A novel drug discovery strategy: Mechanistic investigation of an enantiomeric antitumor agent targeting dual p53 and NF-κB pathways

The p53 and nuclear factor κB (NF-κB) pathways play crucial roles in human cancer development. Simultaneous targeting of both pathways is an attractive therapeutic strategy against cancer. In this study, we report an antitumor molecule that bears a pyrrolo[3,4-c]pyrazole scaffold and functions as an enantiomeric inhibitor against both the p53-MDM2 interaction and the NF-κB activation. It is a first-in-class enantiomeric inhibitor with dual efficacy for cancer therapy. Synergistic effect was observed in vitro and in vivo. Docking and molecular dynamics simulation studies further provided insights into the nature of stereoselectivity.     

NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor

The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.     

The novel NF-κB inhibitor DHMEQ synergizes with celecoxib to exert antitumor effects on human liver cancer cells by a ROS-dependent mechanism

In a previous work of ours dehydroxymethyl-epoxyquinomicin (DHMEQ), an inhibitor of NF-κB, was shown to induce apoptosis through Reactive Oxygen Species (ROS) production in hepatoma cells. The present study demonstrated that DHMEQ cooperates with Celecoxib (CLX) to decrease NF-κB DNA binding and to inhibit cell growth and proliferation more effectively than treatment with these single agents alone in the hepatoma cell lines HA22T/VGH and Huh-6. ROS production induced by the DHMEQ-CLX combination in turn generated the expression of genes involved in endoplasmic reticulum (ER) stress and silencing TRB3 mRNA significantly decreased DHMEQ-CLX-induced cell growth inhibition. Moreover, the DHMEQ-CLX combination was associated with induction of PARP cleavage and down-regulation of the anti-apoptotic proteins Bcl-2, Mcl-1 and survivin, as well as activated Akt. CD95 and CD95 ligand expression increased synergistically in the combination treatment, which was reversed in the presence of NAC. Knockdown of CD95 mRNA expression significantly decreased DHMEQ-CLX-induced cell growth inhibition in both cell lines. These data suggest that the DHMEQ-CLX combination kills hepatoma cells via ROS production, ER stress response and the activation of intrinsic and extrinsic apoptotic pathways.     

The novel NF-κB inhibitor IMD-0354 induces apoptosis in chronic lymphocytic leukemia

Nuclear factor-κB (NF-κB) is an important regulator of cell survival and has been shown to be constitutively active in chronic lymphocytic leukemia (CLL) cells. Recently, a novel NF-κB inhibitor, IMD-0354 (N-(3, 5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide), was shown to specifically inhibit the phosphorylation of IκBα by IkB kinases, thus preventing NF-κB release. In this study, we investigated if IMD-0354 can inhibit NF-κB activation and induce apoptosis in CLL cells in vitro. The rate of increase in apoptosis, drug sensitivity and DNA-binding activity of NF-κB were studied using Annexin V stainings, the fluorometric microculture cytotoxicity assay and electrophoretic mobility shift assay, respectively. Finally, the impact of IMD-0354 treatment on the expression of a set of apoptosis-related genes was investigated. The results clearly show that IMD-0354 induced apoptosis (mean 26%, range 8-48%) in CLL cells, independent of immunoglobulin heavy variable (IGHV) gene mutational status, and showed a dose-dependent cytotoxic effect. IMD-0354 treatment also significantly lowered the DNA-binding activity of NF-κB in CLL cells. In addition, we identified differences in expression levels of pro- and antiapoptotic genes following IMD-0354 treatment. In summary, our novel findings show that IMD-0354 can induce apoptosis in CLL cells, and thus merits further investigation as an anticancer agent in vivo.     

Enhancement of radiosensitivity by a unique novel NF-κB inhibitor, DHMEQ, in prostate cancer

Background:Inducible activation of nuclear factor (NF)-κB is one of the principal mechanisms through which resistant prostate cancer cells are protected from radiotherapy. We hypothesised that inactivation of inducible NF-κB with a novel NF-κB inhibitor, DHMEQ, would increase the therapeutic effects of radiotherapy.Methods:PC-3 and LNCaP cells were exposed to irradiation and/or DHMEQ. Cell viability, cell cycle analysis, western blotting assay, and NF-κB activity were measured. The antitumour effect of irradiation combined with DHMEQ in vivo was also assessed.Results:The combination of DHMEQ with irradiation resulted in cell growth inhibition and G2/M arrest relative to treatment with irradiation alone. Inducible NF-κB activity by irradiation was inhibited by DHMEQ treatment. The expression of p53 and p21 in LNCaP, and of 14-3-3σ in PC-3 cells, was increased in the combination treatment. In the in vivo study, 64 days after the start of treatment, tumour size was 85.1%, 77.1%, and 64.7% smaller in the combination treatment group than that of the untreated control, DHMEQ-treated alone, and irradiation alone groups, respectively.Conclusion:Blockade of NF-κB activity induced by radiation with DHMEQ could overcome radio-resistant responses and may become a new therapeutic modality for treating prostate cancer.     

