GADD45ß stabilized by direct interaction with HSP72 ameliorates insulin resistance and lipid accumulation.

Growth arrest and DNA damage-inducible 45ß (GADD45ß) belongs to the GADD45 family which is small acidic proteins in response to cellular stress. GADD45ß has already been reported to have excellent capabilities against cancer, innate immunity and neurological diseases. However, there is little information regard GADD45ß and non-alcoholic fatty liver disease (NAFLD). In the current work, we found that the expression of GADD45ß was markedly decreased in the livers of NAFLD patients via analyzing Gene Expression Omnibus (GEO) dataset and in mouse model through detecting its mRNA in high-fat-high-fructose diet (HFHFr)-fed mice. Moreover, the results from in vivo experiment demonstrated that overexpression of GADD45ß by AAV8-mediated gene transfer in HFHFr-fed mouse model could reduce the level of serum and hepatic triglyceride (TG), and alleviate insulin resistance. Subsequently, by combining immunoprecipitation (IP) and mass spectrometry, we identified that HSP72 directly interacted with GADD45ß to prevent GADD45ß from being degraded by the proteasome pathway. Finally, the benefits of GADD45ß in regulating key factors of TG synthesis and insulin signaling pathway were abolished after HSP72 knockdown. In conclusion, GADD45ß stabilized by the interaction with HSP72 could alleviate the NAFLD-related pathologies, suggested it might be a potential target for the treatment of NAFLD.

Host relieves lnc-IRAK3-3-sequestered miR-891b to attenuate apoptosis in Enterovirus 71 infection.

Enterovirus 71 (EV71) is an emerging life-threatening pathogen particularly in the Asia-Pacific region. Apoptosis is a major pathogenic feature in EV71 infection. However, which molecular mechanism participating in EV71-induced apoptosis is not completely understood. Long noncoding RNAs (lncRNAs), a newly discovered class of regulatory RNA molecules, govern a wide range of biological functions through multiple regulatory mechanisms. Whether lncRNAs involved in EV71-induced apoptosis was investigated in this study. We conducted an apoptosis-oriented approach by integrating lncRNA and mRNA profilings. lnc-IRAK3-3 is down-regulated in EV71 infection and plays an important role in EV71 infection-induced apoptosis. Compensation of lnc-IRAK3-3 in EV71 infection promoted cell apoptosis wherein GADD45ß expression was increased and further triggered caspase3 and PARP cleavage. Using bioinformatics analysis and functional assays, lnc-IRAK3-3 could functionally sequester miR-891b and GADD45ß 3'UTR whereas miR-891b showed the inhibitory activity on GADD45ß expression. Taken together, lnc-IRAK3-3 has the ability capturing miR-891b to enforce GADD45ß expression and eventually promotes apoptosis. On the contrary, host cells suppress lnc-IRAK3-3 to relieve lnc-IRAK3-3-sequestered miR-891b, restrain GADD45ß, and attenuate apoptosis in EV71 infection that prevent host cells from severe damages. We discover a new molecular mechanism by which host cells counteract EV71-induced apoptosis through the lnc-IRAK3-3/miR-891b/GADD45ß axis partially.

The effects of cucurbitacin E on GADD45ß-trigger G2/M arrest and JNK-independent pathway in brain cancer cells.

Cucurbitacin E (CuE), an active compound of the cucurbitacin family, possesses a variety of pharmacological functions and chemotherapy potential. Cucurbitacin E exhibits inhibitory effects in several types of cancer; however, its anticancer effects on brain cancer remain obscure and require further interpretation. In this study, efforts were initiated to inspect whether CuE can contribute to anti-proliferation in human brain malignant glioma GBM 8401 cells and glioblastoma-astrocytoma U-87-MG cells. An MTT assay measured CuE's inhibitory effect on the growth of glioblastomas (GBMs). A flow cytometry approach was used for the assessment of DNA content and cell cycle analysis. DNA damage 45ß (GADD45ß) gene expression and CDC2/cyclin-B1 disassociation were investigated by quantitative real-time PCR and Western blot analysis. Based on our results, CuE showed growth-inhibiting effects on GBM 8401 and U-87-MG cells. Moreover, GADD45ß caused the accumulation of CuE-treated G2/M-phase cells. The disassociation of the CDC2/cyclin-B1 complex demonstrated the known effects of CuE against GBM 8401 and U-87-MG cancer cells. Additionally, CuE may also exert antitumour activities in established brain cancer cells. In conclusion, CuE inhibited cell proliferation and induced mitosis delay in cancer cells, suggesting its potential applicability as an antitumour agent.

