Discovery of Covalent MKK4/7 Dual Inhibitor.

MKK4/7 are kinases that phosphorylate JNKs and regulate the MAPK signaling pathway. Their overexpression has been associated with tumorigenesis and aggressiveness in cancers such as breast, prostate, non-small cell lung, and pediatric leukemia, making them a potential target for inhibitor development. Here, we report the discovery, development, and validation of a dual MKK4/7 inhibitor, BSJ-04-122, that covalently targets a conserved cysteine located before the DFG motif and displays excellent kinome selectivity. BSJ-04-122 exhibits potent cellular target engagement and induces robust target-specific downstream effects. The combination of the dual MKK4/7 inhibitor with a selective, covalent JNK inhibitor demonstrated an enhanced antiproliferative activity against triple-negative breast cancer cells. Taken together, the results show that BSJ-04-122 represents a pharmacological probe for MKK4/7 and credential covalent targeting as a way to explore the therapeutic potential of these kinases.

Structural basis for producing selective MAP2K7 inhibitors.

Mitogen-activated protein kinase kinase 7 (MAP2K7) in the c-Jun N-terminal kinase signal cascade is an attractive drug target for a variety of diseases. The selectivity of MAP2K7 inhibitors against off-target kinases is a major barrier in drug development. We report a crystal structure of MAP2K7 complexed with a potent covalent inhibitor bearing an acrylamide moiety as an electrophile, which discloses a structural basis for producing selective and potent MAP2K7 inhibitors.

Catalytic Domain Plasticity of MKK7 Reveals Structural Mechanisms of Allosteric Activation and Diverse Targeting Opportunities.

MKK7 (MEK7) is a key regulator of the JNK stress signaling pathway and targeting MKK7 has been proposed as a chemotherapeutic strategy. Detailed understanding of the MKK7 structure and factors that affect its activity is therefore of critical importance. Here, we present a comprehensive set of MKK7 crystal structures revealing insights into catalytic domain plasticity and the role of the N-terminal regulatory helix, conserved in all MAP2Ks, mediating kinase activation. Crystal structures harboring this regulatory helix revealed typical structural features of active kinase, providing exclusively a first model of the MAP2K active state. A small-molecule screening campaign yielded multiple scaffolds, including type II irreversible inhibitors a binding mode that has not been reported previously. We also observed an unprecedented allosteric pocket located in the N-terminal lobe for the approved drug ibrutinib. Collectively, our structural and functional data expand and provide alternative targeting strategies for this important MAP2K kinase.

Down-Regulation of DDR1 Induces Apoptosis and Inhibits EMT through Phosphorylation of Pyk2/MKK7 in DU-145 and Lncap-FGC Prostate Cancer Cell Lines.

BACKGROUND: In cancer cells, re-activation of Epithelial-Mesenchymal Transition (EMT) program through Discoidin Domain Receptor1 (DDR1) leads to metastasis. DDR1-targeted therapy with siRNA might be a promising strategy for EMT inhibition. Therefore, the aim of this study was to investigate the effect of DDR1 knockdown in the EMT, migration, and apoptosis of prostate cancer cells. For this purpose, the expression of DDR1 was down regulated by the siRNA approach in LNcap-FGC and DU-145 prostate cancer cells. METHODS: Immunocytochemistry was carried out for the assessment of EMT. E-cadherin, N-cadherin, Bax, Bcl2, and the phosphorylation level of Proline-rich tyrosine kinase 2 (Pyk2) and Map Kinase Kinase 7 (MKK7) was determined using the western blot. Wound healing assay was used to evaluate cell migration. Flow cytometry was employed to determine the apoptosis rate in siRNA-transfected cancer cells. RESULTS: Our findings showed that the stimulation of DDR1 with collagen-I caused increased phosphorylation of Pyk2 and MKK7 signaling molecules that led to the induction of EMT and migration in DU-145 and LNcap- FGC cells. In contrast, DDR1 knockdown led to significant attenuation of EMT, migration, and phosphorylation levels of Pyk2 and MKK7. Moreover, DDR1 knockdown via induction of Bax expression and suppression of Bcl-2 expression induces apoptosis. CONCLUSION: Collectively, our results indicate that the DDR1 targeting with siRNA may be beneficial for the inhibition of EMT and the induction of apoptosis in prostate cancer.

