Cell-signalling in sperm midpiece ensures quiescence and survival in cauda epididymis

Sperm in most mammalian species including rat, mice and human are kept completely quiescent (motionless) and viable for up to a few weeks in the cauda epididymis before ejaculation. Vigorous motility is initiated almost instantly upon sperm release from cauda during ejaculation. The molecular mechanisms that suppress sperm motility but increase cell-survival during storage in cauda epididymis are not known. Intracellular signalling via phosphorylation cascades are quick events that may regulate motility and survival of transcriptionally inactive sperm. Pathscan® intracellular signalling array provided the preliminary picture of cell-signaling in quiescent and motile rat sperm, indicating upregulation of cell-survival pathways in quiescent sperm, which were downregulated during motility activation. Interactome of signalling-proteins involved in motility activation was constructed by STRING-software, which identified MAPK-p38, AKT, mTOR and their downstream target p70S6K as the key kinases regulating sperm function. Further validation was achieved by western-blotting and pathway activators/inhibitors. Immunofluorescence localized the kinase proteins in the sperm mid-piece region (mitochondria), a known extra-nuclear target for these signalling pathways. Activators of these kinases inhibited sperm motility but increased viability, and vice-versa was true for inhibitors, in most of the cases. Activators and inhibitors also affected sperm mitochondrial membrane potential, ATP content and ROS levels. Data suggest that sperm motility and survival are inversely complementary and critically regulated by intracellular cell signalling. Aberrant cell signalling in caudal sperm may affect cell survival (sperm concentration) and motility of ejaculated sperm.

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Pharmacological Inhibition of Lck is Able to Revert Glucocorticoid Resistance in Pediatric T-ALL

Blood ◽

10.1182/blood.v128.22.838.838 ◽

2016 ◽

Vol 128 (22) ◽

pp. 838-838

Author(s):

Valentina Serafin ◽

Giorgia Capuzzo ◽

Gloria Milani ◽

Silvia Bresolin ◽

Marica Pinazza ◽

...

Keyword(s):

High Risk ◽

Cell Survival ◽

Molecular Mechanisms ◽

Active Form ◽

Resistant Cell ◽

Poor Responders ◽

Downstream Target ◽

Blast Cells ◽

Approved Drugs

Abstract The AIEOP-BFM group has traditionally used the peripheral blood blast cells count after a 7-day glucocorticoids (GC) prephase to classify patients as Prednisone Good Responders (PGR) or Prednisone Poor Responders (PPR). As described by Schrappe M. in 2011, PPR patients tend to have a worse prognosis, despite the fact that all of them are assigned to the High Risk protocol. Little is known about the molecular mechanisms that lead to GC resistance, guiding our research to the identification of new specific molecular targets in order to develop new approaches to improve therapy efficacy in these patients. To this end, we performed a Reverse Phase Protein Analysis (RPPA) of 54 PGR and 33 PPR pediatric T-ALL patients at diagnosis, and studied the activation or expression of 87 proteins involved in key cellular signaling pathways. Interestingly, we found a higher expression of LCK phosphorylated at Y505 (inhibited form) in PGR patients (p=0.001), together with a higher phosphorylation of SRC Y416 (active form) in PPR patients (p=0.01). Total LCK and LCK RNA expression were not differentially expressed in the two subgroups of patients, suggesting an increased activation of LCK in PPR patients. Indeed, in agreement with these results, also LCK downstream target PLCɣ, phosphorylated at Y783, resulted hyperactivated in PPR compared to PGR patients (p=0.05), confirmed also by a positive correlation between PLCɣ Y783 and SRC Y416 (r=0.51, p=0.01). Taken together, these results indicate a hyperactivation of the LCK pathway in PPR patients compared to PGR ones. LCK is part of the TCR multiprotein complex together with the GC receptor. In normal T lymphocytes, after GC treatment the complex is disrupted, LCK activation is decreased and downstream prosurvival signaling inhibited, thus leading to cell death. In this light, in GC resistant patients hyperactivated LCK might sustain cell survival regardless of GC activity. We then tested if FDA-approved or recently developed LCK inhibitors would revert GC resistance in T-ALL cells. GC resistant cell lines ALL-SIL, T-ALL1 and CEM were treated with Dasatinib, Bosutinib, Nintedanib and WH-4-023 alone or in combination with Dexamethasone (Dex). All four inhibitors alone are able to decrease cell survival, and all of them strongly synergize with Dex, bringing to the sensitization of these cells to GC treatment. We also tested these compounds alone or in combination with Dex in 4 PPR T-ALL patients cells derived from xenograft mice. Also in these cases we observed an enhanced sensitization of cells to GC treatment. Finally, corroborating the crucial role of LCK in GC resistance, we observed a strong decrease in cell viability after specific LCK gene silencing and Dex treatment in ALL-SIL cells, together with an increased GC resistance following LCK hyperactivation in P12-ICHIKAWA GC sensitive cells. Thus, our results strongly suggest that the inhibition of LCK using clinically approved drugs could represent a promising new additional therapeutic strategy to revert drug resistance in high-risk pediatric T-ALL patients. Disclosures Indraccolo: OncoMed Pharmaceuticals, Inc.: Research Funding.

