Major Signal Transduction Pathways Research Articles

Abstract 4713: Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2: A route for drug repurposing in glioblastoma

Abstract Glioblastoma, the most common and deadly brain tumor, remains a critical unmet medical need due to the limited effectiveness of current treatments. Drug repurposing has recently emerged as a promising strategy to improve glioblastoma outcomes. Antipsychotic drugs, with their established safety profile and potential to disrupt tumor-neuron interactions, have drawn particular attention. Among these, chlorpromazine, a well-tolerated medication included in the 2021 WHO Model List of Essential Medicines, holds promise due to its therapeutic effects in psychiatric disorders stemming from its non-specific interference with various CNS neurotransmitter receptors. Our recent studies have demonstrated chlorpromazine's ability to inhibit several molecular and cellular processes in glioblastoma cells, suggesting its potential as a novel treatment option for this challenging disease.To elucidate chlorpromazine's mechanism of action as a potential anticancer drug, we employed two proteomics approaches: Reverse-Phase Protein microArrays to evaluate its impact on signal transduction pathways and Activity-Based Protein Profiling followed by mass spectrometry to identify novel molecular targets.Our data revealed that chlorpromazine significantly modulates major signal transduction pathways and implicates pyruvate kinase (PK) M2 as a drug target. PKM2, a PK variant characteristic of many cancers, plays a crucial role in orchestrating metabolic alterations, exemplified by the Warburg effect – the high glucose consumption and lactate production by cancer cells even under oxygen-rich conditions. PKM2 functions as a tetramer in the glycolytic pathway, while its dimeric form acquires nuclear localization, protein kinase activity, and interacts with various transcription factors, contributing to its pro-tumorigenic activity.Consistent with its ability to target PKM2, chlorpromazine promoted PKM2 tetramerization in glioblastoma cells, leading to significant alterations in glioblastoma energy metabolism. Notably, RPE-1 non-cancer neuroepithelial cells showed a reduced response to the drug. Additionally, silencing PKM2 diminished the effects of chlorpromazine. 3D modeling revealed that chlorpromazine interacts with the PKM2 tetramer at the same site involved in binding other small-molecule activators that stabilize the PKM2 tetramer.These findings suggest that chlorpromazine counteracts the Warburg effect and, consequently, malignancy in glioblastoma cells, while sparing non-cancerous RPE-1 cells. This preclinical evidence supports the rationale behind our recently completed multicenter Phase II clinical trial investigating the role of chlorpromazine in glioblastoma treatment. The study is registered as EudraCT #2019-001988-75 and ClinicalTrials.gov Identifier #NCT0422444. Citation Format: Claudia Abbruzzese, Silvia Matteoni, Paola Matarrese, Michele Signore, Barbara Ascione, Elisabetta Iessi, Aymone Gurtner, Andrea Sacconi, Andrea Pace, Veronica Villani, Andrea Polo, Susan Costantini, Alfredo Budillon, Gennaro Ciliberto, Marco G. Paggi. Chlorpromazine affects glioblastoma bioenergetics by interfering with pyruvate kinase M2: A route for drug repurposing in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4713.

Effect of IFN‑γ encapsulated liposomes on major signal transduction pathways in the lymphocytes of patients with lung cancer.

Globally, lung cancer affected 2.2 million individuals and caused 1.8 million deaths in 2021. Lung cancer is caused by smoking, genetics and other factors. IFN-γ has anticancer activity. However, the mechanism by which IFN-γ has an effect on lung cancer is not fully understood. The present study aimed to assess the effect of IFN-γ on the peripheral lymphocytes of patients with lung cancer compared with healthy controls. The efficacy of IFN-γ against oxidative stress was assessed using a comet repair assay and the effects of IFN-γ on p53, PARP1 and OGG1 genes and protein levels in lymphocytes was evaluated by RT-qPCR and western blotting. DNA damage was significantly reduced in the lymphocytes of patients treated with IFN-γ. However, there was no effect in the cells of healthy individuals after treatment with naked IFN-γ [IFN-γ (N)] and liposomal IFN-γ [IFN-γ (L)]. Following treatment with IFN-γ (N) and IFN-γ (L), the p53, PARP1 and OGG1 protein and gene expression levels were significantly increased (P<0.001). It has been suggested that IFN-γ may induce p53-mediated cell cycle arrest and DNA repair in patients. These findings supported the idea that IFN-γ (N) and IFN-γ (L) may serve a significant role in the treatment of lung cancer, via cell cycle arrest of cancer cells and repair mechanisms.

