Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Abstract

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the "reprogramming" of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.

Keywords: Acireductone dioxygenase 1 (ADI1); Iron; Ornithine decarboxylase; Polyamines; S-adenosylmethionine (AdoMet); Spermidine/spermine-N(1)-acetyltransferase 1 (SAT1).

Figure 1.. Schematic illustrating the polyamine metabolic pathway in human cells.

This pathway involves three major arms: biosynthesis (blue), which can further sub-divided into: (1) the methionine salvage pathway; (2) the ODC-Antizyme axis; and (3) the “biosynthetic core”; as well as (4) polyamine catabolism (red) and (5) polyamine transport (green).

Figure 2.. The methionine salvage pathway is altered by cellular iron-depletion.

(A) SK-Mel-28 or (B) MCF-7 cells were incubated for 24 h or 48 h/37°C with either control medium, or this medium containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL). Total protein was then extracted and immunoblotting for TfR1 (positive control for iron-depletion), ADI1, MAT2α, AMD1 and β-actin was performed. Results are typical blots from 3 experiments. The quantitation represents mean ± SD (3 independent experiments). Relative to the respective control: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.. The ODC-Antizyme axis is regulated by cellular iron-depletion.

(A) SK-Mel-28 or (B) MCF-7 cells were incubated for 24 h or 48 h/37°C with either control medium, or this medium containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL). Total protein was then extracted and immunoblotting for TfR1 (positive control for iron-depletion), ODC, Antizyme, AZIN1 and β-actin was performed. Results are typical blots from 3 experiments. The quantitation represents mean ± SD (3 independent experiments). Relative to the respective control: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.. The “biosynthetic core”, composed of SRM and SMS, is regulated by cellular iron-depletion.

(A) SK-Mel-28 or (B) MCF-7 cells were incubated for 24 h or 48 h/37°C with either control medium, or this medium containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL). Total protein was then extracted and immunoblotting for TfR1 (positive control for iron-depletion), SRM, SMS and β-actin was performed. Results are typical blots from 3 experiments. The quantitation represents mean ± SD (3 independent experiments). Relative to the respective control: *p < 0.05, ***p < 0.001.

Figure 5.. Polyamine catabolism is regulated by cellular iron-depletion.

(A) SK-Mel-28 or (B) MCF-7 cells were incubated for 24 h or 48 h/37°C with either control medium, or this medium 45 containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL). Total protein was then extracted and immunoblotting for TfR1 (positive control for iron-depletion), SAT1, PAOX, SMOX and β-actin was performed. Results are typical blots from 3 experiments. The quantitation represents mean ± SD (3 independent experiments). Relative to the respective control: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.. Polyamine transport proteins are regulated by cellular iron-depletion.

(A) SK-Mel-28 or (B) MCF-7 cells were incubated for 24 h or 48 h at 37°C with either control medium, or this medium containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL). Total protein was then extracted and immunoblotting for TfR1 (positive control for iron-depletion), SLC22A16, SLC3A2 and β-actin was performed. Results are typical blots from 3 experiments. The quantitation represents mean ± SD (3 independent experiments). Relative to the respective control: *p < 0.05, ***p < 0.001.

Figure 7.. Intracellular polyamine levels and polyamine transport are regulated by cellular iron-depletion.

The effect of regulating cellular iron levels on the polyamine content was assessed in: (A) SK-Mel-28 and (B) MCF-7 cells. Cells were incubated for 24 h/37°C with either control medium, or this medium containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL). Cells were removed from the substratum, washed and then polyamines were extracted, derivatized and quantitated by HPLC-MS/MS, as described in the Experimental Procedures. Total protein was determined for equivalently treated samples and polyamine levels were normalized to protein content. The quantitation represents mean ± SD (3 independent experiments). Additionally, the effect of cellular iron-depletion on polyamine uptake was assessed in: (C) SK-Mel-28 or (D) MCF-7 cells. Cells were incubated for 24 h/37°C with either control medium, or this medium containing 311 (25 μM), or DFO (100 μM). Cells were then washed and re-incubated with fresh serum-free medium containing 1 μM ³H-spermidine for 3 h/37°C, washed again, and internalization determined using Pronase (see Experimental Procedures). The quantitation represents mean ± SD (3 independent experiments). In the same experiments in (C) and (D), total cell extracts were prepared and immunoblotting performed for SLC22A16 and β-actin. Results are typical of 3 experiments performed. Relative to the respective control: *p < 0.05, **p < 0.01.

Figure 8.. Expression of the key polyamine pathway regulators, c-Myc and p53, is modulated by cellular iron-depletion.