Synthesis, anticancer activity, and molecular modeling of 1,4-naphthoquinones that inhibit MKK7 and Cdc25.

Cell division cycle 25 (Cdc25) and mitogen-activated protein kinase kinase 7 (MKK7) are enzymes involved in intracellular signaling but can also contribute to tumorigenesis. We synthesized and characterized the biological activity of 1,4-naphthoquinones structurally similar to reported Cdc25 and(or) MKK7 inhibitors with anticancer activity. Compound 7 (3-[(1,4-dioxonaphthalen-2-yl)sulfanyl]propanoic acid) exhibited high binding affinity for MKK7 (Kd = 230 nM), which was greater than the affinity of NSC 95397 (Kd = 1.1 µM). Although plumbagin had a lower binding affinity for MKK7, this compound and sulfur-containing derivatives 4 and 6-8 were potent inhibitors of Cdc25A and Cdc25B. Derivative 22e containing a phenylamino side chain was selective for MKK7 versus MKK4 and Cdc25 A/B, and its isomer 22f was a selective inhibitor of Cdc25 A/B. Docking studies performed on several naphthoquinones highlighted interesting aspects concerning the molecule orientation and hydrogen bonding interactions, which could help to explain the activity of the compounds toward MKK7 and Cdc25B. The most potent naphthoquinone-based inhibitors of MKK7 and/or Cdc25 A/B were also screened for their cytotoxicity against nine cancer cell lines and primary human mononuclear cells, and a correlation was found between Cdc25 A/B inhibitory activity and cytotoxicity of the compounds. Quantum chemical calculations using BP86 and ωB97X-D3 functionals were performed on 20 naphthoquinone derivatives to obtain a set of molecular electronic properties and to correlate these properties with cytotoxic activities. Systematic theoretical DFT calculations with subsequent correlation analysis indicated that energy of the lowest unoccupied molecular orbital E(LUMO), vertical electron affinity (VEA), and reactivity index ω of these molecules were important characteristics related to their cytotoxicity. The reactivity index ω was also a key characteristic related to Cdc25 A/B phosphatase inhibitory activity. Thus, 1,4-naphthoquinones displaying sulfur-containing and phenylamino side chains with additional polar groups could be successfully utilized for further development of efficacious Cdc25 A/B and MKK7 inhibitors with anticancer activity.

Characterization of Covalent Pyrazolopyrimidine-MKK7 Complexes and a Report on a Unique DFG-in/Leu-in Conformation of Mitogen-Activated Protein Kinase Kinase 7 (MKK7).

Pyrazolopyrimidines are well-established as covalent inhibitors of protein kinases such as the epidermal growth factor receptor or Bruton's tyrosine kinase, and we recently described their potential in targeting mitogen-activated protein kinase kinase 7 (MKK7). Herein, we report the structure-activity relationship of pyrazolopyrimidine-based MKK7 inhibitors and solved several complex crystal structures to gain insights into their binding mode. In addition, we present two structures of apo-MKK7, exhibiting a DFG-out and an unprecedented DFG-in/Leu-in conformation.

Targeting the MKK7-JNK (Mitogen-Activated Protein Kinase Kinase 7-c-Jun N-Terminal Kinase) Pathway with Covalent Inhibitors.

The protein kinase MKK7 is linked to neuronal development and the onset of cancer. The field, however, lacks high-quality functional probes that would allow for the dissection of its detailed functions. Against this background, we describe an effective covalent inhibitor of MKK7 based on the pyrazolopyrimidine scaffold.

Covalent Docking Identifies a Potent and Selective MKK7 Inhibitor.

