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. 2013 Jun;123:1-10.
doi: 10.1016/j.jinorgbio.2013.02.004. Epub 2013 Feb 21.

Organic Cadmium Complexes as Proteasome Inhibitors and Apoptosis Inducers in Human Breast Cancer Cells

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Organic Cadmium Complexes as Proteasome Inhibitors and Apoptosis Inducers in Human Breast Cancer Cells

Zhen Zhang et al. J Inorg Biochem. .
Free PMC article

Abstract

Although cadmium (Cd) is a widespread environmental contaminant and human carcinogen, our studies indicate an organic Cd complex to be a potent inhibitor of proteasomal chymotrypsin-like (CT-like) activity, further capable of inducing apoptosis in a cancer cell-specific manner. It has been reported that the ligands indole-3-butyric acid (L1) and indole-3-propionic acid (L2) have cancer-fighting effects when tested in a rat carcinoma model. In addition, 3, 5-diaminobenzoic acid o-vanillin Schiff bases (L3) have high antimicrobial activity and a large number of Schiff base complexes have been reported to have proteasome-inhibitory activity. We therefore hypothesized that synthetic forms of Cd in combination with L1, L2 and L3 may have proteasome-inhibitory and apoptosis-inducing activities, which would be cancer cell-specific. To test this hypothesis, we have synthesized three novel Cd-containing complexes: [Cd2(C12H12O2N)4(H2O)2]·2H2O (Cd1), [Cd2(C11H10O2N)4(H2O)2]·2H2O (Cd2) and [Cd(C7H4N2O2)(C8H6O2)2]·2H2O (Cd3), by using these three ligands. We sought out to characterize and assess the proteasome-inhibitory and anti-proliferative properties of these three Cd complexes in human breast cancer cells. Cd1, Cd2 and Cd3 were found to effectively inhibit the chymotrypsin-like activity of purified 20S proteasome with IC50 values of 2.6, 3.0 and 3.3 μΜ, respectively. Moreover, inhibition of cancer cell proliferation also correlated with this effect. As a result of proteasomal shutdown, the accumulation of ubiquitinated proteins and the proteasome target IκB-α protein as well as induction of apoptosis were observed. To account for the cancer specificity of this effect, immortalized, non-tumorigenic breast MCF10A cells were used under the same experimental conditions. Our results indicate that MCF10A cells are much less sensitive to the Cd1, Cd2 and Cd3 complexes when compared to MDA MB 231 breast cancer cells. Therefore, our study suggests that these Cd organic complexes are capable of inhibiting tumor cellular proteasome activity and consequently induce cancer cell-specific apoptotic death.

Figures

Fig. 1
Fig. 1
Chemical structures of M1, M2 and M3 (M=Cd(II), Cu(II), or Zn(II)).
Fig. 2
Fig. 2
Purified 20S proteasome inhibition by Cd1, Cd2 and Cd3, supported by nucleophilic susceptibity analysis. A: Purified human proteasome (35 ng) was incubated with DMSO or various concentrations of Cd1, Cd2 and Cd3 for 2 h, followed by proteasomal chymotrypsin-like activity assay. Points, mean+/−SE of three independent experiments; bars, SD. B, C, D: The nucleophilic susceptibility of Cd1, Cd2 and Cd3 was analyzed by using CAChe software. Molecular orbital energy analysis is shown by drawing an electron density isosurface and the highest susceptibility towards nuceophilic attack was signified by white center.
Fig. 3
Fig. 3
Comparison of cell proliferation and proteasome inhibition in MCF-7 and MDA-MB-231 cells treated with different compounds. MCF-7 and MDA-MB-231 cells were treated with each indicated compound at 40 μM for 24 h, followed by MTT assay (A), the chymotrysin-like activity assay (B), and Western blot analysis of polyubiquitinated proteins, PARP, IκB-α (C, D). Molecular weight of intact PARP is 116 kDa and the cleaved PARP fragment is 85 kDa. Molecular weight of IκB-α is 37 kDa. β-actin was used as a loading control. Columns, mean+/−SE of three independent experiments; bars, SD.
Fig. 4
Fig. 4
Concentration effects of Cd1, Cd2 and Cd3 on breast negative cancer MDA-MB-231 cells. MDA-MB-231 cells were treated with DMSO (as control) or different concentrations of Cd1, Cd2 and Cd3 for 24 h, followed by the chymotrysin-like activity assay (A), Western blot analysis of polyubiquitinated proteins, PARP and IκB-α (B), and cellular morphologic changes (C). Columns, mean+/−SE of three independent experiments; bars, SD.
Fig. 5
Fig. 5
Concentration effects of Cd1, Cd2 and Cd3 on breast cancer MCF-7 cells. MCF-7 cells were treated with DMSO (as control) or different concentrations of Cd1, Cd2 and Cd3 for 24 h, followed by the chymotrysin-like activity assay (A), Western blot analysis of polyubiquitinated proteins, PARP and IκB-α (B), and cellular morphologic changes (C). Columns, mean+/−SE of three independent experiments; bars, SD.
Fig. 6
Fig. 6
Kinetic effect of proteasome inhibition and apoptosis induction by Cd1, Cd2 and Cd3 in MDA-MB-231 cells. MDA-MB-231 cells were exposed to 20 μM of Cd1, Cd2 and Cd3 for the indicated times, followed by the proteasomal chymotrypsin-like activity assay (A), Western blot assay (ubiquitin, PARP, IκB-α and β-actin, B), and the apoptosis morphologic changes (C.) Columns, mean+/−SE of three independent experiments; bars, SD.
Fig. 7
Fig. 7
Differential effects of Cd complexes on cancer and non-tumorigenic breast cells. Breast cancer MDA-MB-231 cells and non-cancer MCF-10A cells treated with 20 μM of Cd1, Cd2, Cd3, CdCl2, DSF and DSF-Cd mixture, or DMSO for 24 h, followed by MTT assay (A) and morphological changes evaluation (B). Columns, mean+/−SE of three independent experiments; bars, SD.

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