Advances in research on the effectiveness and mechanism of Traditional Chinese Medicine formulas for colitis-associated colorectal cancer.

Inflammatory bowel disease (IBD) can progress into colitis-associated colorectal cancer (CAC) through the inflammation-cancer sequence. Although the mechanism of carcinogenesis in IBD has not been fully elucidated, the existing research indicates that CAC may represent a fundamentally different pathogenesis pattern of colorectal cancer. At present, there is no proven safe and effective medication to prevent IBD cancer. In recent years, Chinese medicine extracts and Chinese medicine monomers have been the subject of numerous articles about the prevention and treatment of CAC, but their clinical application is still relatively limited. Traditional Chinese Medicine (TCM) formulas are widely applied in clinical practice. TCM formulas have demonstrated great potential in the prevention and treatment of CAC in recent years, although there is still a lack of review. Our work aimed to summarize the effects and potential mechanisms of TCM formulas for the prevention and treatment of CAC, point out the issues and limitations of the current research, and provide recommendations for the advancement of CAC research in the future. We discovered that TCM formulas regulated many malignant biological processes, such as inflammation-mediated oxidative stress, apoptosis, tumor microenvironment, and intestinal microecology imbalance in CAC, through a review of the articles published in databases such as PubMed, SCOPUS, Web of Science, Embase, and CNKI. Several major signal transduction pathways, including NF-κB, STAT3, Wnt/β-catenin, HIF-1α, and Nrf2, were engaged. TCM formula may be a promising treatment candidate to control the colitis-cancer transformation, however further high-quality research is required.

Wnt4 and ephrinB2 instruct apical constriction via Dishevelled and non-canonical signaling

Apical constriction is a cell shape change critical to vertebrate neural tube closure, and the contractile force required for this process is generated by actin-myosin networks. The signaling cue that instructs this process has remained elusive. Here, we identify Wnt4 and the transmembrane ephrinB2 protein as playing an instructive role in neural tube closure as members of a signaling complex we termed WERDS (Wnt4, EphrinB2, Ror2, Dishevelled (Dsh2), and Shroom3). Disruption of function or interaction among members of the WERDS complex results in defects of apical constriction and neural tube closure. The mechanism of action involves an interaction of ephrinB2 with the Dsh2 scaffold protein that enhances the formation of the WERDS complex, which in turn, activates Rho-associated kinase to induce apical constriction. Moreover, the ephrinB2/Dsh2 interaction promotes non-canonical Wnt signaling and shows how cross-talk between two major signal transduction pathways, Eph/ephrin and Wnt, coordinate morphogenesis of the neural tube.

Regulation of phosphoinositide metabolism in Apicomplexan parasites.

Phosphoinositides are a biologically essential class of phospholipids that contribute to organelle membrane identity, modulate membrane trafficking pathways, and are central components of major signal transduction pathways that operate on the cytosolic face of intracellular membranes in eukaryotes. Apicomplexans (such as Toxoplasma gondii and Plasmodium spp.) are obligate intracellular parasites that are important causative agents of disease in animals and humans. Recent advances in molecular and cell biology of Apicomplexan parasites reveal important roles for phosphoinositide signaling in key aspects of parasitosis. These include invasion of host cells, intracellular survival and replication, egress from host cells, and extracellular motility. As Apicomplexans have adapted to the organization of essential signaling pathways to accommodate their complex parasitic lifestyle, these organisms offer experimentally tractable systems for studying the evolution, conservation, and repurposing of phosphoinositide signaling. In this review, we describe the regulatory mechanisms that control the spatial and temporal regulation of phosphoinositides in the Apicomplexan parasites Plasmodium and T. gondii. We further discuss the similarities and differences presented by Apicomplexan phosphoinositide signaling relative to how these pathways are regulated in other eukaryotic organisms.