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DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

eLife ◽

10.7554/elife.16370 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 18

Author(s):

Daniela Justa-Schuch ◽

Maria Silva-Garcia ◽

Esther Pilla ◽

Michael Engelke ◽

Markus Kilisch ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Cell Survival ◽

Molecular Mechanisms ◽

Cell Signalling ◽

E3 Ligase ◽

Negative Regulator ◽

Filamin A ◽

Ubiquitin E3 Ligase ◽

N Terminus

The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway.

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Abstract WP324: Blocking Pro-Inflammatory Platelet-Activating Factor-Receptors and Activating Cell-Survival Pathways Leads to Neuroprotection After Experimental Ischemic Stroke

Stroke ◽

10.1161/str.51.suppl_1.wp324 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Madigan M Reid ◽

Eric J Knott ◽

Ludmila Belayev ◽

Larissa Khoutorova ◽

Nicolas G Bazan

Keyword(s):

Ischemic Stroke ◽

Cell Survival ◽

Molecular Mechanisms ◽

Neuroprotective Effect ◽

Platelet Activating Factor ◽

Thromboxane B2 ◽

Negative Ion ◽

Neuroprotective Activity ◽

Inflammatory Signaling ◽

Survival Pathways

Objective: Acute ischemic stroke triggers complex neurovascular, neuroinflammatory, and synaptic alterations. Our study tested the prediction that blocking platelet-activating factor-receptors (PAF-Rs)-induced pro-inflammatory signaling plus administering a docosahexaenoic acid (DHA) after middle cerebral artery occlusion (MCAo) would lead to sustained neurological recovery. We used two molecules: a) LAU-0901, an antagonist of the PAF-R that blocks pro-inflammatory signaling and that has shown promising efficacy in a stroke model; and b) DHA, which activates cell-survival pathways and possesses potent anti-inflammatory and neuroprotective activity in the brain. Methods: Sprague-Dawley rats received 2h MCAo. Behavior was evaluated at 3, 4 and 24h. Treatments: LAU-0901 (i.p. 30mg/kg, 2h after onset of stroke), DHA (i.v. 5mg/kg, 3h after onset of stroke), LAU-0901+DHA and vehicle. On day 1, following lipids were extracted: Prostaglandins (PGE2, PGF2-α, 6-keto-PGF1a), Hydroxyoctadecadienoic acid (HODE), 11-dehydro-thromboxane B2, Thromboxane B2 (TXB2), 12-hydroxyeicosatetraenoic acid (12-HETE). Lipidomic analysis was conducted using LC-ESI-MS/MS in negative ion mode and results were normalized to total sample protein. Results: LAU-0901 and DHA treatments alone improved behavioral scores compared to vehicle groups by 30-35%. The neuroprotective effect was enhanced using the LAU-0901+DHA, which resulted in improved behavioral scores up to 47% on day 1. Expression of 12-HETE (proinflammatory marker) was reduced by DHA, LAU, LAU+DHA (83, 67, 72%, respectively). Conclusion: It is remarkable that there is differential expression of eicosanoid pathways and of other lipid mediators, some of them are proinflammatory, and other are modulators of neuroinflammation in the ipsilateral stroke side. We are currently exploring the detailed molecular mechanisms involved in the combination treatment of the PAF-R antagonist, LAU-0901, plus DHA.