Ethyl pyruvate, an anti-inflammatory agent, inhibits tumor angiogenesis through inhibition of the NF-κB signaling pathway

Ethyl pyruvate (EP), simple derivative of pyruvate, has been shown to have anti-inflammatory properties. Here, we demonstrate EP's strong anti-angiogenic activity. EP inhibited in vivo angiogenesis in the mouse Matrigel-plug assay and tumor growth in the mouse Lewis lung carcinoma model. EP also interfered with the angiogenic cascade, including growth, invasion, migration, and tube formation. Activation of NF-κB by vascular endothelial cell growth factor was reduced by EP. Taken together, we suggest that EP may have potential as a new multi-functional drug candidate for anti-angiogenesis and cancer therapy.     

Curcumin analog GO-Y030 is a novel inhibitor of IKKβ that suppresses NF-κB signaling and induces apoptosis

Curcumin is a dietary constituent with tumor-suppressing potential, inhibiting various pathways involved in carcinogenesis. However, because of its low bioavailability, the use of curcumin in in vivo trials has been limited. To overcome this problem, we synthesized more than 50 analogs and identified a monoketone analog, GO-Y030, which has a 30-fold higher potential to suppress tumor cell growth compared with curcumin. We investigated the inhibitory effect of GO-Y030 on NF-κB activation. In thyroid, pancreatic cancers and cholangiocarcinoma cells, in which NF-κB is activated, NF-κB activation was suppressed to 8-62% of the control value following treatment with 1 μM GO-Y030, an effect comparable to that of 10 μM curcumin. Direct inhibition of IKKβ kinase activity and suppression of nuclear translocation of the NF-κB p65 subunit were observed. The 50% growth inhibition concentrations of GO-Y030 ranged from one-11th to one-14th of those of curcumin. GO-Y030 also induced cell death comparable to that induced by curcumin but at a 10-fold lower concentration. In pancreatic and thyroid cancer cells, the growth-inhibitory effect of GO-Y030 was 4- and 15-fold greater, respectively, than that of curcumin. GO-Y030 was a much stronger inducer of apoptosis compared with curcumin. The enhanced potency of GO-Y030 may make it more useful than curcumin, which suffers from low bioavailability. GO-Y030 is a good lead compound for the development of useful compounds for practical cancer chemotherapy.     

AGK enhances angiogenesis and inhibits apoptosis via activation of the NF-κB signaling pathway in hepatocellular carcinoma

High levels of angiogenesis and resistance to apoptosis are major clinical features of hepatocellular carcinoma (HCC), a lethal disease with a high incidence worldwide. However, the precise mechanisms underlying these malignant properties remain unclear. Here, we demonstrated that acylglycerol kinase (AGK) is markedly overexpressed in HCC cell lines and clinical tissues. Immunohistochemical analysis of 245 clinical HCC specimens revealed patients with high levels of AGK expression had poorer overall survival compared to patients with low AGK expression. Furthermore, overexpressing AGK significantly enhanced angiogenesis and inhibited apoptosis in vitro and promoted the tumorigenicity of HCC cells in vivo; silencing endogenous AGK had the opposite effects. Importantly, AGK enhanced angiogenesis and inhibited apoptosis in HCC in part via activation of NF-κB signaling. Our findings provide new evidence that AGK plays an important role in promoting angiogenesis and providing resistance to apoptosis, thus AGK may represent a novel therapeutic target for HCC.     

Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor

Background:

Although T-cell immunity is thought to be involved in the prognosis of epithelial ovarian cancer (EOC) patients, immunosuppressive conditions hamper antitumour immune responses. Thus, their mechanisms and overcoming strategies need to be investigated.

Methods:

The role of NF-κB in human EOC cells and macrophages was evaluated by in vitro production of immunosuppressive IL-6 and IL-8 by EOC cells and in vivo analysis of immune responses in nude mice implanted with human EOC cells using an NF-κB inhibitor DHMEQ.

Results:

In EOC patients, increased plasma IL-6, IL-8, and arginase were observed. The NF-κB inhibitor DHMEQ inhibited the production of IL-6 and IL-8 by EOC cell lines. Immunosuppression of human DCs and macrophages by culture supernatant of EOC cells was reversed with the pretreatment of DHMEQ. Administration of DHMEQ to nude mice implanted with human EOC resulted in the restoration of T-cell stimulatory activity of murine DCs along with the reduction of tumour accumulation and arginase expression of MDSCs. Nuclear factor-κB inhibition in tumour-bearing mice also enhanced antitumour effects of transferred murine naive T cells.