Impact of Sleep Deprivation on Major Neuroinflammatory Signal Transduction Pathways

Impact of Sleep Deprivation on Major Neuroinflammatory Signal Transduction Pathways

Role of the PI3K/Akt signaling pathway in liver ischemia reperfusion injury: a narrative review.

To explore the role of phosphatidylinositol-3-kinase/protein kinase B and alpha serine/threonine protein kinase PI3K/PKB (also known as PI3K/Akt) signaling pathway in liver ischemia reperfusion injury. The PI3K/Akt signaling pathway is one of the major signal transduction pathways that regulates numerous cellular activities in vivo. The main functions of this pathway include induction of stem cell differentiation and metastasis, promotion of cell proliferation, inhibition of apoptosis, and regulation of tissue inflammation, tumor growth, and invasion. Liver ischemia reperfusion injury is an inevitable clinical problem that can occur during liver transplantation, liver resection, and various circulatory shock events, and it is one of the primary reasons for postoperative liver dysfunction, and poor disease outcome and patient prognosis. In recent years, it has been found that PI3K/Akt signaling pathway is closely related to liver ischemia reperfusion injury. In this review, a large number of relevant literatures were collected to explain the biological basis of PI3K/Akt signaling pathway and its role in liver ischemia reperfusion injury. The review was based on a PubMed search using the terms "liver ischemia reperfusion injury", "PI3K/Akt signaling pathway", and "PI3K/Akt signaling pathway AND liver ischemia reperfusion injury", so as to understand the complex interaction between them. Activated PI3K/Akt signaling pathway can exert anti-inflammatory, antioxidant stress, anti-apoptosis and autophagy regulation effects through downstream related targeted pathways and proteins, thus alleviating liver ischemia-reperfusion injury. Therefore, the regulation of PI3K/Akt signaling pathway is expected to become an effective targeted pathway for clinical prevention and alleviation of liver ischemia reperfusion injury.

Reconstruction of dynamic regulatory networks reveals signaling-induced topology changes associated with germ layer specification.

SummaryElucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Unfortunately, existing computational gene regulatory network (GRN) reconstruction methods are imprecise, computationally burdensome, and fail to reveal dynamic regulatory topologies. Here, we present Epoch, a reconstruction tool that uses single-cell transcriptomics to accurately infer dynamic networks. We apply Epoch to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESCs) guided by multiple signaling pathways, and we demonstrate that modulating these pathways drives topological changes that bias cell fate potential. We also find that Peg3 rewires the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRNs, we trace how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we identify regulatory circuits of patterning and axis formation that distinguish in vitro and in vivo mesoderm specification.

Vasodilatory Effect of Guanxinning Tablet on Rabbit Thoracic Aorta is Modulated by Both Endothelium-Dependent and -Independent Mechanism.