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Tocotrienols Modulate a Life or Death Decision in Cancers

International Journal of Molecular Sciences ◽

10.3390/ijms20020372 ◽

2019 ◽

Vol 20 (2) ◽

pp. 372 ◽

Cited By ~ 6

Author(s):

Shiau-Ying Tham ◽

Hwei-San Loh ◽

Chun-Wai Mai ◽

Ju-Yen Fu

Keyword(s):

Cell Death ◽

Cell Survival ◽

Anticancer Agents ◽

Molecular Mechanisms ◽

Treatment Strategies ◽

Promising Alternative ◽

Cancer Cell Death ◽

Survival Pathways ◽

Cell Death Mechanisms ◽

A Minor

Malignancy often arises from sophisticated defects in the intricate molecular mechanisms of cells, rendering a complicated molecular ground to effectively target cancers. Resistance toward cell death and enhancement of cell survival are the common adaptations in cancer due to its infinite proliferative capacity. Existing cancer treatment strategies that target a single molecular pathway or cancer hallmark fail to fully resolve the problem. Hence, multitargeted anticancer agents that can concurrently target cell death and survival pathways are seen as a promising alternative to treat cancer. Tocotrienols, a minor constituent of the vitamin E family that have previously been reported to induce various cell death mechanisms and target several key survival pathways, could be an effective anticancer agent. This review puts forward the potential application of tocotrienols as an anticancer treatment from a perspective of influencing the life or death decision of cancer cells. The cell death mechanisms elicited by tocotrienols, particularly apoptosis and autophagy, are highlighted. The influences of several cell survival signaling pathways in shaping cancer cell death, particularly NF-κB, PI3K/Akt, MAPK, and Wnt, are also reviewed. This review may stimulate further mechanistic researches and foster clinical applications of tocotrienols via rational drug designs.

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Potential Therapeutic Targets of Curcumin, Most Abundant Active Compound of Turmeric Spice: Role in the Management of Various Types of Cancer

Recent Patents on Anti-Cancer Drug Discovery ◽

10.2174/1574892815999201102214602 ◽

2020 ◽

Vol 15 ◽

Author(s):

Saleh A. Almatroodi ◽

Mansoor Ali Syed ◽

Arshad Husain Rahmani

Keyword(s):

Clinical Trials ◽

Cancer Cells ◽

Active Compound ◽

Molecular Mechanisms ◽

Human Subjects ◽

Cell Signalling ◽

Chemopreventive Agents ◽

Signalling Molecules ◽

Cancer Management ◽

Induced Apoptosis

Background:: Curcumin, an active compound of turmeric spice is one of the most-studies natural compounds and have been widely recognized as chemopreventive agents. Several molecular mechanisms have been proven, curcumin and its analogs play a role in cancer prevention through modulating various cell signaling pathways as well as inhibition of carcinogenesis process. Objective:: To study the potential role of curcumin in the management of various types of cancer through modulating cell signalling molecules based on available literature and recent patents. Methods:: A wide-ranging literature survey was performed based on Scopus, PubMed, PubMed central and Google scholar for the implication of curcumin in cancer management along with special emphasis on human clinical trials. Moreover, patents were searched through www.google.com/patents, www.freepatentsonline.com and www.freshpatents.com. Result:: Recent studies based on cancer cells have proven that curcumin have potential effects against cancer cells, prevent the growth of cancer and act as cancer therapeutic agents. Besides, curcumin exerted anticancer effects through inducing apoptosis, activating tumor suppressor genes, cell cycle arrest, inhibiting tumor angiogenesis, initiation, promotion and progression stages of tumor. It was established that co-treatment of curcumin and anti-cancer drugs could induce apoptosis and also play a significant role in the suppression of the invasion and metastasis of cancer cells. Conclusion:: Accumulating evidences suggest that curcumin has potentiality to inhibit cancer growth, induced apoptosis and modulate various cell signalling pathways molecules. Well-designed clinical trials of curcumin based on human subjects are still needed to establish the bioavailability, mechanism of action, efficacy and safe dose in the management of various cancers.

