Targeting Iron in Colon Cancer via Glycoconjugation of Thiosemicarbazone Prochelators

The implication of iron in the pathophysiology of colorectal cancer is documented at both the biochemical and epidemiological levels. Iron chelators are therefore useful molecular tools for the study and potential treatment of this type of cancer characterized by high incidence and mortality rates. We report a novel prochelation strategy that utilizes a disulfide redox switch to connect a thiosemicarbazone iron-binding unit with carbohydrate moieties targeting the increased expression of glucose transporters in colorectal cancer cells. We synthesized three glycoconjugates (GA2TC4, G6TC4, and M6TC4) with different connectivity and/or carbohydrate moieties, as well as an aglycone analog (ATC4). The sugar conjugates present increased solubility in neutral aqueous solutions, and the ester-linked conjugates M6TC4 and G6TC4 compete as effectively as d-glucose for transporter-mediated cellular uptake. The glycoconjugates show improved selectivity compared to the aglycone analog and are 6–11 times more toxic in Caco-2 colorectal adenocarcinoma cells than in normal CCD18-co colon fibroblasts

Prodigiosin Analogue Designed for Metal Coordination: Stable Zinc and Copper Pyrrolyldipyrrins

The pyrrolyldipyrrin motif is found in several naturally occurring prodigiosin pigments. The potential roles of the interactions of prodigiosins with transition metals and the properties of metal-bound pyrrolyldipyrrins, however, have been difficult to assess because of the very limited number of well-characterized stable complexes. Here, we show that the introduction of a <i>meso</i>-aryl substituent and an ethyl ester group during the sequential assembly of the three heterocycles affords a pyrrolyldipyrrin of enhanced coordinating abilities when compared to that of natural prodigiosins. UV–visible absorption studies indicate that this ligand promptly binds Zn­(II) ions with 2:1 ligand-to-metal stoichiometry and Cu­(II) ions with 1:1 stoichiometry. Notably, no addition of base is required for the formation of the resulting stable complexes. The crystal structures reveal that whereas the tetrahedral zinc center engages two nitrogen donors on each ligand, the pseudosquare planar copper complex features coordination of all three pyrrolic nitrogen atoms and employs the ester group as a neutral ligand. This first example of coordination of a redox-active transition metal within a fully conjugated pyrrolyldipyrrin framework was investigated spectroscopically by electron paramagnetic resonance to show that the 1:1 metal-to-ligand ratio found in the crystal structure is also maintained in solution

Prodigiosin Analogue Designed for Metal Coordination: Stable Zinc and Copper Pyrrolyldipyrrins

The pyrrolyldipyrrin motif is found in several naturally occurring prodigiosin pigments. The potential roles of the interactions of prodigiosins with transition metals and the properties of metal-bound pyrrolyldipyrrins, however, have been difficult to assess because of the very limited number of well-characterized stable complexes. Here, we show that the introduction of a <i>meso</i>-aryl substituent and an ethyl ester group during the sequential assembly of the three heterocycles affords a pyrrolyldipyrrin of enhanced coordinating abilities when compared to that of natural prodigiosins. UV–visible absorption studies indicate that this ligand promptly binds Zn­(II) ions with 2:1 ligand-to-metal stoichiometry and Cu­(II) ions with 1:1 stoichiometry. Notably, no addition of base is required for the formation of the resulting stable complexes. The crystal structures reveal that whereas the tetrahedral zinc center engages two nitrogen donors on each ligand, the pseudosquare planar copper complex features coordination of all three pyrrolic nitrogen atoms and employs the ester group as a neutral ligand. This first example of coordination of a redox-active transition metal within a fully conjugated pyrrolyldipyrrin framework was investigated spectroscopically by electron paramagnetic resonance to show that the 1:1 metal-to-ligand ratio found in the crystal structure is also maintained in solution

Polarization dictates the macrophage iron phenotype.

<p>(A) mRNA expression of iron regulated genes FPN, IRP2, TfR, HAMP, CP, and FTL was quantified in primary human macrophages using qRT-PCR. Results were normalized to the unstimulated control. Protein expression of FPN (B) and FTL (C) was determined in macrophages by FACS analysis. (D) Iron content in macrophage supernatants was quantified by AAS measurement. (E) MCF-7 breast cancer cells were stimulated with macrophage conditioned media (CM) and proliferation was analyzed by qRT-PCR of (E) PCNA, Ki-67, (F) Western analysis of PCNA, and (G) xCELLigence. Data are means ± S.D.M, n>6, *p<0.05, **p<0.01, ***p<0.001 vs. control/CM_control.</p

Prochelation approach and stability in cell culture medium.

<p>(A) Scheme of the redox directed chelation system: The disulfide-based prochelator (TC3-S)₂ undergoes intracellular reduction to produce the active chelator TC3-SH, which readily binds iron forming a stable complex. (B) Stability of prochelator (TC3-S)₂ at 12.0 μM in phenol red-free EMEM: relative concentrations over the course of 24 hours were determined by UV-Visible absorption spectroscopy (λ_max = 318 nm, 37°C). No measureable loss of the prochelator is observed over the course of 2 hours, and less than 10% loss is observed over 24 hours.</p

Chelator treatment induces a macrophage phenotype shift towards iron-sequestration.

<p>(A) mRNA expression of iron regulated genes FPN, IRP2, TfR, HAMP, CP, and FTL was quantified using qRT-PCR. Protein expression of FPN (B) and FTL (C) was determined by FACS analysis. (D) Iron content in macrophage supernatants was quantified by AAS measurement. Data are shown as means ± S.D.M, n>6, *p<0.05, **p<0.01, ***p<0.001 vs. DMSO.</p

Iron chelator functionality in primary human macrophages.

<p>(A) Time dependent activation of HIF-1α in primary human macrophages was determined by Western blot analysis. DMOG- as well as (TC3-SH)₂-treated cells were compared to DMSO-stimulated control macrophages. (B) Cell viability was measured by AnnexinV/PI staining using FACS measurement. The percentage of living, apoptotic, and necrotic cells is represented. Staurosporine (Sts) was used as a positive control. Data are shown as mean ± S.D.M, n>4, *p<0.05, **p<0.01, ***p<0.001 vs. DMSO.</p

Functional consequences of chelator-dependent reprogramming of the macrophage iron phenotype in breast cancer cells.

<p>As a functional readout, proliferation of macrophage conditioned media (CM)-stimulated MCF-7 cells was measured by qRT-PCR of (A) PCNA and Ki-67, (B) Western analysis of PCNA, as well as using (C) the xCELLigence real-time analysis system. (D) Migration and (E) adhesion to either collagen I or fibronectin matrix of MCF-7 tumor cells upon MΦ-conditioned media treatment. (F) Data are shown as means ± S.D.M, n>3, *p<0.05, **p<0.01, ***p<0.001 vs. CM_DMSO.</p