Most studies suggest that EF24 impairs cell growth by inducing cell cycle arrest followed by induction of apoptosis, which is accompanied by caspase-3 activation. However, the cell signaling pathway mediating the EF24 effect was not elucidated until 2008 when Kasinski et al. first revealed that EF24 induced cell apoptosis via suppressing NF-κB signaling pathway through direct action on IkB kinase (IKK) (21). NF-κB regulates a wide variety of genes involved in cell proliferation, differentiation, cell cycle control (36), oncogenic activation (37) and metastasis (38). EF24 can inhibit the catalytic activity of IKK protein complex, which blocks IκB phosphorylation and subsequent degradation, and finally prevents the nuclear translocation of p65 subunit of NF-κB. The study provides a molecular explanation for the superior activity of EF24 over curcumin (with a potency about 10 times higher than that of curcumin). Other groups further verified the involvement of EF24 in inhibiting NF-κB signaling pathway in cancer (39–42). EF24 robustly conferred radiation-induced cell death mainly by inhibiting radiation-induced NF-κB signaling in breast cancer (43). In the same year, the same group found that EF24 can suppress the radiation-induced NF-κB-DNA binding activity/promoter activation in genetically varied human neuroblastoma (44). Inhibition of the NF-κB signaling extends the therapeutic application of EF24 to other NF-κB-dependent diseases, such as inflammatory diseases (described below).

By targeting IKK in the NF-κB pathway, EF24 was also able to inhibit the Fanconi anemia (FA) pathway, a multigene DNA damage response network implicated in the repair of DNA lesions. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced Fanconi anemia D2 ubiquitination (FANCD2-Ub) and foci in a cell-cycle independent manner (45). Beside the FA pathway, several other signaling pathways interplay with NF-κB, one of which is the mitogen-activated protein kinase (MAPK) pathway (46, 47). Given the substantial role of EF24 in inhibiting NF-κB, scientists started to notice its role in regulating MAPK pathway. Thomas et al. reported that EF24 drastically induced the upregulation of three major MAPK pathways mediated by ERK, JNK, and p38 (48). On the contrary, Lin et al. reported that EF24 exerts its anti-tumor activity in oral squamous cell carcinoma via deactivation of the MAPK/ERK signaling pathway (49). Therefore, the effect of EF24 on the MAPK pathway is still under debate and needs to be verified in more cancer types in the future.

Another important role of EF24 is to regulate HIF-1α expression which is closely associated with the outcome of chemotherapy in cancer treatment. For example, Liang et al. reported that sorafenib therapy could induce drug resistance in the treatment of hepatocellular carcinoma, in which HIF-1α plays an important role (50). Anti-angiogenic effects of sustained sorafenib therapy caused intratumor hypoxia, which induced HIF-1α and protected cancer cells from sorafenib treatment. EF24 can inhibit HIF-1α by sequestering it in cytoplasm and can promote its degradation by upregulating Von Hippel-Lindau tumor suppressor (VHL). Combination of EF24 and sorafenib showed synergistic effects against metastasis both in vivo and in vitro (50), providing compelling evidence for the potential of clinical application of EF24. Similar to EF24, the parent compound curcumin can also regulate HIF-1α expression (51), albeit through distinct mechanisms. Curcumin inhibited HIF-1α gene transcription, while EF24 exerted the activity by inhibiting HIF-1α post-transcriptionally (22).