Vasodilatory therapy plays an important role in the treatment of cardiovascular diseases, especially hypertension and coronary heart disease. Previous research found that Guanxinning tablet (GXNT), a traditional Chinese compound preparation composed of Salvia miltiorrhiza (Danshen) and Ligusticum chuanxiong (Chuanxiong), increase blood flow in the arteries, but whether vasodilation plays a role in this effect remains unclear. Here, we found that GXNT significantly alleviated the vasoconstriction of isolated rabbit thoracic aorta induced by phenylephrine (PE), norepinephrine (NE), and KCl in a dose-dependent manner with or without endothelial cells (ECs). Changes in calcium ion levels in vascular smooth muscle cells (VSMCs) showed that both intracellular calcium release and extracellular calcium influx through receptor-dependent calcium channel (ROC) declined with GXNT treatment. Experiments to examine potassium channels suggested that endothelium-denuded vessels were also regulated by calcium-activated potassium channels (Kca) and ATP-related potassium channels (KATP) but not voltage-gated potassium channels (kv) and inward rectifying potassium channels (KIR). For endothelium-intact vessels, the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) contents in vascular tissue obviously increased after GXNT treatment, and pretreatment with the NO synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) or guanylyl cyclase inhibitor methylthionine chloride (MB) significantly inhibited vasodilation. An assessment of NO-related pathway protein expression revealed that GXNT enhanced the expression of phosphorylated endothelial NO synthase (eNOS) in a dose-dependent manner but had no effect on total eNOS, p-Akt, Akt, or PI3K levels in human umbilical vein ECs (HUVECs). In addition to PI3K/AKT signaling, Ca2+/calmodulin (CaM)-Ca2+/CaM-dependent protein kinase II (CaMKII) signaling is a major signal transduction pathway involved in eNOS activation in ECs. Further results showed that free calcium ion levels were decreased in HUVECs with GXNT treatment, accompanied by an increase in p-CaMKII expression, implying an increase in the Ca2+/CaM-Ca2+/CaMKII cascade. Taken together, these findings suggest that the GXNT may have exerted their vasodilative effect by activating the endothelial CaMKII/eNOS signaling pathway in endothelium-intact rings and calcium-related ion channels in endothelium-denuded vessels.

LNA -Gapmer Antisense Oligonucleotides Targeting the Oncogenic Lncrna MALAT1 Trigger Anti-Multiple Myeloma Activity By Proteasome Blockade

Dysregulation of non-coding RNAs is emerging as a common feature of human cancer. While short non-coding RNAs have been extensively investigated in multiple myeloma (MM), the biological role and the therapeutic significance of aberrantly expressed long non-coding RNAs (lncRNAs) are yet undetermined. A proprietary custom annotation pipeline of microarray data on lncRNA expression in different plasma cell (PC) dyscrasias, including 20 MGUS, 33 smoldering MM, 170 MM, 36 PC leukemia patients and 9 healthy donors, revealed high expression of MALAT1 in MM PCs. On this finding, we investigated the functional significance of MALAT1 by using novel 16-mer LNA gapmeR synthetic ASOs, that trigger selective RNAse-H dependent degradation of MALAT1. LNA-gapmeR ASOs targeting MALAT1 inhibited proliferation and clonogenicity of MM cell lines (n=7) and patient PCs (n=5), triggering ER-stress response and apoptosis, even in the presence of bone marrow stromal cells, while sparing healthy peripheral blood mononuclear cells. Conversely, MALAT1-enforced expression by lentiviral vectors enhanced cell proliferation and migration, reverted the effect of anti-MALAT1 ASOs and activated major oncogenic signal transduction pathways, such as AKT and MAPK. In vivo, i.p. administration of anti-MALAT1 gapmeRs inhibited the growth of human MM xenografts, with no evidence of organ toxicity. Microarray gene expression profiling indicated that MALAT1 depletion downregulated genes encoding for 20S proteasome β-subunits, as evidenced by gene set enrichment analysis (GSEA).Down-modulation of PSMβ2/4/5/7 mRNA and protein by anti-MALAT1 ASOs was confirmed by qPCR and Western blot respectively, and was associated to reduced trypsine-, chymotrypsine- and caspase-like proteasome activities and to the accumulation of polyubiquitylated proteins. Mechanistically, we demonstrated that proteasome inhibition upon MALAT1 knock-down was due to downmodulation of the major transcriptional activators of proteasome gene expression NRF1 and NRF2. At the same time, NRF1 was found to bind to and transactivate MALAT1 promoter, as demonstrated by chromatin immunoprecipitation and reporter assays, thus establishing a positive feedback-loop with MALAT1. Intriguingly, this loop was overactivated in bortezomib-resistant MM cells. Collectively, our findings highlight an unprecedented role of the lncRNA MALAT1 in the regulation of the proteasome machinery, and provide the first preclinical rationale for using ASO therapeutics targeting dysregulated lncRNAs for MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Pathophysiology of psoriasis: A review.