GADD45ß plays a protective role in acute lung injury by regulating apoptosis in experimental sepsis in vivo.

Sepsis is a systemic inflammatory response syndrome due to microbial infection. Growth arrest and DNA-damage-inducible 45 beta (GADD45ß) are induced by genotoxic stress and inflammatory cytokines. However, the role of GADD45ß during bacterial infection remains unclear. This study was aimed at investigating the role of GADD45ß in sepsis. We used GADD45ß-knockout (KO) mice and C57BL/6J wild-type (WT) mice. Experimental sepsis was induced by lipopolysaccharide (LPS) administration or cecal ligation and puncture (CLP). Sepsis-induced mortality was higher in GADD45ß-KO mice than in WT mice. Histopathological data demonstrated LPS treatment markedly increased lung injury in GADD45ß-KO mice as compared to that in WT mice; however, no significant difference was observed in the liver and kidney. Further, mRNA levels of inflammatory cytokines, such as Il-1ß, Il-6, Il-10, and Tnf-α, were higher in the lungs of LPS-treated GADD45ß-KO mice than in WT mice. Interestingly, plasma levels of these inflammatory cytokines were decreased in LPS-administered GADD45ß-KO mice. A significant increase in lung cell apoptosis was observed at early time points in GADD45ß-KO mice after administration of LPS as compared to that in WT mice. In line with LPS-induced apoptosis, JNK, and p38 activity was higher in the lung of GADD45ß-KO mice at 3 hr after LPS treatment than that in WT mice. In summary, this study is the first to demonstrate the protective role of GADD45ß in sepsis and the results suggest that GADD45ß could be used as a novel therapeutic target to cure sepsis.

Brazilin induces T24 cell death through c-Fos and GADD45ß independently regulated genes and pathways.

Purified Brazilin from Sappan wood extract has been reported with significant antitumor effect, especially on human T24 cells and bladder cancer mouse models. Brazilin can significantly induce expression of c-Fos and GADD45ß and transfection expression of c-Fos and GADD45ß in T24 cells can induce significant cell morphology changes, reduced viability and cell death, while transfection of siRNA-c-Fos and siRNA-GADD45ß can reverse the induced cell death. Co-transfection of both c-Fos and GADD45ß into T24 cells resulted in a significantly additive effect when compared to single transfection with only c-Fos or GADD45ß. Meanwhile, transfection of interfering siRNA-c-Fos or siRNA-GADD45ß can partially rescue the cell viability and siRNA co-transfection showed increased rescue rate. The transfection expression and interference with pcDNA3.1-c-Fos/siRNA-c-Fos or pcDNA3.1-GADD45ß/siRNA-GADD45ß did not affect each other's expression. Moreover, analysis of c-Fos and GADD45ß regulated genes and signal pathways showed that no common regulated genes or pathways were present. All the results indicated that c-Fos and GADD45ß mediate independent Brazilin-inducible genes and pathways. © 2018 IUBMB Life, 70(11):1101-1110, 2018.

Hepatocyte SHP deficiency protects mice from acetaminophen-evoked liver injury in a JNK-signaling regulation and GADD45ß-dependent manner.

Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure. Prolonged c-Jun N-terminal kinase (JNK) activation plays a central role in APAP-induced liver injury; however, growth arrest and DNA damage-inducible 45 beta (GADD45ß) is known to inhibit JNK phosphorylation. The orphan nuclear receptor small heterodimer partner (SHP, NR0B2) acts as a transcriptional co-repressor of various genes. The aim of the present study was to investigate the role of SHP in APAP-evoked hepatotoxicity. We used lethal (750 mg/kg) or sublethal (300 mg/kg) doses of APAP-treated wild-type (WT), Shp knockout (Shp-/-), hepatocyte-specific Shp knockout (Shphep-/-), and Shp and Gadd45ß double knockout (Shp-/-Gadd45ß-/-) mice for in vivo studies. Primary mouse hepatocytes were used for a comparative in vitro study. SHP deficiency protected against APAP toxicity with an increased survival rate, decreased liver damage, and inhibition of prolonged hepatic JNK phosphorylation in mice, which was independent of APAP metabolism regulation. Furthermore, Shphep-/- mice showed diminished APAP hepatotoxicity compared with WT mice. SHP-deficient primary mouse hepatocytes also showed decreased cell death and inhibition of sustained JNK phosphorylation following toxic APAP treatment. While SHP expression declined, GADD45ß expression increased after APAP treatment in WT mice. In Shp-/- mice, APAP-evoked GADD45ß induction was significantly enhanced. Notably, the ameliorative effects of SHP deficiency on APAP-induced liver injury were abolished in Shp-/-Gadd45ß-/- mice. The current study is the first to demonstrate that hepatocyte-specific SHP deficiency protects against APAP overdose-evoked hepatotoxicity in a JNK signaling regulation and GADD45ß dependent manner. SHP is suggested to be a novel therapeutic target for APAP overdose treatment.

Gadd45ß ameliorates L-DOPA-induced dyskinesia in a Parkinson's disease mouse model.

The dopamine precursor 3,4-dihydroxyphenyl-l-alanine (L-DOPA) is currently the most efficacious pharmacotherapy for Parkinson's disease (PD). However, long-term L-DOPA treatment leads to the development of abnormal involuntary movements (AIMs) in patients and animal models of PD. Recently, involvement of growth arrest and DNA damage-inducible 45ß (Gadd45ß) was reported in neurological and neurobehavioral dysfunctions. However, little is known about the role of Gadd45ß in the dopaminergic nigrostriatal pathway or L-DOPA-induced dyskinesia (LID). To address this issue, we prepared an animal model of PD using unilateral 6-hydroxydopamine (6-OHDA) lesions in the substantia nigra of Gadd45ß(+/+) and Gadd45ß(-/-) mice. Dyskinetic symptoms were triggered by repetitive administration of L-DOPA in these 6-OHDA-lesioned mice. Whereas dopamine denervation in the dorsal striatum decreased Gadd45ß mRNA, chronic L-DOPA treatment significantly increased Gadd45ß mRNA expression in the 6-OHDA-lesioned striatum of wild-type mice. Using unilaterally 6-OHDA-lesioned Gadd45ß(+/+) and Gadd45ß(-/-) mice, we found that mice lacking Gadd45ß exhibited long-lasting increases in AIMs following repeated administration of L-DOPA. By contrast, adeno-associated virus-mediated expression of Gadd45ß in the striatum reduced AIMs in Gadd45ß knockout mice. The deficiency of Gadd45ß in LID increased expression of ΔFosB and c-Fos in the lesioned striatum 90 min after the last administration of L-DOPA following 11days of daily L-DOPA treatments. These data suggest that the increased expression of Gadd45ß induced by repeated administration of L-DOPA may be beneficial in patients with PD.

