Soy Isoflavones Induce Cell Death by Copper-Mediated Mechanism: Understanding Its Anticancer Properties

Abstract

Cancer incidence varies around the globe, implying a relationship between food and cancer risk. Plant polyphenols are a class of secondary metabolites that have recently attracted attention as possible anticancer agents. The subclass of polyphenols, known as isoflavones, includes genistein and daidzein, which are present in soybeans and are regarded as potent chemopreventive agents. According to epidemiological studies, those who eat soy have a lower risk of developing certain cancers. Several mechanisms for the anticancer effects of isoflavones have been proposed, but none are conclusive. We show that isoflavones suppress prostate cancer cell growth by mobilizing endogenous copper. The copper-specific chelator neocuproine decreases the apoptotic potential of isoflavones, whereas the iron and zinc chelators desferroxamine mesylate and histidine do not, confirming the role of copper. Reactive oxygen species (ROS) scavengers reduce isoflavone-induced apoptosis in these cells, implying that ROS are cell death effectors. Our research also clearly shows that isoflavones interfere with the expression of the two copper transporter genes, CTR1 and ATP7A, in cancerous cells. Copper levels are widely known to be significantly raised in all malignancies, and we confirm that isoflavones can target endogenous copper, causing prooxidant signaling and, eventually, cell death. These results highlight the importance of copper dynamics within cancer cells and provide new insight into the potential of isoflavones as cancer-fighting nutraceuticals.

Keywords: anticancer; cell death; copper; daidzein; genistein; isoflavones.

Figure 1

Chemical structure of genistein and daidzein.

Figure 2

The effect of genistein and daidzein on the proliferation of prostate cancer cell lines determined by the MTT assay. The LNCaP and DU145 cancer cell lines were grown with genistein and daidzein at the given concentrations for 96 h. The effect on cell proliferation was performed by MTT assay as described in Section 4. Values reported are mean ± S.E of triplicate experiments. * p < 0.01 compared to the untreated control (0 µM of the isoflavone).

Figure 3

Analysis of genistein and daidzein on apoptosis in prostate cancer cell lines. After incubating prostate cancer cell lines for 96 h with increasing doses of both the isoflavones, apoptosis was detected using the Cell Death Detection ELISA Kit (Roche, Palo Alto, CA, USA), as shown in the figure and discussed in Section 4. Values reported are mean ± S.E of three independent experiments. * p value < 0.01 when compared to control.

Figure 4

The effect of different metal chelators on the antiproliferative effects of genistein and daidzein in prostate cancer cell lines. As indicated in the figure, LNCaP and DU145 cancer cells were treated with 50 µM genistein/and daidzein either alone or in the presence of copper chelator neocuproine (Neo), iron chelator desferrioxamine mesylate (DM) or zinc chelator histidine (His). Metal chelators were utilized at a concentration of 50 µM. The MTT assay was done 96 h following treatment, as stated in Section 4. Values reported are mean ± S.E of three independent experiments. * p value < 0.01 when compared to control.

Figure 5

The effect of metal chelators on isoflavone-induced apoptosis in prostate cancer cell lines. LNCaP and DU145 cancer cells were treated with 50 µM genistein/and daidzein alone or in the presence of the copper chelator neocuproine (Neo), iron chelator desferrioxamine mesylate (DM), or zinc chelator histidine (His). The chelators used had a concentration of 50 µM. Apoptosis was detected using the Cell Death Detection ELISA Kit (Roche, Palo Alto, CA, USA). Values reported are mean ± S.E of three independent experiments. * p value < 0.01 when compared to control.

Figure 6

The effect of isoflavones on prostate cancer cell migration in the presence of the copper chelator neocuproine. The assay was carried out as described in Section 4. The cells were cultured with and without genistein/and daidzein (50 µM) and with or without neocuprione (50 µM). Values reported are mean ± S.E. of three independent experiments. * p value < 0.01 when compared to control.

Figure 7

The effect of isoflavones on cell proliferation inhibition in normal prostate epithelial cells (HPrEC) and HPrEC cells cultured in copper-supplemented media (HPrEC-Cu). HPrEC and HPrEC-Cu (normal cells cultured in a medium containing 25 µM CuCl₂) were treated for 96 h with a 50 µM concentration of genistein/and daidzein. The cell proliferation was then assessed using the MTT assay, as indicated in Section 4. Values reported are mean ± S.E. of three independent experiments. * p value < 0.01 when compared to respective control.

Figure 8

The effect of isoflavones on the increased mRNA levels of copper transporters CTR1 and ATP7A in HPrEC-Cu cells relative to parental HPrEC cells. As mentioned in Section 4, CTR1 and ATP7A mRNA expression was measured using real-time PCR. Only HPrEC-Cu cells (regular HPrEC cells cultured in a medium containing 25 µM CuCl₂) with elevated mRNA expression of copper transporters were treated with genistein/and daidzein (50 µM). Values reported are mean ± S.E. of three independent experiments. * p value < 0.01 when compared to control.

Figure 9

The effect of isoflavones on cell proliferation of HPrEC-Cu cells (normal HPrEC cells cultured in a medium containing 25 µM CuCl₂) was compromised after the knock-down of CTR1 and ATP7A. HPrEC-Cu cells were initially treated for 48 h with targeted siRNA against CTR1 (siCTR1) and ATP7A (siATP7A), followed by 96 h with the indicated doses of genistein/and daidzein. Values reported are mean ± S.E. of three independent experiments. * p value < 0.01 when compared to respective control.

Figure 10

A proposed schematic diagram showing the interaction of isoflavones and copper in the down-regulation of CTR1 and ATP7A. In addition, the involvement of redox cycling generates reactive oxygen species, leading to DNA damage and, ultimately, apoptosis.