Conclusions:

NF-κB is involved in the immunosuppression induced by human EOC, and its inhibitor may restore antitumour immune responses, indicating that NF-κB is an attractive target for EOC treatment.     

Antitumor effect of berberine against primary effusion lymphoma via inhibition of NF-κB pathway

Primary effusion lymphoma (PEL) is an infrequent and distinct entity among the aggressive non-Hodgkin B cell lymphomas that occurs predominantly in patients with advanced AIDS. It shows serous lymphomatous effusion in body cavities, and is resistant to conventional chemotherapy with a poor prognosis. Thus, the optimal treatment for PEL is not well defined and there is a need for novel agents. PEL has been recognized as the tumor caused by Kaposi sarcoma-associated herpes virus/human herpes virus-8 (KSHV/HHV-8), and nuclear factor (NF)-κB activation plays a critical role in the survival and growth of PEL cells. In this study, we assessed the antitumor effect of berberine, a naturally occurring isoquinoline alkaloid, on this pathway. The methylthiotetrazole assay showed that cell proliferation in the PEL cell lines was inhibited by berberine. Berberine also induced caspase-dependent apoptosis and suppressed NF-κB activity by inhibiting IκB kinase (IKK) phosphorylation, IκB phosphorylation and IκB degradation, upstream targets of the NF-κB pathway, in PEL cells. In a xenograft mouse model that showed ascites and diffuse organ invasion of PEL cells, treatment with berberine inhibited the growth and invasion of PEL cells significantly compared with untreated mice. These results show that the suppression of NF-κB is a molecular target for treating PEL, and berberine is a potential antitumor agent for PEL.     

Significance of VEGF and NF-κB Expression in Thyroid Carcinoma

Vascular endothelial growth factor (VEGF), a dimeric 42-kd pro- tein, is a multifunctional cytokine that plays a key role in both physiological and pathological angiogenesis.[1] Angiogenesis is known to be a prerequisite for tumor growth and metastasis.VE…     

LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118

Since disturbance of angiogenesis predisposes to ischemic injuries, attempts to promote angiogenesis have been made to improve clinical outcomes of patients with many ischemic disorders. While hypoxia inducible factors (HIFs) stimulate vascular remodeling and angiogenesis, hyperlipidemia impairs angiogenesis in response to various pro-angiogenic factors. However, it remains uncertain how HIFs regulate angiogenesis under hyperlipidemia. Here, we report that exposure to low-density lipoprotein (LDL) suppressed in vitro angiogenesis of human brain microvascular endothelial cells. Whereas LDL exposure diminished expression of HIF-1α and HIF-2α induced by hypoxia, it inhibited DMOG- and TNFα-induced HIF-1α and HIF-2α expression in normoxia. Notably, in both hypoxia and normoxia, LDL markedly reduced expression of HIF-1β, a constitutively stable HIF subunit, an event associated with NF-κB inactivation. Moreover, knockdown of HIF-1β down-regulated HIF-1α and HIF-2α expression, in association with increased HIF-1α hydroxylation and 20S proteasome activity after LDL exposure. Significantly, the proteasome inhibitor BSc2118 prevented angiogenesis attenuation by LDL through restoring expression of HIFs. Together, these findings argue that HIF-1β might act as a novel cross-link between the HIF and NF-κB pathways in suppression of angiogenesis by LDL, while proteasome inhibitors might promote angiogenesis by reactivating this signaling cascade under hyperlipidemia.     

HIV protease inhibitor Lopinavir induces apoptosis of primary effusion lymphoma cells via suppression of NF-κB pathway

Primary effusion lymphoma (PEL) is a non-Hodgkin lymphoma that occurs predominantly in patients with advanced AIDS. In this study, we examined the effect of HIV protease inhibitors, Lopinavir (LPV), Ritonavir (RTV) and Darunavir (DRV) on PEL cell lines in vitro and in vivo. LPV and RTV, but not DRV induced caspase-dependent apoptosis and suppressed NF-κB activity by inhibiting IKK phosphorylation in PEL cells. In a PEL xenograft mouse model, LPV significantly inhibited the growth and invasion of PEL cells. These results suggest that LPV may have promise for the treatment and prevention of PEL, which occurs in HIV/AIDS patients.     

