Regulation of prostate cancer cell invasion by modulation of extra- and intracellular redox balance - PubMed (original) (raw)

Regulation of prostate cancer cell invasion by modulation of extra- and intracellular redox balance

Luksana Chaiswing et al. Free Radic Biol Med. 2012.

Abstract

Recent metabolic profiles of human prostate cancer tissues showed a significant increase in cysteine (Cys) and a significant decrease in reduced glutathione (GSH) during cancer progression from low- to high-grade Gleason scores. Cys is primarily localized extracellularly, whereas GSH is present mostly inside the cell. We hypothesized that extra- or intracellular redox state alterations differentially regulate cell invasion in PC3 prostate carcinoma cells versus PrEC normal prostate epithelial cells. Cells were exposed to media with calculated Cys/CySS redox potentials (E(h)CySS) ranging from -60 to -180mV. After 3h exposure to a reducing extracellular redox state (E(h)CySS=-180mV), matrix metalloprotease (MMP), gelatinase, and NADPH oxidase activities increased, correlating with increases in cell invasion, cell migration, and extracellular hydrogen peroxide levels in PC3 cells but not PrECs. Knockdown of NADPH oxidase or MMP with silencing RNAs during cultivation with E(h)CySS=-180mV medium significantly decreased PC3 cell invasion. Modulation of extra- and intracellular redox states by exposure of PC3 cells to Cys/CySS-free medium (approx E(h)CySS=-87mV) containing 500μMN-acetylcysteine resulted in a more reducing intracellular redox state and a significant decrease in cell invasive ability. The decrease in PC3 cell invasion induced by these conditions correlated with a decrease in MMP activity. Our studies demonstrated that an extracellular redox state that was more reducing than a physiologic microenvironment redox state increased PC3 cancer cell invasive ability, whereas an intracellular redox environmental that was more reducing than an intracellular physiologic redox state inhibited PC3 cell invasive ability.

Published by Elsevier Inc.

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Figures

Figure 1. Comparison of PrEC vs. PC3 cells: Trx1 in culture media, cell invasive ability, and cell growth in response to Cys/CySS

(A) Trx1 protein expression in media cultured with PrEC or PC3 cells for 24 hr; control is RPMI 1640 medium without cultured cells. (B) Comparison of cell invasive ability through Matrigel matrix membranes at 24 hr between PrEC and PC3 cells. (C–D) Effect of 450 μM Cys/6 μM CySS supplemented medium on growth of PrEC cells or PC3 cells. *p-value ≤ 0.05, #p-value = 0.09

Figure 2. Increase in invasive ability and MMP enzymatic activity of PC3 cells but not PrEC cells by incubation with a reducing extracellular redox state

Cells were cultured with Cys/CySS supplemented media for 3 hr. Cell invasive ability through Matrigel matrix membrane and MMP activity were analyzed. (A) PrEC cell invasive ability. (B) PC3 cell invasive ability. (C) Total MMP activity in the media. (D) Zymography assay of MMP9. Purified pro-MMP9 proteins were directly incubated with EhCySS −180 mV medium and zymography enzyme activity was analyzed at different time points. *p-value ≤ 0.05.

Figure 3. Induction of PC3 cell migration ability and gelatinase activity in media with a reducing extracellular redox state

PC3 cells were cultured with either EhCySS −88 or −180 mV media. After culture for 3 hr, in vitro wounding assays were performed at 6 hr, 18 hr, and 24 hr and fluorescent gelatin degradation assays were performed at 18 hr after wounding. (A) Phase-contrast photographs of migration of PC3 cells at 24 hr. (B) Quantitative analysis of migration distance. (C) Photograph of FITC-conjugated gelatin areas at 18 hr that were degraded by gelatinase enzymes (arrows) of PC3 cells. (D) Quantitative analysis of gelatin degradation areas. *p-value ≤ 0.05.

Figure 4. Induction of extracellular H2O2 levels and NADPH oxidase activity after culture in media with a reducing extracellular redox state

Cells were incubated with Cys/CySS supplemented media for 3 hr. Media or cell homogenates were collected for analysis. (A) H2O2 levels in media of PrEC cells. (B) H2O2 levels in media of PC3 cells. (C) NADPH oxidase activity of PC3 cells. *p-value ≤ 0.05.

Figure 5. Effects of decreased NADPH oxidase or MMP9 protein levels on PC3 cell invasion after culture in media with a reducing extracellular redox state

PC3 cells were transfected with NOX1, MMP9, or non-specific siRNAs for 48 hr and continuously cultured for an additional 72 hr. siRNA transfected-PC3 cells were cultured with EhCySS −180 mV medium for 3 hr. Cells were collected for western blot (A and B) and in vitro invasion analysis (C). Mock= PC3 cells that were cultured with EhCySS −180 mV medium for 3 hr. *p-value ≤ 0.05.

Figure 6. Regulation of PC3 cell invasion and MMP activity by modulation of extra- and intracellular redox states

PC3 cells were cultured with regular RPMI 1640 medium ± 500 μM NAC or Cys/CySS-free RPMI 1640 medium ± 500 μM NAC for 3 hr. Cells or conditioned media were collected for redox western blot, cell invasion, and MMP activity assays. (A) Redox western blot assay. Oxy2= fully oxidized (two disulfides) Trx1, Oxy1= mixture of reduced and oxidized Trx1, R= fully reduced Trx1. PC3 cells were treated with DTT or H2O2 for 30 min and were used to determine fully reduced Trx1 or fully oxidized Trx1. (B) Cell invasion assay. (C) MMP9 zymography activity gel. (D) Relative quantification of MMP9 zymography activity gels. Lane 1 = regular RPMI 1640 medium, lane 2 = regular RPMI 1640 medium + NAC, lane 3 = Cys/CySS-free RPMI 1640 medium, lane 4 = Cys/CySS-free RPMI 1640 medium + NAC. *p-value ≤ 0.05 when compared with regular RPMI 1640 medium. **p-value = 0.06 when compared with Cys/CySS-free RPMI 1640 medium. Data represent average of at least three individual experiments

Figure 7. Proposed mechanisms by which extra- or intracellular redox state regulates prostate cancer cell invasion

(A) Extracellular Cys/CySS shift medium redox state towards reducing, resulting in an alteration or protection of the bond between the active Zn2+ site and a Cys residue, and leading to activation of MMP. Alternatively, a reducing extracellular redox state may affect TIMP with resultant inhibition of TIMP protein function. A shift of extracellular redox state towards reducing may activate NADPH oxidase on the cell membrane, resulting in an increase in extracellular H2O2 production. H2O2 may act as a catalytic co-factor and increase MMP activity by altering the Zn2+-Cys bond complex, altering TIMP activity, or activating migration related proteins. (B) NAC and/or extracellular oxidizing redox state shift intracellular redox state towards reducing, which results in a decrease in MMP activity. Intracellular reducing redox state could possibly inhibit MMP secretion or modulate other redox signaling-related proteins involved in invasion processes.

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Regulation of prostate cancer cell invasion by modulation of extra- and intracellular redox balance - PubMed (original) (raw)