Direct Inhibition of the First PDZ Domain of ZO-1 by Glycyrrhizin is a Possible Mechanism of Tight Junction Opening of Caco-2 Cells.

Glycyrrhizin (GL) is known to exhibit a variety of useful pharmacological activities, including anti-inflammation, anti-hepatotoxicity, and enhancement of intestinal drug absorption. GL has been reported to modify the assembly of...

Effect of different concentrations of lithium chloride on p-GSK-3β content in a model of ischemic stroke

Введение. В современном мире проблема инсультов постепенно выходит на лидирующие позиции. Отсутствие эффективных медикаментозных методов коррекции острого нарушения мозгового кровообращения приводит к необходимости поиска новых препаратов с нейропротекторным потенциалом, способных если не предотвратить, то значимо минимизировать последствия и тяжесть ишемического инсульта. Цель исследования - оценка влияния различных доз хлорида лития на фосфорилирование GSK-3β и выживаемость животных на модели ишемического инсульта. Методика. В исследовании были использованы беспородные крысы - самцы, разделённые на 5 групп: ложнооперированные (n=9), контрольная группа (ишемический инсульт с введением раствора NaCl 0,9% в объеме, эквивалентном вводимым лекарственным средствам в других группах, n=5), и группы с введением хлорида лития в дозах 4,2 мг/кг (n=5), 21 мг/кг (n=5) и 63 мг/кг (n=5). Ишемический инсульт моделировали по методу Лонга. По истечении 7 сут от начала эксперимента животные подвергались гуманной эвтаназии с извлечением головного мозга и дальнейшим определением уровня фосфорилированной формы GSK-3β (p-GSK-3β) методом вестерн-блоттинга. Нейропротекторный эффект солей лития реализуется благодаря прямому ингибированию ключевой киназы аптотического механизма клеточной сигнализации - гликоген-синтазы киназы-3β (GSK-3β) с переводом её в фосфорилированую форму (p-GSK-3β). На 7-е сут также был проведен анализ показателей летальности в группах. Для множественных сравнений рассчитывали критический уровень значимости при использовании поправки Бонферрони. Результат. Хлорид лития в дозе 4,2 мг/кг оказывал минимальное влияние как на уровень p-GSK-3β (p=0,8), так и на летальность по отношению к контрольной группе (p>0,017). Доза 21 мг/кг, в свою очередь, значимо повышала уровень p-GSK-3β (p=0,008), но не снижала летальность (p>0,017) по отношению к группе контроля. При использовании дозировки 63 мг/кг уровень p-GSK-3β был максимально приближен к группе ложнооперированных животных (p=0,007), а летальность на 7 сут была значимо ниже (p>0,017). Заключение. Хлорид лития обладает отчётливым дозозависимым нейропротекторным эффектом. Нейропротекторный эффект солей лития реализуется благодаря прямому ингибированию ключевой киназы аптотического механизма клеточной сигнализации - гликоген-синтазы киназы-3β (GSK-3β) с переводом её в фосфорилированую форму (p-GSK-3β) Реализация нейропротекторного эффекта данного препарата потенциально способна улучшить прогнозы течения ишемического инсульта. Background. Ischemic stroke is becoming a major medical concern worldwide. Reasons for this include the aging population, which experiences an increasing frequency of cardiovascular problems. Additionally, social factors, e.g., smoking, fatigue, substance abuse, lead to strokes in young and middle-aged people. The lack of effective medical methods for correcting acute cerebral circulatory disorders underscores the need for new drugs whose neuroprotective potential can prevent or significantly minimize the consequences and severity of ischemic stroke. Aim. To evaluate the effect of different doses of lithium chloride on GSK-3ß phosphorylation and on animal survival in a model of ischemic stroke. Methods. 29 male rats were divided into five groups: Sham-operated (n=9); control, ischemic stroke with administration of a volume of 0.9% NaCl solution equivalent to the volume of the administered drugs in other groups (n=5); and groups with administration of lithium chloride at doses of 4.2 mg/kg (n=5), 21 mg/kg (n=5), and 63 mg/kg (n=5). Ischemic stroke was produced by the Long method. After 7 days, the animals were subjected to humane euthanasia. The brain was excised, and the phosphorylated form of GSK-3β (p-GSK-3β) was measured by Western blotting. The neuroprotective effect of lithium salts occurs due to a direct inhibition of the key kinase of the apoptotic mechanism of cell signaling, glycogen-synthase kinase (GSK-3β), that is transformed into a phosphorylated form. Also, the group mortality rates were analyzed on day 7. For multiple comparisons, a critical level of significance was calculated using the Bonferroni correction. Results. Lithium chloride, 4.2 mg/kg, had a minimal effect on both p-GSK-3ß (p=0.8) and mortality compared to the control group (p>0.017). A dose of 21 mg/kg significantly increased p-GSK-3ß (p=0.008), but did not reduce mortality (p>0.017), relative to the control group. At a dose of 63 mg/kg, p-GSK-3ß was similar to that of the sham operated animals (p=0.007), and the mortality on day 7 was significantly lower (p>0.017). Conclusion. Lithium chloride produces a dose-dependent, neuroprotective effect. This protective effect occurs due to a direct inhibition of the key kinase of the apoptotic mechanism of cell signaling, glycogen-synthase kinase (GSK-3β), that is transformed into a phosphorylated form. This neuroprotection is potentially able to improve the prognosis of ischemic stroke.

