Historically, natural products are important starting materials in the lead discovery phase of the drug discovery process and have been a major source for new chemical entities [24
]. More recently, combinatorial chemistry has become an alternative choice. However, the number of lead optimization candidates yielded by combinatorial chemistry has been much less than expected [25
]. The underlying reason might be that chemical structures obtained through combinatorial approaches lack essential lead-like properties [24
]. Because of these problems, and the fact that CK2 overexpression is associated with multiple human cancers and may therefore be a promising target for cancer therapy, we decided to screen the natural product library obtained from the NCI to identify novel CK2 inhibitors. For this purpose, we used a cell-free kinase assay to screen the libraries. Coumestrol was identified as a promising CK2 inhibitor. Kinetic assays in our study also showed that coumestrol is an ATP competitive and reversible inhibitor toward CK2. The results, combined with those from a kinetic study, led us to identify and validate coumestrol as a novel CK2 kinase inhibitor.
To the best of our knowledge, our study is the first to show that coumestrol is a CK2 kinase inhibitor in both cell-free assay and cancer cells. The cell-free IC50
value of coumestrol (0.23 μM) on CK2 kinase activity is comparable to that of several well established CK2 inhibitors, such as 2-Dimethylamino-4,5,6,7-tetrabromo-1H
-benzimidazole (DMAT) (0.15 μM), [5-oxo-5, 6-dihydroindolo-(1, 2-a) quinazolin-7-yl] acetic acid (IQA) (0.39 μM), 4,5,6,7-tetrabromo benzotriazole (TBB) (0.50 μM) [22
] and 1, 3, 8-trihydroxyanthraquinone (emodin) (0.89 μM) [26
We also showed that coumestrol triggered apoptosis in cancer cells. Previous studies suggest that CK2 plays an essential role in suppressing apoptosis. Overexpression of CK2 in cancer cells protects cells from etoposide- and diethylstilbestrol-induced apoptosis [27
], resulting in suppressed apoptosis mediated through tumor necrotic factor-alpha (TNF-α), TRAIL and Fas L, and augments apoptosis in cells sensitive to these ligands [28
]. Treatment of a variety of cancer cells with cell-permeable CK2 inhibitors such as TBB, IQA and DMAT has been shown to induce activation of caspases and then apoptosis [22
]. In our study, coumestrol inhibited Akt/PKB Ser129 phosphorylation in cancer cells. Akt/PKB is activated by CK2 and ensures cell survival via activation of anti-apoptotic pathways, including the NF-κB pathway and suppression of caspase activities [31
]. Thus, coumestrol induces apoptosis in cancer cells at least partially by inhibiting the Akt/PKB pathway by down regulation of CK2 kinase and then decreased phosphorylation of Akt/PKB Ser129.
Coumestrol belongs to the class of phytoestrogens that includes isoflavones and coumestans. It is the most prevalent derivative of coumestan [34
], which can be found in leguminous plants serving as food sources for humans. Coumestrol intake in the Asian population is 10 times greater than that of the non-Asian population [35
]. The half-lives of plasma genistein and daidzein, compounds from the same family of coumestrol, were found to be 8.36 and 5.79 hr, respectively, in humans [36
]. A pharmacokinetic study of soy-derived phytoestrogens in rats suggested that genestein has a half-life of 4.3 hr, daidzein 2.3 hr and coumestrol 5.5 hr, almost equal to 5.6 hr observed for zearalenone [37
A specific dietary supplement, selected vegetables (SV), which contains coumestrol, was studied in tumor-bearing mice and in stage IIIB and IV non-small cell lung cancer patients [37
]. The study found 53-74% inhibition of tumor growth in mice, but more strikingly, patients in stage IIIB and IV NSCLC who took SV daily for 2–46 months had prolonged survival and attenuation of the normal pattern of progression compared to patients not taking SV [38
Soy isoflavones, because they are estrogen-like compounds, are thought to have potential side effects on patients with ER-positive breast cancer. The structure of coumestrol is similar to that of estradiol, and coumestrol reportedly can bind to two estrogen receptor subtypes (ERα and ERβ) but with lower binding affinity than that of estradiol [39
]. Despite the structural similarities, soy isoflavones bind to ER differently than estrodiol does and are thought to exhibit only beneficial effects of estrogen [40
]. High consumption of soy foods may reduce the risk of breast cancer [44
]. However, whether the use of coumestrol as a cancer treatment may have side effects related to estrogen receptors requires further study.
Coumestrol is a relatively small molecule (MW 268), which provides room for physical/chemical activity modifications. Tumors that overexpress CK2 could be potentially treated with coumestrol or coumestrol derivatives that have better drug-like properties [15
]. Coumestrol and its derivatives can also potentially target several key signaling pathways such as the Akt pathway, a particular example being EGFR mutations [46
]. Thus, coumestrol may represent a new class of targeted treatments for cancer.