There are two major NF-κB signaling pathways, canonical pathway (or classical) and the non-canonical pathway (or alternative pathway) [25
]. The canonical pathway is mainly activated by TNF-α, IL-1 and LPS. Non-canonical pathway is activated by LTα/β, CD40 ligand and Blys/BAFF, but not by TNF-α, IL-1 and LPS [25
]. In the present study, we have identified many drugs in human clinical use that inhibit the canonical NF-κB pathway, by screening a comprehensive collection of approximately 2,800 small molecule drugs that have been approved by regulatory agencies for human use or clinical trials. Some of these drugs are known or suspected NF-κB inhibitors used in the treatment of cancer, but others are used for other indications where NF-κB activity would be neither expected nor necessarily desired. Virtually all of the drugs identified as NF-κB inhibitors produced cytotoxicity on cervical cancer cells, in similar rank order of potency to their NF-κB inhibition. These drugs also induced LDH release from cervical cancer cells indicating the cell death mostly via cell necrosis. Approximately over half of the drugs activated caspase 3/7 and disrupted mitochondrial membrane potential, and only a subset inhibited IκBα phosphorylation, suggesting additional mechanisms for NF-κB inhibition. Among the 19 NF-κB inhibiting drugs identified, ectinascidin 743, digitoxin, ouabain, bortezomib and chromomycin A3 were the most potent NF-κB inhibitors, and ectinascidin 743, bortezomib, chromomycin A3, emetine, daunorubicinum, and lestaurtinib were most potent in the caspase 3/7 and cytotoxicity assays. Emetine, fluorosalan, narasin, lestaurtinib, tribromsalan and bithionol were shown to inhibit IκBα phosphorylation. These results provide new information on activities and mechanisms of action of approved drugs, which may suggest mechanisms of potential novel applications in cancer treatment.
Ectinascidin 743, a marine-derived alkaloid, is approved by the European Commission and US FDA for the treatment of ovarian cancer [26
]. It is also in phase II clinical trials for the treatment of prostate and breast cancers, and pediatric sarcomas [26
]. There are three tetrahydroisoquinoline moieties in ectinascidin 743 structure. The central carbinolamine of three moieties of ectinascidin 743 covalently binds to DNA by alkylating the N2 aminogroup of quanines at specific sequences, which induces the cancer cells to die [26
]. Our data suggest that inhibition of NF-κB signaling, activation of caspase 3/7 and induction of LDH release are mechanisms by which ectinascidin 743 acts.
, a naturally occurring antibiotic isolated from S. griseus
], was also identified as an NF-κB inhibitor from this study. The anticancer property of chromomycin A3
includes inhibiting DNA-dependent RNA synthesis and is clinically used for the treatment of various diseases including chronic myelogenous leukemia, testicular carcinoma and Paget’s disease [27
]. We found that chromomycin A3
is similar to ectinascidin 743 in its activation of caspase 3/7, induction of LDH release and growth inhibition of cervical cancer cells. These compounds inhibited the NF-κB signaling pathway in a concentration-dependent manner, but had weak or no effect on IκBα phosphorylation, which suggests that these drug may affect other targets such as degradation of IκBα.
Bortezomib, a proteasome inhibitor, has been approved for the treatment of relapse/refractory multiple myeloma and mantle-cell lymphoma [28
]. Bortezomib’s inhibitory effect on myeloma cell growth and survival is mediated through NF-κB signaling because it inhibits the 26S subunit of the proteasome which leads to the inability of IκB degradation and NF-κB activation [28
]. This mechanism of action of bortezomib is confirmed in our study where bortezomib inhibited the NF-κB signaling pathway, but had no effect on IκBα phosphorylation.