The c-Jun NH2-terminal kinase (JNK) signaling pathway is central to the cell response to stress, inflammatory signals, and toxins. While selective inhibitors are known for JNKs and for various upstream MAP3Ks, no selective inhibitor is reported for MKK7--one of two direct MAP2Ks that activate JNK. Here, using covalent virtual screening, we identify selective MKK7 covalent inhibitors. We optimized these compounds to low-micromolar inhibitors of JNK phosphorylation in cells. The crystal structure of a lead compound bound to MKK7 demonstrated that the binding mode was correctly predicted by docking. We asserted the selectivity of our inhibitors on a proteomic level and against a panel of 76 kinases, and validated an on-target effect using knockout cell lines. Lastly, we show that the inhibitors block activation of primary mouse B cells by lipopolysaccharide. These MKK7 tool compounds will enable better investigation of JNK signaling and may serve as starting points for therapeutics.

Probing the interaction interface of the GADD45ß/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry.

GADD45ß is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45ß physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45ß/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45ß-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45ß and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45ß and MKK7 largely occurs between GADD45ß loop 2 (region 103-117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45ß/MKK7 interaction by contacting the MKK7 peptides, 113-136 and 259-274. Accordingly, an MKK7_KD Δ(101-136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45ß and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45ß/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.

The role of MAP2 kinases and p38 kinase in acute murine liver injury models.

c-Jun N-terminal kinase (JNK) mediates hepatotoxicity through interaction of its phospho-activated form with a mitochondrial outer membrane protein, Sh3bp5 or Sab, leading to dephosphorylation of intermembrane Src and consequent impaired mitochondrial respiration and enhanced ROS release. ROS production from mitochondria activates MAP3 kinases, such as MLK3 and ASK1, which continue to activate a pathway to sustain JNK activation, and amplifies the toxic effect of acetaminophen (APAP) and TNF/galactosamine (TNF/GalN). Downstream of MAP3K, in various contexts MKK4 activates both JNK and p38 kinases and MKK7 activates only JNK. The relative role of MKK4 versus 7 in liver injury is largely unexplored, as is the potential role of p38 kinase, which might be a key mediator of toxicity in addition to JNK. Antisense oligonucleotides (ASO) to MKK4, MKK7 and p38 (versus scrambled control) were used for in vivo knockdown, and in some experiments PMH were used after in vivo knockdown. Mice were treated with APAP or TNF/GalN and injury assessed. MKK4 and MKK7 were expressed in liver and each was efficiently knocked down with two different ASOs. Massive liver injury and ALT elevation were abrogated by MKK4 but not MKK7 ASO pretreatment in both injury models. The protection was confirmed in PMH. Knockdown of MKK4 completely inhibited basal P-p38 in both cytoplasm and mitochondria. However, ALT levels and histologic injury in APAP-treated mice were not altered with p38 knockdown versus scrambled control. p38 knockdown significantly increased P-JNK levels in cytoplasm but not mitochondria after APAP treatment. In conclusion, MKK4 is the major MAP2K, which activates JNK in acute liver injury. p38, the other downstream target of MKK4, does not contribute to liver injury from APAP or TNF/galactosamine.

Heme Oxygenase-1 Inhibits Neuronal Apoptosis in Spinal Cord Injury through Down-Regulation of Cdc42-MLK3-MKK7-JNK3 Axis.