Psoriasis is a complex chronic inflammatory skin disease caused by the dynamic interplay between multiple genetic risk foci, environmental risk factors, and excessive immunological abnormalities. Psoriasis affects approximately 2% of the population worldwide, and dramatic advances have been achieved in the understanding and treatment options for psoriasis. Recent progress in biological therapies has revealed the fundamental roles of tumor necrosis factor-α, interleukin (IL)-23p19, and the IL-17A axis together with skin-resident immune cells and major signal transduction pathways in the pathogenesis of psoriasis. In addition to IL-17-producing T helper17 cells, innate lymphoid cell (ILC)3 induces psoriasis rashes directly without T-cell/antigen interaction in response to the released antimicrobial peptides from activated keratinocytes and inflammatory cytokines. ILC3 typically expresses retinoic acid receptor-related orphan receptor gamma t in the nucleus, matures in the presence of IL-7 and IL-23, and produces IL-17 and IL-22. The number of ILC3s is increased in the blood, psoriasis rash, and even in nonrash areas of psoriatic skin. Psoriasis is significantly associated with cardiovascular disease, metabolic syndrome, and inflammatory disorders, particularly the severe type. The similarity of enterobacteria in the psoriasis gut to that in diabetic patients may be related to its pathogenesis. In the current review, we focus on the pathophysiology of psoriasis in the accelerated immunological inflammatory loop, danger signal from keratinocytes, and cytokines, particularly IL-17 and IL-23p19. In addition, pathophysiological speculation with regard to morphology has been supplemented. Finally, the differences and similarities between psoriasis and atopic dermatitis are discussed.

Protein Phosphatase Type 2C Functions in Phytohormone-Dependent Pathways and in Plant Responses to Abiotic Stresses.

Plants, as sessile organisms, are susceptible to a myriad of stress factors, especially abiotic stresses. Over the course of evolution, they have developed multiple mechanisms to sense and transduce environmental stimuli for appropriate responses. Among those, phosphorylation and dephosphorylation, regulated by protein kinases and protein phosphatases, respectively, are considered crucial signal transduction mechanisms. Regarding the latter group, protein phosphatases type 2C (PP2Cs) represent the largest division of PPs. In addition, the discovery of regulatory functions of PP2Cs in the abscisic acid (ABA)-signaling pathway, the major signal transduction pathway in abiotic stress responses, indicates the significant importance of PP2C members in plant adaptation to adverse environmental factors. In this review, current understanding of the roles of PP2Cs in different phytohormone-dependent pathways related to abiotic stress is summarized, highlighting the crosstalk between the ABA-signaling pathway with other hormonal pathways via certain ABA-related PP2Cs. We also updated the progress of in planta characterization studies of PP2Cs under abiotic stress conditions, providing knowledge of PP2C manipulation in developing abiotic stress-tolerant crops.

Proteoglycan 4 Reduces Neuroinflammation and Protects the Blood-Brain Barrier after Traumatic Brain Injury.

Neuroinflammation and dysfunction of the blood–brain barrier (BBB) are two prominent mechanisms of secondary injury in neurotrauma. It has been suggested that Toll-like receptors (TLRs) play important roles in initiating and propagating neuroinflammation resulting from traumatic brain injury (TBI), but potential beneficial effects of targeting these receptors in TBI have not been broadly studied. Here, we investigated the effect of targeting TLRs with proteoglycan 4 (PRG4) on post-traumatic neuroinflammation and BBB function. PRG4 is a mucinous glycoprotein with strong anti-inflammatory properties, exerting its biological effects by interfering with TLR2/4 signaling. In addition, PRG4 has the ability to inhibit activation of cluster of differentiation 44 (CD44), a cell-surface glycoprotein playing an important role in inflammation. Using the controlled cortical impact model of TBI in rats, we showed a rapid and prolonged upregulation of message for TLR2/4 and CD44 in the injured cortex. In the in vitro model of the BBB, recombinant human PRG4 (rhPRG4) crossed the endothelial monolayers through a high-capacity, saturable transport system. In rats sustaining TBI, PRG4 delivery to the brain was enhanced by post-traumatic increase in BBB permeability. rhPRG4 injected intravenously at 1 h post-TBI potently inhibited post-traumatic activation of nuclear factor kappa B and extracellular signal-regulated kinases 1/2, the two major signal transduction pathways associated with TLR2/4 and CD44, and curtailed the post-traumatic influx of monocytes. In addition, PRG4 restored normal BBB function after TBI by preventing the post-traumatic loss of tight junction protein claudin 5 and reduced neuronal death. Our observations provide support for therapeutic strategies targeting TLRs in TBI.

