Natural prenylated xanthones as potential inhibitors of PI3k/Akt/mTOR pathway in triple negative breast cancer cells

Three prenylated xanthones, garcinone E (1), bannaxanthone D (2) and bannanxanthone E (3) were isolated from the leaves of Garcinia mckeaniana Graib. Their structures were elucidated by spectral methods and compared with literature data. To evaluate their anti-proliferative effects in tumor cells, firstly, cisplatin was used as a positive control and the effects of compound 1-3 were determined by performing MTT assay in MDA-MB-231, CNE-2 and A549 cancer cells. The results showed compound 1-3 exhibited stronger inhibitory effect than cisplatin in MDA-MB-231. Further effects of compound 1-3 in TNBC MDA-MB-231 and MDA-MB-468 cells were examined by performing cell cycle and apoptosis assays. The results indicated that compound 1-3 had ability to arrest cell cycle at G2/M phase and induce apoptosis. Furthermore, compound 2 significantly down-regulated PI3K, Akt and mTOR levels in both total proteins and phosphorylated form, which is its potential anti-cancer mechanism. These findings indicated that those prenylated xanthones might serve as promising leading compounds for the development of anticancer drug for TNBC.

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 Phosphorylated Form of the Histone H2AX (γH2AX) in the Brain from Embryonic Life to Old Age

The γ phosphorylated form of the histone H2AX (γH2AX) was described more than 40 years ago and it was demonstrated that phosphorylation of H2AX was one of the first cellular responses to DNA damage. Since then, γH2AX has been implicated in diverse cellular functions in normal and pathological cells. In the first part of this review, we will briefly describe the intervention of H2AX in the DNA damage response (DDR) and its role in some pivotal cellular events, such as regulation of cell cycle checkpoints, genomic instability, cell growth, mitosis, embryogenesis, and apoptosis. Then, in the main part of this contribution, we will discuss the involvement of γH2AX in the normal and pathological central nervous system, with particular attention to the differences in the DDR between immature and mature neurons, and to the significance of H2AX phosphorylation in neurogenesis and neuronal cell death. The emerging picture is that H2AX is a pleiotropic molecule with an array of yet not fully understood functions in the brain, from embryonic life to old age.

An intrinsic temporal order of c-Jun N-terminal phosphorylation regulates its activity by orchestrating co-factor recruitment

Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-Jun proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-Jun multisite phosphorylation has remained elusive. Here we show that c-Jun phosphorylation by JNK exhibits a defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identified the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis revealed three c-Jun phosphorylation states: unphosphorylated c-Jun recruits the Mbd3 repressor, serine63/73 doubly-phosphorylated c-Jun binds to the Tcf4 co-activator, whereas the fully phosphorylated form disfavours Tcf4 binding attenuating JNK signalling. Thus, c-Jun phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.

MKP-1 modulates ubiquitination/phosphorylation of TLR signaling

Ubiquitination and phosphorylation are reversible posttranslational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK MAPKs. We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1−/− showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1, and an increased K63 polyubiquitination on TRAF6. Increased K63 polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, ubiquitin-specific protease-13 (USP13), which cleaves polyubiquitin-chains on client proteins, was substantially enhanced in murine MKP-1–deficient BMDMs. An inhibitor of USP13 decreased the K63 polyubiquitination on TRAF6, TAK1 phosphorylation, IL-1β, and TNF-α induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

Potential Use of Biomarker γ-H2AX on Peripheral Blood Patient Who Underwent Radioiodine Ablation Treatment in Indonesia

Thyroid cancer is one of the most common endocrine malignancies. Although the 10-year survival rate of differentiated thyroid cancer (DTC) is about 90% after conventional treatments, a small proportion of patients still suffer from tumor recurrence or drug resistance. DNA doublestrand breaks (DSBs) are important cellular lesions that can result from ionizing radiation exposure. The biomarker for DSB formation is the phosphorylated form of the histone H2 variant H2AX (γ-H2AX). We propose the use of γ-H2AX as a DNA DSB biomarker in thyroid cancer patients receiving radioiodine treatment as a possibility to detect the potential of instability genome after receiving the treatment. Evaluating DNA DSB damage with γ-H2AX biomarker might be important in managing thyroid cancer.

EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis

Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. In this work, we have identified a novel EGFR inhibitor, which we have named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. We show that EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. We also find that elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, we propose that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. We suggest that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.

Expression of STAT6 and Phosphorylated STAT6 in Primary Central Nervous System Lymphomas

The signal transducer and activator of transcription 6 (STAT6) is implicated in the pathogenesis of some lymphomas including primary central nervous system lymphomas (PCNSLs). The aim of this study was to investigate STAT6 expression and clinicopathologic features in 25 PCNSLs using immunohistochemistry with 2 different anti-STAT6 antibodies. One (YE361) recognizes the C-terminus domain of the STAT6 protein and the other (Y641) recognizes the phosphorylated form of the protein. The phosphorylated STAT6 form was not expressed in any of the cases studied whereas the YE361 STAT6 showed only cytoplasmic expression in 14 (56%) cases. This expression did not correlate with age, prognostic score, multiplicity, invasion of deep structures, response to treatment, disease recurrence, overall survival, or BCL6, BCL2, PD-L1, and CD8 expression. A STAT6 expression score showed a trend for correlating with clinical performance status. It also showed a positive correlation with MYC expression. Thus, the phosphorylated form of STAT6 was not found in the current series, while the YE361 STAT6 showed only cytoplasmic expression and was associated with expression of MYC.

Expression of Cancer Stem Cell Markers EpCAM and CD90 Is Correlated with Anti- and Pro-Oncogenic EphA2 Signaling in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser897, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.