The mechanism by which spinal cord injury (SCI) induces neuronal death has not been thoroughly understood. Investigation on the molecular signal pathways involved in SCI-mediated neuronal apoptosis is important for development of new therapeutics for SCI. In the current study, we explore the role of heme oxygenase-1 (HO-1) in the modulation of mixed lineage kinase 3/mitogen-activated protein kinase kinase/cJUN N-terminal kinase 3 (MLK3/MKK7/JNK3) signaling, which is a pro-apoptotic pathway, after SCI. We found that MLK3/MKK7/JNK3 signaling was activated by SCI in a time-dependent manner, demonstrated by increase in activating phosphorylation of MLK3, MKK7, and JNK3. SCI also induced HO-1 expression. Administration of HO-1-expressing adeno-associated virus before SCI introduced expression of exogenous HO-1 in injured spinal cords. Exogenous HO-1 reduced phosphorylation of MLK3, MKK7, and JNK3. Consistent with its inhibitory effect on MLK3/MKK7/JNK3 signaling, exogenous HO-1 decreased SCI-induced neuronal apoptosis and improved neurological score. Further, we found that exogenous HO-1 inhibited expression of cell division cycle 42 (Cdc42), which is crucial for MLK3 activation. In vitro experiments indicated that Cdc42 was essential for neuronal apoptosis, while transduction of neurons with HO-1-expressing adeno-associated virus significantly reduced neuronal apoptosis to enhance neuronal survival. Therefore, our study disclosed a novel mechanism by which HO-1 exerted its neuroprotective efficacy. Our discovery might be valuable for developing a new therapeutic approach for SCI.

Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury.

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45ß (GADD45ß-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK's pathological activation. GADD45ß-I was engineered by optimizing the domain of the GADD45ß, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45ß-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen-glucose deprivation in vitro. Moreover, GADD45ß-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45ß-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45ß-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.

Protection of rat intestinal epithelial cells from ischemia/reperfusion injury by (D-Ala2, D-Leu5)-enkephalin through inhibition of the MKK7-JNK signaling pathway.

Previous studies have demonstrated that (D­Ala2, D­Leu5)­enkephalin (DADLE) protects rats from hepatic ischemia/reperfusion (I/R) injury. In the present study, DADLE was also observed to alleviate IR­induced intestinal epithelial cell injury in rats by inhibiting mitogen­activated protein kinase kinase 7 (MKK7)­c­Jun N­terminal kinase (JNK) pathway signaling. To investigate the protective effect of DADLE on hypoxia/reoxygenation injury in rat intestinal epithelial cells, rat intestinal epithelial cells were treated with different concentrations of DADLE, following which the cell survival rate was determined using a tetrazolium (MTT) colorimetric assay, and apoptosis was determined using flow cytometry. To confirm whether the protective effect of DADLE was due to its effect on MKK7­JNK signaling, the phosphorylation levels of MKK7 and JNK were analyzed using western blot analysis following treatment with different concentrations of DADLE. The results demonstrated that, following treatment with DADLE, the survival rate of the rat intestinal cells subjected to I/R­induced injury increased significantly and the apoptotic rate decreased in a concentration­dependent manner. In addition, the levels of phosphorylated MKK7 and JNK decreased in a concentration­dependent manner following treatment with DADLE. Silencing the gene expression of MKK7 using small interfering RNA prior to DADLE treatment resulted in a reduction in the protective effects of DADLE on the rat intestinal epithelial cells subjected to I/R injury. Collectively, the results of the present study demonstrated that the protective effects of DADLE in I/R injury in rat intestinal cells occurred through inhibition of the MKK7­JNK pathway.

Reduced parasite motility and micronemal protein secretion by a p38 MAPK inhibitor leads to a severe impairment of cell invasion by the apicomplexan parasite Eimeria tenella.

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.

Cancer-selective targeting of the NF-κB survival pathway with GADD45ß/MKK7 inhibitors.

Constitutive NF-κB signaling promotes survival in multiple myeloma (MM) and other cancers; however, current NF-κB-targeting strategies lack cancer cell specificity. Here, we identify the interaction between the NF-κB-regulated antiapoptotic factor GADD45ß and the JNK kinase MKK7 as a therapeutic target in MM. Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45ß/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells. DTP3 has similar anticancer potency to the clinical standard, bortezomib, but more than 100-fold higher cancer cell specificity in vitro. Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses. Hence, cancer-selective targeting of the NF-κB pathway is possible and, at least for myeloma patients, promises a profound benefit.

A Protoberberine derivative HWY336 selectively inhibits MKK4 and MKK7 in mammalian cells: the importance of activation loop on selectivity.