Emetine, a crystalline alkaloid derived from ipecac root, is known as a protein synthesis inhibitor and DNA interacting agent [29
] and is clinically used in the treatment of protozoan infection. Recently the anti-cancer properties of emetine have been shown. Boon-Unge et al [30
] reported that the specific mechanism of emetine is to target malignant cells via up regulation of the Bcl-xS splicing variant which promotes apoptosis. We also found that emetine inhibited both NF-κB signaling and IκBα phosphorylation, as well as induced caspase 3/7 activity and had cytotoxicity on in cervical cancer cells.
Several tyrosine kinase inhibitors, such as lestaurtinib, sorafenib tosylate and sunitinib malate, were identified as NF-κB antagonists in this study. These compounds have a broad spectrum of kinase inhibition and are shown to inhibit vascular endothelial growth factor recept and FMS-like tyrosine kinase 3 (FLT3) [31
]. Lestaurtinib (CEP-701), sorafenib (BAY-43-9006) and sunitinib (SU-11248) have been in phase I-II clinical trials for treatment of acute myeloid leukemia by targeting VEFGR and FLT3 [32
]. In this study we found that lestaurtinib is the most potent NF-κB blocker among these three tyrosine kinase inhibitors. The inhibitory effect of lestaurtinib on NF-κB signaling is via the inhibition of IκBα phosphorylation. In human cervical cancer cells, lestaurtinib induced apoptosis by activating caspase 3/7 and suppressed the growth of these cancer cells. By contrast, sorafenib tosylate had only a moderate inhibitory effect on NF-κB signaling and IκBα phosphorylation. Sorafenib tosylate disrupted mitochondrial membrane potential, but did not activate caspase 3/7, suggesting that the apoptosis induced by this drug may be independent of caspase activation.
Two cardiac glycoside drugs, digitoxin and ouabain, were found to inhibit NF-κB signaling in the NF-κB bla
assay and were potent inhibitors of cancer cell growth, but had no effect on IκBα phosphorylation and did not induce caspase 3/7(). These drugs also had no effect on mitochondrial membrane potential (Supplemental Table 1
), suggesting that these drugs may not induce cellular apoptosis. We also found that digoxin, another cardiac glycoside drug in the NPC, inhibited NF-κB signaling in the NF-κB bla
assay (data not shown). These results suggest that these drugs may affect other targets in the NF-κB signaling pathway. It has been recently demonstrated that digitoxin and related cardiac glycoside drugs commonly act through blocking TNF-α-dependent binding of the TNF receptor to the TNF receptor-associated death domain in the TNF-α/ NF-κB signaling pathway [33
]. Clinically, cardiac glycosides have been used to treat congestive heat failure and arrhythmias via the mechanism of inhibiting Na+
ATPase, which results in increased intracellular calcium concentration [34
]. Additionally, these cardiac glycoside drugs had an inhibitory effect on tumor cell proliferation in many types of cancers including lung, breast, colon, prostate and liver [34
]. The potential anti-cancer mechanisms of these drugs have been linked to the inhibition of HIF-1α (hypoxia inducible factor 1α) synthesis in Hep3B cells, human liver carcinoma cells [35
], the reduction of the p53 levels via the inhibition of p53 protein synthesis in lung cancer cells [36
], the inhibition of activation of the TNF-α/ NF-κB signaling pathway [33
], and the inhibition of DNA topoisomerases I and II in breast cancer MCF-7 cells [37
There is considerable ongoing interest in the development of NF-κB inhibitors for cancer treatment, and many new chemical entities that work on various nodes in the NF-κB pathway have been or are being developed. However, the long, expensive, and unpredictable process of new drug development has led to increasing interest in finding new clinical applications for drugs already approved for another indication. In the present study, we have identified several NF-κB inhibitors previously approved for other clinical uses, and several anti-cancer agents not previously appreciated to inhibit the NF-κB pathway. Our identification of novel NF-κB inhibitors among the existing pharmacopeia provides insight into the mechanisms of therapeutic action of existing drugs, as well as suggesting possible new uses for these drugs in diseases characterized by NF-κB pathway overactivity.