Nutrient sensing and cAMP signaling in yeast: G-protein coupled receptor versus transceptor activation of PKA.

A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the Gap1 amino acid transceptor and the catalytic subunits of PKA, Tpk1, 2 and 3. In addition, we reveal a conserved consensus motif in the nutrient transceptors that is also present in Bcy1, the regulatory subunit of PKA. This suggests that nutrient transceptor activation of PKA may be mediated by direct release of bound PKA catalytic subunits, triggered by the conformational changes occurring during transport of the substrate by the transceptor. Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA.

Adolescent Fluoxetine Exposure Induces Persistent Gene Expression Changes in the Hippocampus of Adult Male C57BL/6 Mice.

Mood-related disorders have a high prevalence among children and adolescents, posing a public health challenge, given their adverse impact on these young populations. Treatment with the selective serotonin reuptake inhibitor fluoxetine (FLX) is the first line of pharmacological intervention in pediatric patients suffering from affect-related illnesses. Although the use of this antidepressant has been deemed efficacious in the juvenile population, the enduring neurobiological consequences of adolescent FLX exposure are not well understood. Therefore, we explored for persistent molecular adaptations, in the adult hippocampus, as a function of adolescent FLX pretreatment. To do this, we administered FLX (20 mg/kg/day) to male C57BL/6 mice during adolescence (postnatal day [PD] 35-49). After a 21-day washout period (PD70), whole hippocampal tissue was dissected. We then used qPCR analysis to assess changes in the expression of genes associated with major intracellular signal transduction pathways, including the extracellular signal-regulated kinase (ERK), the phosphatidylinositide-3-kinase (PI3K)/AKT pathway, and the wingless (Wnt)-dishevelled-GSK3β signaling cascade. Our results show that FLX treatment results in long-term dysregulation of mRNA levels across numerous genes from the ERK, PI3K/AKT, and Wnt intracellular signaling pathways, along with increases of the transcription factors CREB, ΔFosB, and Zif268. Lastly, FLX treatment resulted in persistent increases of transcripts associated with cytoskeletal integrity (β-actin) and caspase activation (DIABLO), while decreasing genes associated with metabolism (fucose kinase) and overall neuronal activation (c-Fos). Collectively, these data indicate that adolescent FLX exposure mediates persistent alterations in hippocampal gene expression in adulthood, thus questioning the safety of early-life exposure to this antidepressant medication.

The effect and associated mechanism of action of phosphodiesterase 4 (PDE4) inhibitor on CD4+ lymphocyte proliferation.