A protoberberine derivative library was used to search for selective inhibitors against kinases of the mitogen-activated protein kinase (MAPK) cascades in mammalian cells. Among kinases in mammalian MAPK pathways, we identified a compound (HWY336) that selectively inhibits kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7). The IC50 of HWY336 was 6 µM for MKK4 and 10 µM for MKK7 in vitro. HWY336 bound to both kinases reversibly via noncovalent interactions, and inhibited their activity by interfering with access of a protein substrate to its binding site. The binding affinity of HWY336 to MKK4 was measured by surface plasmon resonance to determine a dissociation constant (Kd) of 3.2 µM. When mammalian cells were treated with HWY336, MKK4 and MKK7 were selectively inhibited, resulting in inhibition of c-Jun NH2-terminal protein kinases in vivo. The structural model of HWY336 bound to either MKK4 or MKK7 predicted that HWY336 was docked to the activation loop, which is adjacent to the substrate binding site. This model suggested the importance of the activation loop of MKKs in HWY336 selectivity. We verified this model by mutating three critical residues within this loop of MKK4 to the corresponding residues in MKK3. The mutant MKK4 displayed similar kinase activity as wild-type kinase, but its activity was not inhibited by HWY336 compared to wild-type MKK4. We propose that the specific association of HWY336 to the activation loop of MKK4/MKK7 is responsible for its selective inhibition.

Isoangustone A, a novel licorice compound, inhibits cell proliferation by targeting PI3K, MKK4, and MKK7 in human melanoma.

Licorice root is known to possess various bioactivities, including anti-inflammatory and anticancer effects. Glycyrrhizin, a triterpene compound, is the most abundant constituent of dried licorice root. However, high intake or long-term consumption of glycyrrhizin causes several side effects, such as hypertension, hypertensive encephalopathy, and hypokalemia. Therefore, finding additional active compounds other than glycyrrhizin in licorice that exhibit anticancer effects is worthwhile. We found that isoangustone A (IAA), a novel flavonoid from licorice root, suppressed proliferation of human melanoma cells. IAA significantly blocked cell-cycle progression at the G1-phase and inhibited the expression of G1-phase regulatory proteins, including cyclins D1 and E in the SK-MEL-28 human melanoma cell line. IAA suppressed the phosphorylation of Akt, GSK-3ß, and JNK1/2. IAA also bound to phosphoinositide 3-kinase (PI3K), MKK4, and MKK7, strongly inhibiting their kinase activities in an ATP-competitive manner. Moreover, in a xenograft mouse model, IAA significantly decreased tumor growth, volume, and weight of SK-MEL-28 xenografts. Collectively, these results suggest that PI3K, MKK4, and MKK7 are the primary molecular targets of IAA in the suppression of cell proliferation. This insight into the biologic actions of IAA provides a molecular basis for the potential development of a new chemotherapeutic agent.

Manipulating JNK signaling with (--)-zuonin A.

Recently, in a virtual screening strategy to identify new compounds targeting the D-recruitment site (DRS) of the c-Jun N-terminal kinases (JNKs), we identified the natural product (-)-zuonin A. Here we report the asymmetric synthesis of (-)-zuonin A and its enantiomer (+)-zuonin A. A kinetic analysis for the inhibition of c-Jun phosphorylation by (-)-zuonin A revealed a mechanism of partial competitive inhibition. Its binding is proposed to weaken the interaction of c-Jun to JNK by approximately 5-fold, without affecting the efficiency of phosphorylation within the complex. (-)-Zuonin A inhibits the ability of both MKK4 and MKK7 to phosphorylate and activate JNK. The binding site of (-)-zuonin A is predicted by docking and molecular dynamics simulation to be located in the DRS of JNK. (+)-Zuonin A also binds JNK but barely impedes the binding of c-Jun. (-)-Zuonin A inhibits the activation of JNK, as well as the phosphorylation of c-Jun in anisomycin-treated HEK293 cells, with the inhibition of JNK activation being more pronounced. (-)-Zuonin A also inhibits events associated with constitutive JNK2 activity, including c-Jun phosphorylation, basal Akt activation, and MDA-MB-231 cell migration. Mutations in the predicted binding site for (-)-zuonin A can render it significantly more or less sensitive to inhibition than wild type JNK2, allowing for the design of potential chemical genetic experiments. These studies suggest that the biological activity reported for other lignans, such as saucerneol F and zuonin B, may be the result of their ability to impede protein-protein interactions within MAPK cascades.

