The VR23 Antitumor Compound Also Shows Strong Anti-Inflammatory Effects in a Human Rheumatoid Arthritis Cell Model and Acute Lung Inflammation in Mice.

We previously found that the novel VR23 proteasome inhibitor not only possesses an effective antitumor activity without causing any ill effects to animals but also reduces side effects caused by a partner drug when used in combination. In this article, we report that VR23, unlike other proteasome inhibitors, exhibits potent anti-inflammatory activity. In the LPS-induced THP-1 monocyte model, VR23 downregulates proinflammatory cytokines IL-1ß, TNF-α, IL-6, and IL-8 at a similar efficacy to dexamethasone. In contrast, two well-known proteasome inhibitors, bortezomib and carfilzomib, do not effectively downregulate these proinflammatory cytokines. Data from a study with SW982 synovial cell line and primary human synoviocytes showed that VR23 not only effectively downregulates IL-6 but also inhibits cell migration. Interestingly, the IL-6 downregulation by VR23 was significantly more pronounced in the primary synovial cells from rheumatoid arthritis patients than those from healthy donors, suggesting that VR23 can be selective against rheumatoid arthritis. Finally, VR23 effectively reduces neutrophil migration, TNF-α secretion, and tissue inflammation in mice (female BALB/c strain) with an LPS-induced acute lung injury. Thus, our current data indicate that VR23 can be effective on both acute and chronic inflammatory conditions. Taken together with our previous work, VR23 is not only effective on inflammatory conditions but also applicable to different aspects of cancer control, including the treatment and prevention of tumor development by chronic inflammatory responses.

Novel quinolone chalcones targeting colchicine-binding pocket kill multidrug-resistant cancer cells by inhibiting tubulin activity and MRP1 function.

Agents targeting colchicine-binding pocket usually show a minimal drug-resistance issue, albeit often associated with high toxicity. Chalcone-based compounds, which may bind to colchicine-binding site, are found in many edible fruits, suggesting that they can be effective drugs with less toxicity. Therefore, we synthesized and examined 24 quinolone chalcone compounds, from which we identified ((E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-17) and ((E)-6-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-20) as promising leads. In particular, CTR-20 was effective against 65 different cancer cell lines originated from 12 different tissues, largely in a cancer cell-specific manner. We found that both CTR-17 and CTR-20 reversibly bind to the colchicine-binding pocket on ß-tubulin. Interestingly however, both the CTRs were highly effective against multidrug-resistant cancer cells while colchicine, paclitaxel and vinblastine were not. Our study with CTR-20 showed that it overcomes multidrug-resistance through its ability to impede MRP1 function while maintaining strong inhibition against microtubule activity. Data from mice engrafted with the MDA-MB-231 triple-negative breast cancer cells showed that both CTR-17 and CTR-20 possess strong anticancer activity, alone or in combination with paclitaxel, without causing any notable side effects. Together, our data demonstrates that both the CTRs can be effective and safe drugs against many different cancers, especially against multidrug-resistant tumors.

VR23: A Quinoline-Sulfonyl Hybrid Proteasome Inhibitor That Selectively Kills Cancer via Cyclin E-Mediated Centrosome Amplification.

The proteasome is clinically validated as a target for cancer therapeutics. However, proteasome-inhibitory agents that are cancer selective have yet to be developed. In this study, we report the identification of a safe and effective proteasome inhibitor with selective anticancer properties. We screened a chemical library constructed using a hybrid approach that incorporated a 4-piperazinylquinoline scaffold and a sulfonyl phamarcophore. From this library, we identified 7-chloro-4-(4-(2,4-dinitrophenylsulfonyl)piperazin-1-yl)quinoline (VR23) as a small molecule that potently inhibited the activities of trypsin-like proteasomes (IC50 = 1 nmol/L), chymotrypsin-like proteasomes (IC50 = 50-100 nmol/L), and caspase-like proteasomes (IC50 = 3 µmol/L). Data from molecular docking and substrate competition assays established that the primary molecular target of VR23 was ß2 of the 20S proteasome catalytic subunit. Notably, VR23 was structurally distinct from other known proteasome inhibitors and selectively killed cancer cells by apoptosis, with little effect on noncancerous cells. Mechanistic investigations showed that cancer cells exposed to VR23 underwent an abnormal centrosome amplification cycle caused by the accumulation of ubiquitinated cyclin E. In combinations with the clinically approved chymotrypsin-like proteasome inhibitor bortezomib, VR23 produced a synergistic effect in killing multiple myeloma cells, including those that were resistant to bortezomib. VR23 was effective in vivo in controlling multiple myelomas and metastatic breast cancer cells, in the latter case also enhancing the antitumor activity of paclitaxel while reducing its side effects. Overall, our results identify VR23 as a structurally novel proteasome inhibitor with desirable properties as an anticancer agent.

Bortezomib induces nuclear translocation of IκBα resulting in gene-specific suppression of NF-κB--dependent transcription and induction of apoptosis in CTCL.

Cutaneous T-cell lymphoma (CTCL) is characterized by constitutive activation of nuclear factor κB (NF-κB), which plays a crucial role in the survival of CTCL cells and their resistance to apoptosis. NF-κB activity in CTCL is inhibited by the proteasome inhibitor bortezomib; however, the mechanisms remained unknown. In this study, we investigated mechanisms by which bortezomib suppresses NF-κB activity in CTCL Hut-78 cells. We demonstrate that bortezomib and MG132 suppress NF-κB activity in Hut-78 cells by a novel mechanism that consists of inducing nuclear translocation and accumulation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which then associates with NF-κB p65 and p50 in the nucleus and inhibits NF-κB DNA binding activity. Surprisingly, however, while expression of NF-κB-dependent antiapoptotic genes cIAP1 and cIAP2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1 and cIAP2 promoters are occupied by NF-κB p65/50 heterodimers, whereas Bcl-2 promoter is occupied predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib function in CTCL and suggest that the inhibition of NF-κB-dependent gene expression by bortezomib is gene specific and depends on the subunit composition of NF-κB dimers recruited to NF-κB-responsive promoters.

