliferation of DU145 cells and the expression of c-Myc and cyclin D1 were evaluated in NaB (5 mM) alone, and co-treatment with KN-93, NAC, or Colivelin for indicated time. (B) The cell viability and the levels of Glo1 and Stat3 in RWPE-1 cells were examined after treatment with NaB for 48 h. Results are expressed as the mean ± SEM. **P<0.01 and ***P<0.001 vs. the Control group. (C) The proposed schematic diagram of NaB-mediated Nrf2/Glo1/MGO pathway and apoptosis in PCa cells. NaB inhibits the expression and activity of Nrf2 through PERK inactivation and accumulation of nuclear Bach1. Then, Glo1expression is attenuated via suppression of Janus kinase 2/Stat3 pathway, leading to accumulation of MGO and apoptotic cell death. The elevated MGO further downregulates Nrf2, Glo1 and HO-1. The CaMKII-mediated activation of MAPKs and Stat1 also contributes to NaB-induced cell death. NaB, sodium butyrate; NAC, N-acetyl-cysteine; Glo1, glyoxalase1; Stat3, signal transducer and activator of transcription 3; Nrf2, nuclear factor erythroid 2-related factor 2; MGO, cytotoxic methylglyoxal; PERK, eukaryotic translation initiation factor 2 alpha kinase 3; Bach1, BTB domain and CNC homolog 1; HO-1, heme oxygenase-1; CaMKII, calcium/calmodulin dependent protein kinase II gamma.