Alpinetin suppresses proliferation of human hepatoma cells by the activation of MKK7 and elevates sensitization to cis-diammined dichloridoplatium.

Alpinetin is a type of novel plant flavonoid derived from Alpinia katsumadai Hayata, found to possess strong anti-hepatoma effects. However, the detailed antitumor mechanism of alpinetin remains unclear. Mitogen-activated protein kinase kinase-7 (MKK7) can regulate cellular growth, differentiation and apoptosis. The aim of this study was to investigate the role of MKK7 in the anti-hepatoma effect mediated by alpinetin. HepG2 cells were treated with alpinetin at various doses and for different times, and the levels of phosphorylated MKK7 (p-MKK7) and total MKK7 were tested by RT-PCR and western blotting. Following transient transfection with RNA interference, cell viability and cell cycle stage were determined using methyl thiazolyl tetrazolium assay and flow cytometry, in order to assess the antitumor action of alpinetin. In addition, chemosensitization to cis-diammined dichloridoplatium (CDDP) by alpinetin was assessed by cell counting array and the cell growth inhibitory rate was calculated. The results showed that alpinetin suppressed HepG2 cell proliferation and arrested cells in the G0/G1 phase by up-regulating the expression levels of p-MKK7. On the contrary, inhibiting the expression of MKK7 reversed the antitumor effect of alpinetin. Moreover, alpinetin enhanced the sensitivity of HepG2 hepatoma cells to the chemotherapeutic agent CDDP. Taken together, our studies indicate that activation of MKK7 mediates the anti-hepatoma effect of alpinetin. MKK7 may be a putative target for molecular therapy against hepatoma and alpinetin could serve as a potential agent for the development of hepatoma therapy.

Constitutive NF-kappaB activation confers interleukin 6 (IL6) independence and resistance to dexamethasone and Janus kinase inhibitor INCB018424 in murine plasmacytoma cells.

Myeloma cells are dependent on IL6 for their survival and proliferation during the early stages of disease, and independence from IL6 is associated with disease progression. The role of the NF-κB pathway in the IL6-independent growth of myeloma cells has not been studied. Because human herpesvirus 8-encoded K13 selectively activates the NF-κB pathway, we have used it as a molecular tool to examine the ability of the NF-κB pathway to confer IL6 independence on murine plasmacytomas. We demonstrated that ectopic expression of K13, but not its NF-κB-defective mutant or a structural homolog, protected plasmacytomas against IL6 withdrawal-induced apoptosis and resulted in emergence of IL6-independent clones that could proliferate long-term in vitro in the absence of IL6 and form abdominal plasmacytomas with visceral involvement when injected intraperitoneally into syngeneic mice. These IL6-independent clones were dependent on NF-κB activity for their survival and proliferation but were resistant to dexamethasone and INCB018424, a selective Janus kinase 1/2 inhibitor. Ectopic expression of human T cell leukemia virus 1-encoded Tax protein, which resembles K13 in inducing constitutive NF-κB activation, similarly protected plasmacytoma cells against IL6 withdrawal-induced apoptosis. Although K13 is known to up-regulate IL6 gene expression, its protective effect was not due to induction of endogenous IL6 production but instead was associated with sustained expression of several antiapoptotic members of the Bcl2 family upon IL6 withdrawal. Collectively, these results demonstrate that NF-κB activation cannot only promote the emergence of IL6 independence during myeloma progression but can also confer resistance to dexamethasone and INCB018424.