Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

Abstract

Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1-5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47(phox)). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity.

Figures

Figure 1. NOX expression is associated with disease progression in TRAMP mice.

(A,B) NOX1 and p67phox expression was analyzed by IHC in TRAMP prostate tissue with different stages of the disease as well as in non-transgenic mice prostate tissue as negative control. Representative photographs are presented at 100x; inset represent further magnification of a part of the photograph. Immunoreactivity (represented by brown staining) of NOX1 and p67phox was scored as described in methods. Abbreviations: NC: Negative control; LG: Low-grade; HG: High-grade; PIN: Prostate intra-epithelial neoplasia; WD: Well differentiated; MD: Moderately differentiated; PD: Poorly differentiated. *p ≤ 0.001.

Figure 2. Comparison of various parts of Graviola for cytotoxicity against PCa cells.

(A,B) LNCaP and PC3 cells were seeded at the density of 5 × 104 cells/well in 6-well plates. After 24 h of seeding, cells were treated with 25 and 50 μg/ml concentrations of Graviola extracts (D1: seeds extract; D2: pulp extract; D3: exocarp extract; D4: leaves extract; D5: twigs extract) for 48 h. At the end of 48 h, cells were harvested and counted as mentioned in ‘Materials and Methods’, and total cell number and percentage of dead cells are presented as mean ± SEM. *p ≤ 0.001; #p ≤ 0.01.

Figure 3. GPE inhibits NOX activity in human PCa cells.

(A–C) Human PCa 22Rv1, LNCaP and PC3 cells were seeded at the density of 4 × 105 cells/60 mm culture dishes. After 24 h of seeding, cells were treated with 1, 2.5 and 5 μg/ml concentrations of GPE under hypoxic condition (1% O2) for 24 h. In each case, cells cultured under normoxic condition (21% O2) served as relevant control. At the end of 24 h, cells were harvested and NOX activity was measured as mentioned in ‘Materials and Methods’ and represented as rlu/mg protein. (D) Direct inhibition of NOX activity by GPE was assessed by pre-incubating GPE (1.0–5.0 μg/ml) with cellular homogenates and NOX activity was measured as mentioned in ‘Materials and Methods’ and represented as rlu/mg protein. Each value represents mean ± SEM of three samples for each treatment. $p ≤ 0.05; #p ≤ 0.01; *p ≤ 0.001.

Figure 4. GPE inhibits the expression of members of NOX system and HIF-1α in human PCa 22Rv1 cells.

(A) 22Rv1 cells were treated with GPE (1–10 μg/ml) under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. Thereafter, cells were collected and analyzed for mRNA expression of NOX1, p47phox and HIF-1α by RT-PCR using gene specific primers as mentioned in ‘Materials and Methods’. β-actin served as an endogenous internal standard. (B) 22Rv1 cells were treated with GPE (1–5 μg/ml) under hypoxic (1% O2) condition for 24 h. Thereafter, cells were collected and cytosolic and membrane fractions were prepared and analyzed for levels of NOX1, NOX2, p67phox and p47phox by immunoblotting. β-actin and E-cadherin were used to check the purity of cytoplasmic and membrane fractions as well as for loading control. Densitometry data presented below the bands are ‘fold change’ as compared with control (DMSO treated) after normalization with respective loading control. (C) 22Rv1 cells were treated with DPI (20 μM) and GPE (10 and 20 μg/ml) under hypoxic (1% O2) condition for 6 h. Cells cultured under normoxic condition (21% O2) served as relevant control. Thereafter, cells were collected and total cell lysates were prepared and analyzed for HIF-1α, HIF-1β, HIF-2α and tubulin. Densitometry data presented below the bands are ‘fold change’ as compared with hypoxia control (DMSO treated) after normalization with respective loading control. (D) 22Rv1 cells were treated with GPE (10 and 20 μg/ml) under hypoxic (1% O2) condition for 6 h. Cells cultured under normoxic condition (21% O2) served as relevant control. Thereafter, cells were collected and nuclear/cytoplasmic fractions were prepared and analyzed for HIF-1α expression. Membranes were also probed for TBP and tubulin as loading control for nuclear and membrane fractions, respectively. Densitometry data presented below the bands are ‘fold change’ as compared with hypoxia control (DMSO treated) after normalization with respective loading control. Abbreviations: N: Normoxic; H: Hypoxic; GPE: Graviola pulp extract; DPI: Diphenyleneiodonium.

Figure 5. GPE inhibits the clonogenicity of human PCa cells under normoxic and hypoxic conditions.

(A,B) Human PCa 22Rv1 and PC3 cells (~1 × 103 cells per well) were cultured in 6-well plates and treated with GPE (1–5 μg/ml) under normoxic (21% O2) or hypoxic (1% O2) conditions for 48 hrs. Thereafter, cells were maintained under normoxic conditions and clones were counted after 7 days. Each value represents mean ± SEM of three samples for each treatment. #p ≤ 0.01; *p ≤ 0.001.

Figure 6. GPE reduces cell viability of human PCa cells under normoxic and hypoxic conditions.

(A,B) 22Rv1, LNCaP, PC3 and PWR-1E cells were seeded at the density of 5 × 104 cells/well in six well plates. After 24 h of seeding, cells were treated with 2.5–20 μg/ml concentrations of GPE under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 and 48 h. At the end of each time point, cells were harvested and counted as mentioned in ‘Materials and Methods’, and total cell number and percentage of dead cells are shown. Each value represents mean ± SEM of three samples for each treatment. $p ≤ 0.05; #p ≤ 0.01; *p ≤ 0.001.

Figure 7. Chemical characterization of GPE.

Sample was analyzed as detailed in the methods. (A) The total ion chromatogram of the GPE. (B) The extracted ion chromatograms, detailed to the right, overlaid with each other. The color coding subdivides the acetogenins based upon their molecular ions.

Figure 8. Example characterization of acetogenins.

The MS/MS spectrum of the peak at 4.58 min (coded blue in Fig. 7). These data illustrate the common fragmentation patterns observed with acetogenins. In particular, note the fragments adjacent to hydroxy moieties, thereby facilitating the elucidation of the positioning of the THF ring and each hydroxy group. Based on these data and the associated literature, this compound is tentatively identified as annonacin.