The oncogenic factor HIF-1α is implicated in regulating aerobic glycolysis and the expression of Glut1 (52). Since, EF24 was able to inhibit HIF-1α activity, it likely affects glucose metabolism, thereby regulating cancer cell survival. Actually, unlike normal cells which produce energy through mitochondrial oxidative phosphorylation, most cancer cells produce their energy predominantly through a high rate of glycolysis followed by lactic acid fermentation, even in the presence of abundant oxygen, which is termed the Warburg effect (53). Although less efficient than oxidative phosphorylation in terms of ATP production, aerobic glycolysis generates additional metabolites that may help cancer cells to proliferate and growth. EF24 has been reported to block glucose uptake and the rate of glycolysis, and thereby inhibits cell migration and invasion in ovarian cancer cells (54). Another notable anti-tumor mechanism of EF24 is regulation of ROS production. Elevated oxidative status has been found in many types of cancer cells, which contributes to carcinogenesis (55–57). In 2014, Roy et al. reported that EF24 could protect protein disulfide isomerase (PDI), an important endoplasmic reticulum-resident oxidoreductase chaperone, from ROS-induced damage (58). In human ovarian cancer cells, EF24 suppressed ROS generation and activated antioxidant response element (ARE)-dependent gene transcription (28). Similarly, EF24 suppressed the level of superoxide in combination with SN38 in cancerous tissue (30). On the contrary, some other studies found that EF24 induces ROS production. The authors who first designed and synthesized EF24 observed that it was able to induce ROS production in MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells (19). Similarly, EF24 was reported to induce ROS production in gastric cancer cells (59), and show synergistic anti-tumor activity with rapamycin (60) or Akt inhibitor (61). The same group reported a similar effect of EF24 on inducing ROS in human colon cancer lines (HCT-116 and SW-620 cells), but moderate effects in HT-29 cells (62). Therefore, it seems that the role of EF24 in ROS induction may be cell type-dependent, and more evidence is needed to clarify these contradictory results. Interestingly, Skoupa et al. recently concluded that apoptosis induced by the EF-24 is not mediated by oxidative stress-related in human leukemia cells (20). A redox-dependent-mediated mechanism only marginally contributes to the EF24-induced apoptosis in K562 cells. Therefore, the effects of EF24 on ROS and ROS-mediated apoptosis in cancer cells remains to be confirmed in the further studies.

EF24 was also found to target microRNAs to regulate the tumor progress. MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate gene expression. Dysregulation of miRNA expression in cancer has been well-established (63). Yang et al. reported that EF24 was able to downregulate miR-21 and thereby enhance the expression of its target genes PTEN and PDCD4, which inhibit tumor growth and metastasis (64). Another group reported that EF24 suppressed melanoma metastasis by upregulating miR-33b and concomitantly reducing HMGA2 expression (65). The role of EF24 in regulating miRNAs provides a novel mechanism for its anti-cancer function.

Since the development of EF24, scientists have compared its activity with its parent compound curcumin. Most of the studies were focused on the anti-cancer activity. Adams et al. synthesized a series of curcumin analogs including EF24, and submitted them to the NCI anti-cancer cell line screen. The results showed that EF24 was effective against all of the cell lines. The mean panel GI₅₀ (concentration at which the drug inhibits tumor cell growth by 50%) was 10-fold better than curcumin and cisplatin (18). The authors also submitted the analogs to an in vitro anti-angiogenesis screen and revealed that EF24 was more active than curcumin in the assay. Their in vivo experiment suggested that EF24 was well tolerated by mice and much safer than the chemotherapy drug cisplatin (18). Later, Subramaniam et al. further compared the potency of EF24 to curcumin in gastrointestinal cancer cells and demonstrated that EF24 was more potent than curcumin. For example, 1 μmol/L of EF24 significantly suppressed proliferation and colony formation of the colon and gastric cancer cell lines while at the same dose of curcumin had no effect (29). Consistently, EF24 exhibited IC₅₀ values 10 to 20 times lower than that of curcumin in multiple cancer cell lines, including lung, ovarian, cervical, breast, prostate cancer cells and cholangiocarcinoma cells (21, 22, 28, 40, 48). Similarly, in human osteogenic sarcoma cells (Saos2), EF24 was 3-fold more potent than curcumin (31). This evidence collectively suggests that EF24 displays much more potent anti-cancer activity than its parent compound in vitro. Although EF24 shared many anti-cancer mechanisms with its parent compound curcumin, such as inhibiting NF-κB and HIF-1α, it exerts its effects in different ways, for example, in how it regulates HIF-1α activity (described above). In addition, curcumin efficiently inhibited proteasome activity. By contrast, EF24 was 20-fold less active than curcumin for proteasome inhibition (45). Likewise, curcumin can regulate the STAT3, while EF24 has no effect on STAT activation (64).