Metformin ameliorates acetaminophen hepatotoxicity via Gadd45ß-dependent regulation of JNK signaling in mice.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure. Prolonged c-Jun N-terminal kinase (JNK) activation plays a central role in APAP-induced liver injury and growth arrest, and DNA damage-inducible 45 beta (Gadd45ß) is known to inhibit JNK phosphorylation. Metformin has recently been shown to have hepatoprotective effects. The aim of the present study is to investigate whether metformin mitigates APAP-induced hepatotoxicity and to ascertain the molecular basis of this effect. METHODS: We used APAP- and/or metformin-treated Gadd45ß knockout (KO) mice and wild type (WT) C57BL/6J control mice. Primary mouse hepatocytes were isolated from WT and Gadd45ß KO mice were used for in vitro study. RESULTS: Metformin pretreatment protected against APAP toxicity with decreased liver damage, and inhibited APAP-induced prolonged hepatic JNK phosphorylation in WT mice. Gadd45ß expression was increased after APAP treatment, and the expression of Gadd45ß was further enhanced by metformin. The effects of metformin on APAP-induced liver injury and JNK phosphorylation were abolished in Gadd45ß KO mice. Notably, subtoxic doses of APAP caused cell death and sustained JNK phosphorylation in Gadd45ß-deficient primary hepatocytes. In parallel, APAP increased mortality, severe liver injury, and JNK activation in Gadd45ß KO mice. Interestingly, metformin administered after APAP treatment protected against APAP-evoked hepatotoxicity in WT mice, but not in Gadd45ß KO mice. CONCLUSIONS: This study is the first to demonstrate that metformin shows protective and therapeutic effects against APAP overdose-evoked hepatotoxicity via Gadd45ß-dependent JNK regulation. Metformin would be a promising therapeutic strategy for treatment of APAP overdose.

Downregulation of Gadd45ß alleviates osteoarthritis by repressing lipopolysaccharide-induced fibroblast-like synoviocyte inflammation, proliferation and migration.

OBJECTIVE: Gadd45ß have a regulatory role in cellular inflammation, proliferation and migration. However, the role of Gadd45ß in synovial inflammation in osteoarthritis (OA) remains to be explored. This study aimed to ascertain whether Gadd45ß is involved in OA synovial inflammation. METHODS: The rat model was induced by sodium iodoacetate and the cellular model was constructed with lipopolysaccharide (LPS)-induced fibroblast-like synoviocytes (FLSs). siRNA was applied to interfere with the expression of intracellular Gadd45ß. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression of Gadd45ß mRNA and protein. The inflammation, proliferation, and migration of OA-FLSs were detected by enzyme-linked immunosorbent assay, cell scratch assay, 5-ethynyl-2'-deoxyuridine assay, etc. The effect of downregulation of Gadd45ß on the nuclear factor-κB (NF-κB) pathway was investigated. RESULTS: Expression of Gadd45ß in OA rat synovial tissues and OA-FLSs was increased, and LPS treatment promoted cell proliferation and enhanced cell migration. Gadd45ß interference inhibited the inflammation, proliferation and migration of cells induced by LPS. LPS promoted P65 expression in the nucleus and activated the NF-κB signaling pathway, whereas si-Gadd45ß reversed this situation. CONCLUSIONS: si-Gadd45ß inhibited the inflammatory response, proliferation and migration of FLSs, and activation of the NF-κB signaling pathway, which could delay the progression of OA. Hence, it may become a potential therapeutic target for OA.

Enriched environment treatment promotes neurofunctional recovery by regulating the ALK5/Smad2/3/Gadd45ß signaling pathway in rats with cerebral ischemia /reperfusion injury.

It has been demonstrated that an enriched environment (EE) treatment can alter neuroplasticity in neurodegenerative diseases. However, the role of EE treatment in ischemic stroke remains unclear. Previous findings have revealed that EE treatment can promote cerebral activin-receptor-like-kinase-5 (ALK5) expression after cerebral ischemia/reperfusion (I/R) injury. ALK5 has been identified as a potential mediator of neuroplasticity through its modulation of Smad2/3 and Gadd45ß. Therefore, the aim of this study was to investigate whether EE treatment could promote neurofunctional recovery by regulating the ALK5/Smad2/3/Gadd45ß pathway. The study utilized the rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). The ALK5/Smad2/3/Gadd45ß signaling pathway changes were evaluated using western blotting (WB). Brain injury was assessed by infarct volume and neurobehavioral scores. The effect of EE treatment on neurogenesis was evaluated using Doublecortin (DCX) and Nestin, axonal plasticity with biotinylated dextran amine (BDA) nerve tracing, and dendritic plasticity was assessed using Golgi-Cox staining. EE treatment has been demonstrated to modulate the Smad2/3/Gadd45ß pathway by regulating the expression of ALK5. The protective effects of EE treatment on brain infarct volume, neurological function, newborn neurons, dendritic and axonal plasticity following cerebral I/R injury were counteracted by ALK5 silencing. EE treatment can enhance neurofunctional recovery after cerebral I/R injury, which is achieved by regulating the ALK5/Smad2/3/Gadd45ß signaling pathway to promote neuroplasticity.