The Effect of Direct and Indirect EZH2 Inhibition in Rhabdomyosarcoma Cell Lines

Enhancer of Zeste homolog 2 (EZH2) is involved in epigenetic regulation of gene transcription by catalyzing trimethylation of histone 3 at lysine 27. In rhabdomyosarcoma (RMS), increased EZH2 protein levels are associated with poor prognosis and increased metastatic potential, suggesting EZH2 as a therapeutic target. The inhibition of EZH2 can be achieved by direct inhibition which targets only the enzyme activity or by indirect inhibition which also affects activities of other methyltransferases and reduces EZH2 protein abundance. We assessed the direct inhibition of EZH2 by EPZ005687 and the indirect inhibition by 3-deazaneplanocin (DZNep) and adenosine dialdehyde (AdOx) in the embryonal RD and the alveolar RH30 RMS cell line. EPZ005687 was more effective in reducing the cell viability and colony formation, in promoting apoptosis induction, and in arresting cells in the G1 phase of the cell cycle than the indirect inhibitors. DZNep was more effective in decreasing spheroid viability and size in both cell lines than EPZ005687 and AdOx. Both types of inhibitors reduced cell migration of RH30 cells but not of RD cells. The results show that direct and indirect inhibition of EZH2 affect cellular functions differently. The alveolar cell line RH30 is more sensitive to epigenetic intervention than the embryonal cell line RD.

SMER28 attenuates PI3K/mTOR signaling by direct inhibition of PI3K p110 delta

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and consequently growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction of phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3Kδ, and to a lesser extent p110γ. The biological relevance of our observations was underscored by interference of SMER28 with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor typrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited.

Direct Inhibition of Microglia Activation by Pretreatment With Botulinum Neurotoxin A for the Prevention of Neuropathic Pain