Combination chemotherapy of serine protease inhibitor nafamostat mesilate with oxaliplatin targeting NF-κB activation for pancreatic cancer

In this study, we assessed if nafamostat mesilate may enhance anti-tumor effects of oxaliplatin on Panc-1 cells and pancreatic cancer mouse model. In combination treatment with nafamostat mesilate and oxaliplatin, NF-κB activation was inhibited by suppressing IκBα phosphorylation, and caspase-8-mediated apoptosis was more prominent than that treated with oxaliplatin alone, both in vitro and in vivo. Nafamostat mesilate reduced proliferation rate of Panc-1 cells as compared with oxaliplatin alone in vitro and enhanced oxaliplatin-induced tumor growth inhibition in vivo. Combination chemotherapy using nafamostat mesilate and oxaliplatin induces synergistic cytotoxicity in pancreatic cancer and could be a novel strategy for treatment.     

Activation of the NF-κB pathway as a mechanism of alcohol enhanced progression and metastasis of human hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is the third leading cause of cancer-related death in human. Alcohol is a known risk factor for HCC. However it is still unclear whether and how alcohol enhances the progression and metastasis of existing HCC.

Methods and results

We first retrospectively investigated 52 HCC patients (24 alcohol-drinkers and 28 non-drinkers), and found a positive correlation between alcohol consumption and advanced Tumor-Node-Metastasis (TNM) stages, higher vessel invasion and poorer prognosis. In vitro and in vivo experiments further indicated that alcohol promoted the progression and migration/invasion of HCC. Specifically, in a 3-D tumor/endothelial co-culture system, we found that alcohol enhanced the migration/invasion of HepG2 cells and increased tumor angiogenesis. Consistently, higher expression of VEGF, MCP-1 and NF-κB was observed in HCC tissues of alcohol-drinkers. Alcohol induced the accumulation of intracellular reactive oxygen species (ROS) and the activation of NF-κB signaling in HepG2 cells. Conversely, blockage of alcohol-mediated ROS accumulation and NF-κB signaling inhibited alcohol-induced expression of VEGF and MCP-1, the tumor growth, angiogenesis and metastasis.

Conclusion

This study suggested that chronic moderate alcohol consumption may promote the progression and metastasis of HCC; the oncogenic effect may be at least partially mediated by the ROS accumulation and NF-ĸB-dependent VEGF and MCP-1 up-regulation.     

The diverse and complex roles of NF-κB subunits in cancer

It is only recently that the full importance of nuclear factor-κB (NF-κB) signalling to cancer development has been understood. Although much attention has focused on the upstream pathways leading to NF-κB activation, it is now becoming clear that the inhibitor of NF-κB kinases (IKKs), which regulate NF-κB activation, have many independent functions in tissue homeostasis and normal immune function that could compromise the clinical utility of IKK inhibitors. Therefore, if the NF-κB pathway is to be properly exploited as a target for both anticancer and anti-inflammatory drugs, it is appropriate to reconsider the complex roles of the individual NF-κB subunits.     

Antitumor effect of dehydroxymethylepoxyquinomicin, a small molecule inhibitor of nuclear factor-κB, on glioblastoma

Glioblastoma is the most malignant type of brain tumor. Despite recent advances in therapeutic modalities, the prognosis of glioblastoma remains very poor. Recent studies have indicated that RelA/nuclear factor (NF)-κB is consistently activated in human glioblastoma. In this study, we searched for a new treatment modality for glioblastoma, by examining the effects of dehydroxymethylepoxyquinomicin (DHMEQ), a unique small molecule inhibitor of NF-κB. Addition of DHMEQ to cultured human glioblastoma cells inhibited the nuclear translocation of RelA. It also reduced the growth rate of human glioblastoma cells significantly in 6 cell lines and modestly in 3 among 10 cell lines examined. Then, we performed further analyses using 3 sensitive cell lines (U87, U251, and YKG-1). The growth retardation was accompanied by G2/M arrest in vitro. Increased apoptosis was observed in U87 and YKG-1, but not U251 cells after DHMEQ treatment. Then, we tested the efficacy of DHMEQ in chemoprevention through the use of a nude mouse model. Subcutaneous tumors formed by U87 or U251 cells were reduced by ～40% in size by intraperitoneal administration of DHMEQ started immediately after implantation of the cells. DHMEQ treatment achieved statistically significant improvements in survival curves of mice intracranially implanted with U87 or U251 cells. Histological analysis revealed increased areas of necrosis, increased numbers of collapsed microvessels, decreased nuclear immunoreactivity of RelA, and decreased immunoreactivity of urokinase-type plasminogen activator in the DHMEQ-treated U87 tumor tissues. These results suggest that the targeting of NF-κB by DHMEQ may serve as a promising treatment modality in glioblastoma.