Gadd45ß is critical for regulation of type I interferon signaling by facilitating G3BP-mediated stress granule formation.

Stress granules (SGs) constitute a signaling hub that plays a critical role in type I interferon responses. Here, we report that growth arrest and DNA damage-inducible beta (Gadd45ß) act as a positive regulator of SG-mediated interferon signaling by targeting G3BP upon RNA virus infection. Gadd45ß deficiency markedly impairs SG formation and SG-mediated activation of interferon signaling in vitro. Gadd45ß knockout mice are highly susceptible to RNA virus infection, and their ability to produce interferon and cytokines is severely impaired. Specifically, Gadd45ß interacts with the RNA-binding domain of G3BP, leading to conformational expansion of G3BP1 via dissolution of its autoinhibitory electrostatic intramolecular interaction. The acidic loop 1- and RNA-binding properties of Gadd45ß markedly increase the conformational expansion and RNA-binding affinity of the G3BP1-Gadd45ß complex, thereby promoting assembly of SGs. These findings suggest a role for Gadd45ß as a component and critical regulator of G3BP1-mediated SG formation, which facilitates RLR-mediated interferon signaling.

GADD45ß Regulates Hepatic Gluconeogenesis via Modulating the Protein Stability of FoxO1.

Increased hepatic gluconeogenesis is one of the main contributors to the development of type 2 diabetes. Recently, it has been reported that growth arrest and DNA damage-inducible 45 beta (GADD45ß) is induced under both fasting and high-fat diet (HFD) conditions that stimulate hepatic gluconeogenesis. Here, this study aimed to establish the molecular mechanisms underlying the novel role of GADD45ß in hepatic gluconeogenesis. Both whole-body knockout (KO) mice and adenovirus-mediated knockdown (KD) mice of GADD45ß exhibited decreased hepatic gluconeogenic gene expression concomitant with reduced blood glucose levels under fasting and HFD conditions, but showed a more pronounced effect in GADD45ß KD mice. Further, in primary hepatocytes, GADD45ß KD reduced glucose output, whereas GADD45ß overexpression increased it. Mechanistically, GADD45ß did not affect Akt-mediated forkhead box protein O1 (FoxO1) phosphorylation and forskolin-induced cAMP response element-binding protein (CREB) phosphorylation. Rather it increased FoxO1 transcriptional activity via enhanced protein stability of FoxO1. Further, GADD45ß colocalized and physically interacted with FoxO1. Additionally, GADD45ß deficiency potentiated insulin-mediated suppression of hepatic gluconeogenic genes, and it were impeded by the restoration of GADD45ß expression. Our finding demonstrates GADD45ß as a novel and essential regulator of hepatic gluconeogenesis. It will provide a deeper understanding of the FoxO1-mediated gluconeogenesis.

The Screening of Combinatorial Peptide Libraries for Targeting Key Molecules or Protein-Protein Interactions in the NF-κB Pathway.

Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of cancer and metabolic and cardiovascular diseases, which are all areas of high interest to the pharmaceutical industry. The global market for peptide therapeutics was valued at about 25 billion USD in 2018 and is estimated to reach 57.2 billion USD by the end of 2027. Here, we describe a method for the screening and deconvolution of combinatorial peptide libraries to discover compounds that target discrete signaling components of the NF-κB pathway. Recently, we used this approach to specifically disrupt the interaction between the JNK-activating kinase, MKK7, and the NF-κB-regulated antiapoptotic factor, GADD45ß, in multiple myeloma (MM). We showed that the GADD45ß/MKK7 complex is a functionally critical survival module downstream of NF-κB in MM cells and as such provides an attractive therapeutic target to selectively inhibit NF-κB antiapoptotic signaling in cancer cells. By integrating the library screening and deconvolution methods described here with a rational chemical optimization strategy, we developed the first-in-class GADD45ß/MKK7 inhibitor, DTP3 (a D-tripeptide), which is now being trialed in MM and diffuse large B-cell lymphoma (DLBCL) patients. The same drug discovery approach may be generally applied to therapeutically target other key components of the NF-κB pathway in cancers beyond MM and DLBCL, as well as in non-malignant NF-κB-driven diseases.