Peripheral injection of botulinum neurotoxin A (BoNT/A) has been demonstrated to have a long-term analgesic effect in treating neuropathic pain. Around peripheral nerves, BoNT/A is taken up by primary afferent neurons and inhibits neuropeptide release. Moreover, BoNT/A could also be retrogradely transported to the spinal cord. Recent studies have suggested that BoNT/A could attenuates neuropathic pain by inhibiting the activation of spinal glial cells. However, it remains unclear whether BoNT/A directly interacts with these glial cells or via their interaction with neurons. Our aim here is to determine the direct effect of BoNT/A on primary microglia and astrocytes. We show that BoNT/A pretreatment significantly inhibits lipopolysaccharide (LPS) -induced activation and pro-inflammatory cytokine release in primary microglia (1 U/mL BoNT/A in medium), while it has no effect on the activation of astrocytes (2 U/mL BoNT/A in medium). Moreover, a single intrathecal pre-administration of a low dose of BoNT/A (1 U/kg) significantly prohibited the partial sciatic nerve ligation (PSNL)- induced upregulation of pro-inflammatory cytokines in both the spinal cord dorsal horn and dorsal root ganglions (DRGs), which in turn prevented the PSNL-induced mechanical allodynia and thermal hyperalgesia. In conclusion, our results indicate that BoNT/A pretreatment prevents PSNL-induced neuropathic pain by direct inhibition of spinal microglia activation.

Targeting the Oncogenic Functions of an RNA-Binding Protein Involved in Hematologic Malignancies

hnRNP K (heterogeneous ribonucleoprotein K) is an RNA-binding protein that binds to conserved poly-C rich tracks in RNA and influences a diverse set of molecular pathways involved in tumorigenesis. Our previous studies identified hnRNP K overexpression in patients with diffuse large B-cell lymphoma (46/75, 61%) and acute myeloid leukemia (45/160, 28%). This overexpression correlates with dismal clinical outcomes and a lack of therapeutic responses to standard treatment. To explore hnRNP K's in vivo functions, we generated Hnrnpk-transgenic mouse models. These mice develop lymphoma phenotypes through activation of the c-Myc pathway. In pre-clinical settings, bromodomain inhibitors disrupted hnRNP K-mediated c-Myc activation, demonstrating that hnRNP K overexpression mediated-pathways are amenable to therapeutic intervention. To further our studies, we used IP-mass spectrometry, RNA-sequencing, RNA immunoprecipitation, reverse phase protein analyses, and polysome profiling to identify novel pathways associated with changes in hnRNP K expression. Here, we observed that alterations in hnRNP K expression result in an impairment of ribosomal biogenesis and activation of pathways directly responsible for global translation. Using both knockdown and overexpression systems, we observed a direct correlation between hnRNP K expression and expression of S6, S6K, phosphorylated S6, eIF and mTOR pathways and uncovered defects in rRNA splicing. Collectively, these data indicate that impairment of cap-dependent loading and alterations in ribogenesis may be a driving force in the clinical manifestations of hnRNP K-driven malignancies. Furthermore, these results suggest that translational-inhibitors may be useful in exploiting hnRNP K-dependent vulnerabilities. To examine this aspect, we are currently using FDA-approved translation inhibitors and disruptors of ribogenesis (e.g. homoharringtonineand mTOR-inhibitors) and KTP- compounds, respectively. While these indirect targeting strategies are interesting, our results indicate that hnRNP K also regulates cellular programs outside of translation. Thus, potential therapies that effectively target hnRNP K overexpression will require direct inhibition of its RNA binding functions. To this end, we used several screening assays including fluorescence anisotropy (FA), surface plasmon resonance, SYPRO-orange thermal shift assays, and cell proliferation assays to screen 80,000 small molecule compounds which led to the identification of 9 candidates that disrupt hnRNP K-mRNA interactions and cause cell death in an hnRNP K-dependent manner. Further, cellular thermal shift assays revealed these lead compounds engage hnRNP K within cells and most critically, result in reduced expression of hnRNP K targets in vivo. These candidate compounds as well as potentially more potent structural analogs are currently being evaluated. Collectively, our results demonstrate that the oncogenic functions of hnRNP K are amenable to both indirect therapeutic intervention using FDA-approved agents as well as direct inhibition through newly identified small molecule compounds, signifying that there may be a roadmap to effective therapies for hnRNP K-dependent malignancies. Disclosures No relevant conflicts of interest to declare.