The in vivo activity of a compound relies on the bioavailability of the compound at the site of the tumor. Dietary curcumin is poorly absorbed through the intestinal tract, therefore curcumin does not have a therapeutic effect at low doses (29). By contrast, EF-24 has higher oral bioavailability (60%) in mice (66), explaining to some extent the improved in vivo activity of EF24 compared to curcumin.

In addition to the improvement of anti-cancer activity, EF24 shows low toxicity to normal cells. EF24 has been shown to induce apoptosis in cancer cells and inhibit the growth of human breast tumors in a mouse xenograft model but showed low toxicity (18). Subramaniam et al. reported that EF24 inhibited intestinal cancer cell proliferation, but did not affect the proliferation of normal mouse embryonic fibroblasts cells, suggesting that EF24 is not toxic to normal cells (29). Similarly, EF24 inhibits tumor growth in human cholangiocarcinoma while displaying low toxicity levels. As a sensitizer, co-treatment of EF24 and rapamycin selectively enhances the cytotoxicity in gastric cancer cells but not in normal cells (60). Above all, multiple molecular targets, wide-spectrum potency, enhanced bioavailability as well as low toxicity to normal cells confer EF24 a series of advantages in clinical applications.

To further improve the anti-cancer activity, some groups have moved forward to design and synthesize new analogs of EF24. In 2012, Lagisetty et al. synthesized a hydrazinonicotinic acid conjugate using an amine derivative of EF24. The derivative has improved aqueous solubility compared to EF24, and showed significant anti-tumor effects (88). Wu et al. synthesized and purified 20 EF24 analogs, from which they identified one to have greater activity than EF24, showing potent anti-migration and anti-proliferative activity against A549 cells (89). Xie et al. designed and synthesized four series of EF24 analogs, from which they found one displaying excellent inhibition of both IKKβ activity and pancreatic cancer (PC) development and progression (90). A group from Germany synthesized 14 EF24 analogs and revealed that they have promising anti-proliferative activity against eight cancer cell lines with low IC₅₀ values, and showed superior anti-angiogenic and vascular-disruptive effects (91). Last year, a research group from China synthesized a series of EF24 analogs and identified one to have much greater inhibitory activity against IKKβ and to induce apoptosis and cell cycle arrest in multiple cancer cell lines (92). Recently, a series of EF24 analogs were synthesized and screened, resulting in one with good potency and selectivity in killing cancer cells (93).

Improving the bioavailability of chemotherapy drugs can greatly increase the therapeutic effect while reducing the side effects. To achieve this aim, specific drug delivery systems (DDS) have been investigated. For example, scientists observed that the tissue factor is aberrantly and abundantly expressed in many cancer cells (94, 95), and based on this expression, the group that first designed and synthesize EF24 later proposed a new drug delivery system that associated with tissue factor on the surface of cancer cells, releasing the cytotoxic agent into the cytoplasm. This system displayed a greater effect than EF24 alone in human breast and melanoma cell lines (96). Later, it was reported that the conjugation of EF24 with coagulation factor VIIa induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice. The targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects (97). Although EF24 has shown promising in vitro therapeutic efficacy in various human cancer cells, increasing water solubility and systemic bioavailability will be beneficial for its clinical applications. Agashe et al. designed EF24-liposomes and showed their anti-proliferative activity to be superior to that of EF24 alone (98). Bisht et al. designed nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24), evaluated the particles in pancreatic cancer models, and observed good therapeutic efficacy and favorable toxicity profile (99), which provide evidence for development of future combinatorial therapeutic regimens against pancreatic cancer.