Insights into the Interaction Mechanism of DTP3 with MKK7 by Using STD-NMR and Computational Approaches.

GADD45ß/MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45ß/MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect; however, its mode of action is only partially understood. In this work, we have investigated the molecular determinants underlying the MKK7 interaction with DTP3 by combining computational, NMR, and spectroscopic methods. Data gathered by fluorescence quenching and computational approaches consistently indicate that the N-terminal region of MKK7 is the optimal binding site explored by DTP3. These findings further the understanding of the selective mode of action of GADD45ß/MKK7 inhibitors and inform potential mechanisms of drug resistance. Notably, upon validation of the safety and efficacy of DTP3 in human trials, our results could also facilitate the development of novel DTP3-like therapeutics with improved bioavailability or the capacity to bypass drug resistance.

Preclinical toxicology and safety pharmacology of the first-in-class GADD45ß/MKK7 inhibitor and clinical candidate, DTP3.

Aberrant NF-κB activity drives oncogenesis and cell survival in multiple myeloma (MM) and many other cancers. However, despite an aggressive effort by the pharmaceutical industry over the past 30 years, no specific IκBα kinase (IKK)ß/NF-κB inhibitor has been clinically approved, due to the multiple dose-limiting toxicities of conventional NF-κB-targeting drugs. To overcome this barrier to therapeutic NF-κB inhibition, we developed the first-in-class growth arrest and DNA-damage-inducible (GADD45)ß/mitogen-activated protein kinase kinase (MKK)7 inhibitor, DTP3, which targets an essential, cancer-selective cell-survival module downstream of the NF-κB pathway. As a result, DTP3 specifically kills MM cells, ex vivo and in vivo, ablating MM xenografts in mice, with no apparent adverse effects, nor evident toxicity to healthy cells. Here, we report the results from the preclinical regulatory pharmacodynamic (PD), safety pharmacology, pharmacokinetic (PK), and toxicology programmes of DTP3, leading to the approval for clinical trials in oncology. These results demonstrate that DTP3 combines on-target-selective pharmacology, therapeutic anticancer efficacy, favourable drug-like properties, long plasma half-life and good bioavailability, with no target-organs of toxicity and no adverse effects preclusive of its clinical development in oncology, upon daily repeat-dose administration in both rodent and non-rodent species. Our study underscores the clinical potential of DTP3 as a conceptually novel candidate therapeutic selectively blocking NF-κB survival signalling in MM and potentially other NF-κB-driven cancers.

Gadd45ß silencing impaired viability and metastatic phenotypes in cholangiocarcinoma cells by modulating the EMT pathway.

Growth arrest and DNA damage-inducible-ß (Gadd45ß) is a stress-response protein involved in a number of processes, including cell cycle control, DNA repair, survival and death control, and stress signaling, depending on its interactions. Gadd45ß expression is dysregulated in numerous types of cancer, functioning as either a tumor promoter or a tumor suppressor. However, the functions of Gadd45ß in cholangiocarcinoma (CCA), particularly in metastasis, has not been studied. The immunohistochemical analysis of Gadd45ß expression revealed that 75% of histological specimens from patients with CCA expressed high levels of Gadd45ß, and that high Gadd45ß expression was associated with metastasis. The role of Gadd45ß in CCA was examined using siRNA-mediated gene knockdown in HuCCA-1, a human CCA cell line established from a Thai patient. The effects of Gadd45ß downregulation upon cell viability and death, invasion, migration, matrix metalloproteinase (MMP) activity and epithelial-mesenchymal transition (EMT) marker expression were investigated. Gadd45ß knockdown impaired cell viability, which was associated with the induction of apoptosis. In addition, there was a marked reduction in invasion and migration, although MMP activity was unaffected. Impairment of these metastatic properties was accompanied by the decreased expression of EMT markers, including Slug, vimentin, claudin-1 and zona occludens protein 1, whereas E-cadherin expression was increased. The present study suggests that Gadd45ß is involved in regulating the viability and the metastatic potential of CCA cells, which may be mediated by the modulation of the EMT pathway.