Gene-specific repression of proinflammatory cytokines in stimulated human macrophages by nuclear IκBα.

We have previously shown that increased nuclear accumulation of IkappaBalpha inhibits NF-kappaB activity and induces apoptosis in human leukocytes. In this study, we wanted to explore the possibility that the nucleocytoplasmic distribution of IkappaBalpha can be used as a therapeutic target for the regulation of NF-kappaB-dependent cytokine synthesis. Treatment of LPS-stimulated human U937 macrophages with an inhibitor of chromosome region maintenance 1-dependent nuclear export, leptomycin B, resulted in the increased nuclear accumulation of IkappaBalpha and inhibition of NF-kappaB DNA binding activity, caused by the nuclear IkappaBalpha-p65 NF-kappaB interaction. Surprisingly, however, whereas mRNA expression and cellular release of TNF-alpha, the beta form of pro-IL-1 (IL-1beta), and IL-6 were inhibited by the leptomycin B-induced nuclear IkappaBalpha, IL-8 mRNA expression and cellular release were not significantly affected. Analysis of in vivo recruitment of p65 NF-kappaB to NF-kappaB-regulated promoters by chromatin immunoprecipitation in U937 cells and human PBMCs indicated that although the p65 recruitment to TNF-alpha, IL-1beta, and IL-6 promoters was inhibited by the nuclear IkappaBalpha, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation analyses using IkappaBalpha and S536 phosphospecific p65 NF-kappaB Abs demonstrated that although the newly synthesized IkappaBalpha induced by postinduction repression is recruited to TNF-alpha, IL-1beta, and IL-6 promoters but not to the IL-8 promoter, S536-phosphorylated p65 is recruited to IL-8 promoter, but not to TNF-alpha, IL-1beta, or IL-6 promoters. Together, these data indicate that the inhibition of NF-kappaB-dependent transcription by nuclear IkappaBalpha in LPS-stimulated macrophages is gene specific and depends on the S536 phosphorylation status of the recruited p65 NF-kappaB.

Proteasome inhibitors induce apoptosis of prostate cancer cells by inducing nuclear translocation of IkappaBalpha.

Proteasome inhibitors are known to suppress the proteasome-mediated degradation of IkappaBalpha in stimulated cells. This results in the cytoplasmic retention of NFkappaB and its reduced nuclear transcriptional activity. In this study, we show that in the metastatic prostate cancer cells, the proteasome inhibitors exhibit a novel, previously unrecognized effect: they increase the cellular levels of IkappaBalpha, which then translocates to the nucleus, associates with the nuclear p65 NFkappaB, thus inhibiting the constitutive NFkappaB DNA binding activity and inducing apoptosis. The proteasome inhibition-induced nuclear translocation of IkappaBalpha is dependent on de novo protein synthesis, occurs also in other cell types, and does not require IkappaBalpha phosphorylation on Ser-32. Since NFkappaB activity is constitutively increased in many human cancers as well as in inflammatory disorders, the proteasome inhibition-induced nuclear translocation of IkappaBalpha could thus provide a new therapeutic strategy aimed at the specific inhibition of NFkappaB activity by the nuclear IkappaBalpha.

NFkappaB is persistently activated in continuously stimulated human neutrophils.

Increased activation of the transcription factor NFkappaB in the neutrophils has been associated with the pathogenesis of sepsis, acute lung injury (ALI), bronchopulmonary dysplasia (BPD), and other neutrophil-mediated inflammatory disorders. Despite recent progress in analyzing early NFkappaB activation in human neutrophils, activation of NFkappaB in persistently stimulated neutrophils has not been previously studied. Because it is the persistent NFkappaB activation that is thought to be involved in the host response to sepsis and the pathogenesis of ALI and BPD, we hypothesized that continuously stimulated human neutrophils may exhibit a late phase of NFkappaB activity. The goal of this study was to analyze the NFkappaB activation and expression of IkappaB and NFkappaB proteins during neutrophil stimulation with inflammatory signals for prolonged times. We demonstrate that neutrophil stimulation with lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFalpha) induces, in addition to the early activation at 30-60 min, a previously unrecognized late phase of NFkappaB activation. In LPS-stimulated neutrophils, this NFkappaB activity typically had a biphasic character, whereas TNFalpha-stimulated neutrophils exhibited a continuous NFkappaB activity peaking around 9 h after stimulation. In contrast to the early NFkappaB activation that inversely correlates to the nuclear levels of IkappaBalpha, however, in continuously stimulated neutrophils, NFkappaB is persistently activated despite considerable levels of IkappaBalpha present in the nucleus. Our data suggest that NFkappaB is persistently activated in human neutrophils during neutrophil-mediated inflammatory disorders, and this persistent NFkappaB activity may represent one of the underlying mechanisms for the continuous production of proinflammatory mediators.

TNFalpha release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export.

Tumor necrosis factor-alpha (TNFalpha) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNFalpha, and thus control of TNFalpha release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNFalpha release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNFalpha is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNFalpha from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNFalpha could thus provide a novel therapeutic approach for disorders involving excessive TNFalpha release.