PDE4 inhibitors are involved in anti-inflammatory and immunomodulatory responses. Recently, they have been getting attention as a new class of drugs treating inflammatory airway diseases. The T lymphocyte is a major cell type present in the inflammatory infiltrate in the airway wall in patients with chronic obstructive pulmonary disease (COPD), and a previous study found that treatment with a PDE4 inhibitor significantly suppressed T cell proliferation. However, the mechanism of action of PDE4 inhibitors has not been elucidated. The present study aimed to investigate major signal transduction pathways of T lymphocyte and identify the phase, during which PDE4 inhibitors affect T cell proliferation. Isolated splenic CD4+ T cells were grown under stimulation with an anti-CD3/CD28 antibody, and/or treated with roflumilast-n-oxide (RNO). A western blot assay was performed using major antibodies including anti-p-38, anti-p-PI3K, anti-p-JNK, anti-p-ERK 1/2, anti-NFAT1 (NFATc2), and anti-NF-kB antibodies. Additional experiments conducted on the pathway showed significant change following RNO treatment, thus providing further evidence for signal transduction pathway concerning PDE4 inhibitors. T cell proliferation was suppressed by RNO treatment. In the pathways involved in T cell proliferation, only expression of anti-NFAT1 antibody was suppressed by RNO treatment. In additional experiments on the NFAT pathway, the very first phase (TCR signalling) remained unchanged on treatment with RNO, but RNO treatment increased IP3R expression and suppressed calcineurin activity. Calcineurin activity, reduced by RNO, increased on treatment with an IP3 receptor agonist. PED4 inhibitor, roflumilast is speculated to suppress T cell proliferation by interfering with IP3-IP3R binding to inhibit calcium emission, blocking pathway activation from this phase onward, eventually decreasing the level of a growth factor for T cell proliferation, IL-2.

A Framework of Major Tumor-Promoting Signal Transduction Pathways Implicated in Melanoma-Fibroblast Dialogue.

Simple SummaryMelanoma cells reside in a complex stromal microenvironment, which is a critical component of disease onset and progression. Mesenchymal or fibroblastic cell type are the most abundant cellular element of tumor stroma. Factors secreted by melanoma cells can activate non-malignant associated fibroblasts to become melanoma associate fibroblasts (MAFs). MAFs promote tumorigenic features by remodeling the extracellular matrix, supporting tumor cells proliferation, neo-angiogenesis and drug resistance. Additionally, environmental factors may contribute to the acquisition of pro-tumorigenic phenotype of fibroblasts. Overall, in melanoma, perturbed tissue homeostasis contributes to modulation of major oncogenic intracellular signaling pathways not only in tumor cells but also in neighboring cells. Thus, targeted molecular therapies need to be considered from the reciprocal point of view of melanoma and stromal cells.The development of a modified stromal microenvironment in response to neoplastic onset is a common feature of many tumors including cutaneous melanoma. At all stages, melanoma cells are embedded in a complex tissue composed by extracellular matrix components and several different cell populations. Thus, melanomagenesis is not only driven by malignant melanocytes, but also by the altered communication between melanocytes and non-malignant cell populations, including fibroblasts, endothelial and immune cells. In particular, cancer-associated fibroblasts (CAFs), also referred as melanoma-associated fibroblasts (MAFs) in the case of melanoma, are the most abundant stromal cells and play a significant contextual role in melanoma initiation, progression and metastasis. As a result of dynamic intercellular molecular dialogue between tumor and the stroma, non-neoplastic cells gain specific phenotypes and functions that are pro-tumorigenic. Targeting MAFs is thus considered a promising avenue to improve melanoma therapy. Growing evidence demonstrates that aberrant regulation of oncogenic signaling is not restricted to transformed cells but also occurs in MAFs. However, in some cases, signaling pathways present opposite regulation in melanoma and surrounding area, suggesting that therapeutic strategies need to carefully consider the tumor–stroma equilibrium. In this novel review, we analyze four major signaling pathways implicated in melanomagenesis, TGF-β, MAPK, Wnt/β-catenin and Hyppo signaling, from the complementary point of view of tumor cells and the microenvironment.

Respiratory CO2 Combined With a Blend of Volatiles Emitted by Endophytic Serendipita Strains Strongly Stimulate Growth of Arabidopsis Implicating Auxin and Cytokinin Signaling.

