Fig 1.
Chemical structures and the polyamine pathway.
(A) Chemical structure of curcumin (diferuloylmethane). (B) Chemical structures of the natural polyamines spermine, spermidine, and putrescine, the polyamine analogue bis(ethyl)norspermine (BENSpm), and the ODC inhibitor difluoromethylornithine (DFMO). (C) The mammalian polyamine pathway: ornithine decarboxylase (ODC) converts ornithine into putrescine, which acquires sequential aminopropyl groups to form spermidine followed by spermine. The aminopropyl group is derived from decarboxylated S-adenosylmethionine (dcSAM), and its addition onto putrescine or spermidine is mediated by spermidine and spermine synthases (SRM and SMS, respectively). Two routes exist for spermine catabolism: (1) it can be oxidized by spermine oxidase (SMOX) to produce spermidine, which is accompanied by H2O2 and aldehyde generation, or (2) it can undergo acetylation by spermidine/spermine N1-acetyltransferase (SSAT). Acetylated spermine is either exported from the cell or oxidized back to spermidine by peroxisomal N1-acetylpolyamine oxidase (PAOX). Spermidine also undergoes this 2-step acetylation/oxidation back to putrescine via an N1-acetylspermidine intermediate.
Fig 2.
Effects of curcumin on polyamines and their biosynthetic enzymes in AGS cells.
AGS gastric carcinoma cells were treated for 48 h with curcumin followed by analyses of mRNA expression and enzyme activity levels of the polyamine biosynthetic enzymes ODC (A) and SAMDC (encoded by the AMD1 gene) (B). (C) Curcumin decreased intracellular polyamine pools and inhibited growth of AGS cells (SPM: spermine; SPD: spermidine; PUT: putrescine). (Inset) Linear regression of spermine concentration versus % of viable cells remaining relative to untreated cells in response to increasing doses of curcumin (p < 0.001). Graphs depict the means of data collected from at least 2 separate biological experiments, each measured in triplicate (duplicate for HPLC), with error bars indicating standard error of the mean. *p < 0.05 relative to untreated.
Fig 3.
Curcumin induces spermine catabolism through spermine oxidation in AGS cells.
AGS cells were treated for 48 hours and analyzed for SMOX (A) and SSAT (D) mRNA expression via qRT-PCR; (B-C) SMOX and SSAT protein levels by Western blot; and (E) SSAT enzyme activity. Graphs depict the mean values of at least 2 individual experiments, each measured in triplicate. Error bars = SEM; *p < 0.05. A representative western blot is presented in (C).
Fig 4.
Effects of curcumin on polyamine metabolism in HCT116 colon cancer cells.
RNA was collected from HCT116 cells treated with curcumin for 24 hours and used for qRT-PCR of genes encoding (A) the biosynthetic enzymes ODC and SAMDC (AMD1), or (C) the catabolic enzymes SMOX and SSAT (n = 3 biological experiments, each measured in triplicate). Total cell lysates were also collected and used for ODC enzyme activity assays (B; n = 2 independent experiments, each measured in triplicate) and Western blots of the catabolic enzymes (D). In (E), cells were treated for 48 hours with curcumin for either cellular proliferation assays or analysis of intracellular polyamine concentrations by HPLC (n ≥ 2, each measured in duplicate). All columns represent the means with error bars indicating SEM. *p < 0.05 relative to untreated.
Fig 5.
The effects of SMOX induction by curcumin on DNA damage and growth inhibition.
(A) AGS cells were treated for 48 h with curcumin in the presence or absence of 250 μM MDL72527, a polyamine oxidase inhibitor. (B) SMOX knockout or control AGS cells were treated for 48 h with increasing concentrations of curcumin. Lysates were analyzed for changes in γH2AX by Western blot. (C) MTS assays were conducted with the same conditions as those in (A) and (B) to determine the effect of SMOX on curcumin-induced growth inhibition. (D) Intracellular spermine (SPM) and spermidine (SPD) levels of SMOX KO cells lines following curcumin exposure for 48 h. Data points indicate the means; error bars represent SEM; n = 3. CT1 and CT2 = CRISPR control AGS cell lines.
Fig 6.
AGS (A) or HCT116 (B) cells were treated with curcumin for 48 hours in the presence or absence of spermidine or spermine and the bovine serum amine oxidase inhibitor aminoguanidine. MTS assays revealed that exogenous polyamines failed to protect cells from the growth inhibitory effects of curcumin. Data points indicate the means of at least 2 individual experiments, each measured in triplicate, with error bars representing SEM. (C) HPLC analysis of cotreated AGS cells confirmed uptake of spermine and spermidine into the cells, indicating that curcumin did not interfere with polyamine transport. Columns depict the means; error bars indicate SEM; n = 2, each measured in duplicate.
Fig 7.
Curcumin cooperates with DFMO to inhibit cell growth.
MTS cell proliferation assays revealed enhanced growth inhibition when combining curcumin with the ODC inhibitor DFMO in AGS (A) and HCT116 (B) cells. (C) In HCT116 cells, curcumin combined with DFMO for 24 hours enhanced ODC enzyme inhibition beyond that of single agents. Untreated and curcumin-treated ODC values (~7500 and 1600 pmol/hr/mg protein, respectively) are presented in Fig 4B. *p < 0.05 relative to DFMO alone; n = 2 individual experiments, each performed in triplicate; error bars = SEM. (D) Changes in individual and total polyamine pools following combination treatment of HCT116 cells for 24 h with 1 mM DFMO and 4 μg/mL curcumin. *p < 0.05 relative to SPD concentration following DFMO or curcumin alone. n = 2 individual experiments, each measured in duplicate; error bars = SEM. In (A), *p < 0.05 for combinations with 4 μg/mL curcumin and 1 or 5 mM DFMO versus the single agents. In (B), *p < 0.005 for the combination of 6 μg/mL curcumin and 1 mM DFMO, versus single agents. Data points indicate the means of at least 3 independent experiments; error bars = SEM.
Fig 8.
Proposed mechanisms of the combinatorial effects of curcumin with DFMO or BENSpm.
Treatment with curcumin alone down regulates the biosynthetic enzymes ODC and SamDC, while up regulating SMOX. The result is decreased putrescine and the conversion of spermine back to spermidine. SSAT activity is lowered, likely due to the decrease in spermine. (A) Adding DFMO further inhibits ODC, reducing the spermidine generated through biosynthesis. Compensatory uptake of polyamines from the extracellular environment would likely be increased by both DFMO and curcumin. (B) Uptake of BENSpm through the polyamine transporter is stimulated by the curcumin-mediated decrease in biosynthesis. BENSpm induces catabolism through SSAT, thereby increasing the depletion of spermine and catabolizing the spermidine accumulated by the induction of SMOX by curcumin. As no accumulation of putrescine is evident, the acetylated polyamines are presumably exported from the cell.
Fig 9.
Curcumin cooperates with BENSpm to inhibit AGS cell growth.
(A) MTS cell proliferation assays demonstrated enhanced growth inhibition over 72 h when combining curcumin with the polyamine analogue BENSpm in AGS cells. (B) HPLC analyses revealed increased accumulation of BENSpm in the presence of curcumin, resulting in significant decreases in spermidine and spermine pools. (C) SSAT activity is increased with curcumin/BENSpm cotreatment. (D) Representative Western blot indicating SMOX and SSAT protein levels following combination treatment. (E and F) Infrared imaging-based quantification of SSAT and SMOX Western blots, respectively, relative to actin. In A-F, data points indicate the means of at least 2 independent experiments, measured ≥ 2 times; error bars = SEM. *p < 0.05 relative to single agent treatments.