(A) SK-Mel-28 or (A) MCF-7 cells were incubated for 24 h or 48 h/37°C with either control medium, or this medium containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL). Total protein was then extracted and immunoblotting for TfR1 (positive control for iron-depletion), c-Myc, p53 and β-actin was performed. Results are typical blots from 3 experiments. The quantitation represents mean ± SD (3 independent experiments). Relative to the respective control: *p < 0.05, ***p < 0.001.

Figure 9.. Silencing of c-Myc in (A) SK-Mel-28 cells and (B) MCF-7 cells impacts on iron-dependent regulation of the polyamine pathway.

Cells were transiently transfected for 48 h/37°C with a single siRNA specific for c-Myc (si-Myc) or non-targeting pool (NTP) control siRNA (si-NC), all at a final concentrations of either 5 nM (SK-Mel-28) or 2.5 nM (MCF-7). The cells were then incubated with control medium, or this medium containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL) for 48 h/37°C. Total protein was then extracted and expression of TfR1 (positive control for iron-depletion), c-Myc, ADI, ODC, Antizyme, AZIN1 and β-actin were then assessed by immunoblot analysis. Results are typical blots from 3 experiments. The quantitation represents mean ± SD (3 independent experiments). Relative to the respective control: *p < 0.05, **p < 0.01, ***p < 0.001; Relative to the respective siNC condition: # p < 0.05; ## p < 0.01; ### p < 0.001.

Figure 10.. Silencing of p53 in (A) SK-Mel-28 cells and (B) MCF-7 cells impacts on iron-dependent regulation of the polyamine pathway.

Cells were transiently transfected for 48 h/37°C with esiRNA pools specific for p53, or non-targeting pool (NTP) control siRNA (siNC*), all at a final concentration of 20 nM. The cells were then incubated with control medium, or this medium containing 311 (25 μM), DFO (100 μM), or FAC (100 μg/mL) for 48 h/37°C. Total protein was then extracted and expression of TfR1, p53, ADI, ODC, Antizyme, AZIN1, SAT1 and β-actin were then assessed by immunoblot analysis. Results are typical blots from 3 experiments. The quantitation represents mean ± SD (3 independent experiments). Relative to the respective control: *p < 0.05, **p < 0.01, ***p < 0.001; Relative to the respective siNC* condition: # p < 0.05; ## p < 0.01; ### p < 0.001.

Figure 11.. Supplementation with exogenous putrescine or spermidine rescues the proliferation of iron-depleted cells.

(A) SK-Mel-28 and (B) MCF-7 cells were incubated for 72 h/37 °C with 311 (0.02–25 μM) or DFO (0.1–100 μM) in the absence or presence of putrescine (100 μM or 1 mM), spermidine (100 μM or 1 mM), or spermine (100 μM or 1 mM). Cellular proliferation was measured using the MTT proliferation assay and IC₅₀ values were then determined. Results are typical of three independent experiments with data analysis representing mean ± SD (n = 3). Relative to the respective 311- or DFO-treated control: *p < 0.05, **p < 0.01, ***p < 0.001. Relative to the DFO-treated control: ###p < 0.001.

Figure 12.. Schematic summarizing the “reprogramming” of polyamine metabolism by iron-depletion in: (A) SK-Mel-28 human melanoma and (B) MCF-7 human breast cancer cells.

Iron depletion leads to: (1) alterations in the expression of c-Myc and p53, where iron-depletion decreases c-Myc expression in both cell-types, while decreasing expression of mutant p53 (SK-Mel-28) and increasing wild-type p53 (MCF-7); (2) changes in the expression of polyamine pathway proteins involved in catabolism/efflux and biosynthesis/uptake (increased protein expression is marked by red arrows, while decreased protein expression is marked by blue arrows); (3) conserved alterations in the expression of polyamine pathway proteins in SK-Mel-28 and MCF-7 cells, some of which are downstream of the changes in c-Myc and p53; (4) decreases in key polyamine metabolites, AdoMet and spermine, in SK-Mel-28 cells; and decreases in AdoMet, spermidine and spermine in MCF-7 cells; and (5) decreases in proliferation, which can be partially rescued by supplementation with putrescine and spermidine. Collectively, these data demonstrate that cellular iron levels are a potent regulator of polyamine metabolism at multiple levels. These results have crucial ramifications for understanding the integrated role of iron and polyamine metabolism in the proliferation of cancer cells. that cellular iron levels are a potent regulator of polyamine metabolism at multiple levels. These results have crucial ramifications for understanding the integrated role of iron and polyamine metabolism in the proliferation of cancer cells.