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.

NF-κB in the crosshairs: Rethinking an old riddle.

Constitutive NF-κB signalling has been implicated in the pathogenesis of most human malignancies and virtually all non-malignant pathologies. Accordingly, the NF-κB pathway has been aggressively pursued as an attractive therapeutic target for drug discovery. However, the severe on-target toxicities associated with systemic NF-κB inhibition have thus far precluded the development of a clinically useful, NF-κB-targeting medicine as a way to treat patients with either oncological or non-oncological diseases. This minireview discusses some of the more promising approaches currently being developed to circumvent the preclusive safety liabilities of global NF-κB blockade by selectively targeting pathogenic NF-κB signalling in cancer, while preserving the multiple physiological functions of NF-κB in host defence responses and tissue homeostasis.

Growth arrest and DNA-damage-inducible 45 beta (GADD45ß) deletion suppresses testosterone-induced prostate hyperplasia in mice.

AIM: Growth arrest and DNA-damage-inducible 45 beta (GADD45ß) is a member of the gene family associated with cell growth control, apoptosis, and DNA damage repair. The aim of present study was to determine the potential effects of GADD45ß deletion on prostate hyperplasia progression. MAIN METHODS: LNCaP cells were incubated with testosterone propionate (1 µM) for 48 h and specific siRNA used to suppress GADD45ß expression in vitro. For in vivo experiments, testosterone (3 mg/kg, IP) was injected into wild-type (WT) and GADD45ß knockout (GADD45ß-/-) C57BL/6J mice for 21 consecutive days, and serum and prostate tissues subjected to biological and histochemical analyses. KEY FINDINGS: GADD45ß-silenced LNCaP cells showed suppressed testosterone-induced 5α-reductase 2 and androgen receptor expression compared to control LNCaP cells. Moreover, after 21 days of testosterone treatment, prostate weight and stromal tissue increment were relatively lower in GADD45ß-/- than WT counterpart mice. Inhibition of testosterone-induced 5α-reductase 2 and proliferating cell nuclear antigen expression in the GADD45ß-/- group was confirmed via immunohistochemistry analyses. SIGNIFICANCE: Although the exact correlation between GADD45ß and prostate hyperplasia remains to be established, the present GADD45ßdeletion suppressed testosterone-induced prostate hyperplasia which was accompanied by inhibition of 5α-reductase 2-related protein expression.

Methylation mediated Gadd45ß enhanced the chemosensitivity of hepatocellular carcinoma by inhibiting the stemness of liver cancer cells.

BACKGROUND: Defects of the growth arrest DNA damage-inducible gene 45ß (Gadd45ß) play an important role in the progression of tumor and confer resistance to chemotherapy. However, the role of Gadd45ß in the apoptosis of hepatocellular carcinoma is still not clear. Purpose of this study was to explore the effect of Gadd45ß on the apoptosis of liver cancer cells, and the possible mechanism was examined. RESULT: In this study, we first confirmed the decreased expression of Gadd45ß in human liver cancer tissues and human liver cancer cell lines, when compared to the peri-tumor liver tissue and normal liver cells. And, it was found that Gadd45ß could inhibit the stemness of liver cancer cells, enhancing the apoptosis of cancer cells induced by chemotherapy. Furthermore, the results showed that HCC tissues and cell lines showed a higher methylation status in Gadd45ß promoter than that in peri-tumor tissues and normal liver cells. Methylation was then reversed by pretreatment of SMMC-7721 and Hep-3B with 5-azacytidine which is the DNA methyltransferase inhibitor. And the 5-azacytidine decreased the stemness of SMMC-7721 and Hep-3B, enhanced the sensitivity of SMMC-7721 and Hep-3B to cisplatin. CONCLUSIONS: Methylation mediated Gadd45ß expression inhibited the stemness of liver cancer cells, promoting the chemotherapy-induced apoptosis. Thus Gadd45ß may be the potential target for enhancing the chemosensitivity of human hepatocellular carcinoma.