Rhizospheric microorganisms can alter plant physiology and morphology in many different ways including through the emission of volatile organic compounds (VOCs). Here we demonstrate that VOCs from beneficial root endophytic Serendipita spp. are able to improve the performance of in vitro grown Arabidopsis seedlings, with an up to 9.3-fold increase in plant biomass. Additional changes in VOC-exposed plants comprised petiole elongation, epidermal cell and leaf area expansion, extension of the lateral root system, enhanced maximum quantum efficiency of photosystem II (Fv/Fm), and accumulation of high levels of anthocyanin. Notwithstanding that the magnitude of the effects was highly dependent on the test system and cultivation medium, the volatile blends of each of the examined strains, including the references S. indica and S. williamsii, exhibited comparable plant growth-promoting activities. By combining different approaches, we provide strong evidence that not only fungal respiratory CO2 accumulating in the headspace, but also other volatile compounds contribute to the observed plant responses. Volatile profiling identified methyl benzoate as the most abundant fungal VOC, released especially by Serendipita cultures that elicit plant growth promotion. However, under our experimental conditions, application of methyl benzoate as a sole volatile did not affect plant performance, suggesting that other compounds are involved or that the mixture of VOCs, rather than single molecules, accounts for the strong plant responses. Using Arabidopsis mutant and reporter lines in some of the major plant hormone signal transduction pathways further revealed the involvement of auxin and cytokinin signaling in Serendipita VOC-induced plant growth modulation. Although we are still far from translating the current knowledge into the implementation of Serendipita VOCs as biofertilizers and phytostimulants, volatile production is a novel mechanism by which sebacinoid fungi can trigger and control biological processes in plants, which might offer opportunities to address agricultural and environmental problems in the future.

Octominin Inhibits LPS-Induced Chemokine and Pro-inflammatory Cytokine Secretion from RAW 264.7 Macrophages via Blocking TLRs/NF-κB Signal Transduction.

Inflammation is a well-organized innate immune response that plays an important role during the pathogen attacks and mechanical injuries. The Toll-like receptors (TLR)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a major signal transduction pathway observed in RAW 264.7 macrophages during the inflammatory responses. Here, we investigated the anti-inflammatory effects of Octominin; a bio-active peptide developed from Octopus minor in RAW 264.7 macrophages in vitro. Octominin was found to inhibit lipopolysaccharides (LPS)-stimulated transcriptional activation of NF-κB in RAW 264.7 cells and dose-dependently decreased the mRNA expression levels of TLR4. Specifically, in silico docking results demonstrated that Octominin has a potential to inhibit TLR4 mediated inflammatory responses via blocking formation of TLR4/MD-2/LPS complex. We also demonstrated that Octominin could significantly inhibit LPS-induced secretion of pro-inflammatory cytokine (interleukin-β; IL-1β, IL-6, and tumor necrosis factor-α) and chemokines (CCL3, CCL4, CCL5, and CXCL10) from RAW 264.7 cells. Additionally, Octominin repressed the LPS-induced pro-inflammatory mediators including nitric oxide (NO), prostaglandin E2, inducible NO synthase, and cyclooxygenase 2 in macrophages. These results suggest that Octominin is a potential inhibitor of TLRs/NF-κB signal transduction pathway and is a potential candidate for the treatment of inflammatory diseases.

AKT-Dependent Phosphorylation of ADAR1p110 and ADAR2 Represents a New and Important Link Between Cell Signaling and RNA Editing.

RNA editing is a process by which nascent RNA transcripts are covalently modified, thus enhancing the complexity of the transcriptome. The most common modifications are deaminations of adenosine to inosine at sites of complex RNA secondary structure, a process that is carried out by the adenosine deaminase acting on double-strand RNA (ADAR) family of RNA editases. Although much has been learned about the ADAR family members since their discovery, very little information on their post-transcriptional regulation has been reported. Similar to most proteins, the ADAR family members are post-translationally modified at multiple sites. We recently reported that members of the AKT kinase family directly phosphorylate ADAR1p110 and ADAR2 on a conserved threonine within the catalytic domain of the protein. Phosphorylation was observed to differentially inhibit the enzymatic activity of the ADAR proteins toward known RNA substrates. The direct downstream involvement of the AKT kinases in multiple major signaling pathways associated with cell survival, growth, glucose metabolism (insulin signaling), and differentiation is well established; thus, the AKT kinases represent a link between ADAR-dependent A-to-I editing and major signal transduction pathways that are necessary for cell maintenance and development.