The dietary phytochemical indole-3-carbinol is a natural elastase enzymatic inhibitor that disrupts cyclin E protein processing

Abstract

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, induces a G 1 cell-cycle arrest of human breast cancer cells, although the direct cellular targets that mediate this process are unknown. Treatment of highly invasive MDA-MB-231 breast cancer cells with I3C shifted the stable accumulation of cyclin E protein from the hyperactive lower-molecular-mass 35-kDa form that is associated with cancer cell proliferation and poor clinical outcomes to the 50-kDa cyclin E form that typically is expressed in normal mammary tissue. An in vitro cyclin E processing assay, in combination with zymography, demonstrated that I3C, but not its natural dimer, 3,3 -diindolylmethane, disrupts proteolytic processing of the 50-kDa cyclin E into the lower-molecular-mass forms by direct inhibition of human neutrophil elastase enzymatic activity. Analysis of elastase enzyme kinetics using either cyclin E or N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanalide as substrates demonstrated that I3C acts as a noncompetitive inhibitor of elastase activity with an inhibitory constant of Ϸ12 M. Finally, siRNA ablation of neutrophil elastase protein production in MDA-MB-231 cells mimicked the I3C-disrupted processing of the 50-kDa cyclin E protein and the indole-induced cell-cycle arrest. Taken together, our results demonstrate that elastase is the first identified specific target protein for I3C and that the direct I3C inhibition of elastase enzymatic activity implicates the potential use of this indole, or related compounds, in targeted therapies of human breast cancers where high elastase levels are correlated with poor prognosis.A critical challenge in controlling breast cancer is the identification of therapeutic agents that can effectively control the growth of both estrogen-responsive and nonresponsive breast cancer cells with reduced side effects, especially during prolonged treatments. Epidemiological studies show that frequent consumption of certain vegetables is associated with a lower incidence of cancers at various sites (1). For example, the consumption of Brassica (cruciferous) vegetables, such as cabbage, broccoli, and Brussels sprouts, is directly associated with decreased risk of reproductive tissue cancers in humans (2, 3) and reduced tumor incidence in experimental animals (4). These studies implicate the existence of specific biologically active phytochemicals that represent a largely untapped source of potent chemotherapeutic agents. One such promising molecule is indole-3-carbinol (I3C), a natural compound derived from glycobrassicin in Brassica vegetables, which has been shown to exhibit potent anticarcinogenic properties in a wide range of cancers such as lung, liver, colon, cervical, endometrial, prostate, and breast cancer (5-7). In addition, out of broad spectrum of analyzed phytochemicals, I3C was 1 of the few that tested positive as a chemopreventative agent in a panel of short-term bioassays relevant to carcinogen-induced DNA damage, tumor initiat...

Targeting of the Akt-Nuclear Factor-κB Signaling Network by [1-(4-Chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), a Novel Indole-3-Carbinol Derivative, in a Mouse Model of Hepatocellular Carcinoma

“…In light of the pleiotropic mode of action of indole-3-carbinol in targeting multiple pathways, the present study, however, could not exclude the involvement of Akt-and NF-B-independent signaling targets in OSU-A9's antitumor effects. For example, a recent study has identified intracellular elastase as one possible direct target of indole-3-carbinol that leads to cell cycle arrest as a consequence of altered proteolytic processing of cyclin E in MDA-MB-231 breast cancer cells (Nguyen et al, 2008). This possible mechanism is currently under investigation in OSU-A9-treated HCC cells.…”

Dietary Habits Contributing to Breast Cancer Risk Among Iranian Women

“…Some studies reported the no significant associations between vegetable or fruit intake and breast cancer risk (Van Gils et al, 2005, Jung et al, 2013, but others revealed a reduction in risk of breast cancer by high intake of fruits, vegetable (Sangrajrang et al, 2013)and fruits and vegetables combined (Aune et al, 2012) especially among women who were low or most physically active (Kruk, 2014). It has been suggested that phytochemicals in vegetables could decrease the level of cyclin E (Nguyen et al, 2008) and nuclear factor-kappaB (Johnson, 2007;Ahmad A et al, 2011) and consequently, reduce the risk of developing breast cancer.…”

Post-translational modification and stability of low molecular weight cyclin E

“…Specifically, the authors show that treatment of highly invasive MDA-MB-231 breast cancer cells with I3C shifted the stable accumulation of cyclin E protein from the hyperactive LMW form to the 50-kDa form. They also use an in vitro cyclin E processing assay to demonstrate that I3C disrupts the proteolytic processing of the 50-kDa cyclin E into the LMW forms by direct inhibition of human neutrophil elastase enzymatic activity (Nguyen et al, 2008). This study provides direct evidence that elastase uses cyclin E as a substrate to generate the LMW forms of this protein.…”

“…The LMW isoforms of cyclin E are produced through aberrant proteolysis of full length cyclin E by the protease elastase (Porter et al, 2001), although there exists some evidence that the protease calpain 2 may be both upregulated by cyclin E and responsible for also producing these isoforms (Libertini et al, 2005). However, recently a specific inhibitor of elastase indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, was shown to induce a G(1) cell cycle arrest of human breast cancer cells by targeting the generation of the LMW isoforms of cyclin E (Nguyen et al, 2008). Specifically, the authors show that treatment of highly invasive MDA-MB-231 breast cancer cells with I3C shifted the stable accumulation of cyclin E protein from the hyperactive LMW form to the 50-kDa form.…”