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MiR-597 Targeting 14-3-3σ Enhances Cellular Invasion and EMT in Nasopharyngeal Carcinoma Cells

Current Molecular Pharmacology ◽

10.2174/1874467212666181218113930 ◽

2019 ◽

Vol 12 (2) ◽

pp. 105-114 ◽

Cited By ~ 1

Author(s):

Lisha Xie ◽

Tao Jiang ◽

Ailan Cheng ◽

Ting Zhang ◽

Pin Huang ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Cell Lines ◽

Western Blotting ◽

Molecular Mechanisms ◽

Epithelial Mesenchymal Transition ◽

Suppressor Gene ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Downstream Target ◽

Before And After

Background: Alterations in microRNAs (miRNAs) are related to the occurrence of nasopharyngeal carcinoma (NPC) and play an important role in the molecular mechanism of NPC. Our previous studies show low expression of 14-3-3σ (SFN) is related to the metastasis and differentiation of NPC, but the underlying molecular mechanisms remain unclear. Methods: Through bioinformatics analysis, we find miR-597 is the preferred target miRNA of 14-3-3σ. The expression level of 14-3-3σ in NPC cell lines was detected by Western blotting. The expression of miR-597 in NPC cell lines was detected by qRT-PCR. We transfected miR-597 mimic, miR-597 inhibitor and 14-3-3σ siRNA into 6-10B cells and then verified the expression of 14-3-3σ and EMT related proteins, including E-cadherin, N-cadherin and Vimentin by western blotting. The changes of migration and invasion ability of NPC cell lines before and after transfected were determined by wound healing assay and Transwell assay. Results: miR-597 expression was upregulated in NPC cell lines and repaired in related NPC cell lines, which exhibit a potent tumor-forming effect. After inhibiting the miR-597 expression, its effect on NPC cell line was obviously decreased. Moreover, 14-3-3σ acts as a tumor suppressor gene and its expression in NPC cell lines is negatively correlated with miR-597. Here 14-3-3σ was identified as a downstream target gene of miR-597, and its downregulation by miR-597 drives epithelial-mesenchymal transition (EMT) and promotes the migration and invasion of NPC. Conclusion: Based on these findings, our study will provide theoretical and experimental evidences for molecular targeted therapy of NPC.

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Tea bioactive components prevent carcinogenesis via anti‐pathogen, anti‐inflammation and cell survival pathways

IUBMB Life ◽

10.1002/iub.2445 ◽

2020 ◽

Author(s):

Xiangqi Fan ◽

Xiangjun Xiao ◽

Xiangbing Mao ◽

Daiwen Chen ◽

Bing Yu ◽

...

Keyword(s):

Cell Survival ◽

Bioactive Components ◽

Survival Pathways ◽

Anti Inflammation

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LONP1 and ClpP cooperatively regulate mitochondrial proteostasis for cancer cell survival

Oncogenesis ◽

10.1038/s41389-021-00306-1 ◽

2021 ◽

Vol 10 (2) ◽

Author(s):

Yu Geon Lee ◽

Hui Won Kim ◽

Yeji Nam ◽

Kyeong Jin Shin ◽

Yu Jin Lee ◽

...

Keyword(s):

Cell Death ◽

Cell Growth ◽

Cell Survival ◽

Cancer Cell ◽

Stress Responses ◽

Molecular Mechanisms ◽

Tca Cycle ◽

Mitochondrial Matrix ◽

Mitochondrial Functions ◽

Cancer Cell Survival

AbstractMitochondrial proteases are key components in mitochondrial stress responses that maintain proteostasis and mitochondrial integrity in harsh environmental conditions, which leads to the acquisition of aggressive phenotypes, including chemoresistance and metastasis. However, the molecular mechanisms and exact role of mitochondrial proteases in cancer remain largely unexplored. Here, we identified functional crosstalk between LONP1 and ClpP, which are two mitochondrial matrix proteases that cooperate to attenuate proteotoxic stress and protect mitochondrial functions for cancer cell survival. LONP1 and ClpP genes closely localized on chromosome 19 and were co-expressed at high levels in most human cancers. Depletion of both genes synergistically attenuated cancer cell growth and induced cell death due to impaired mitochondrial functions and increased oxidative stress. Using mitochondrial matrix proteomic analysis with an engineered peroxidase (APEX)-mediated proximity biotinylation method, we identified the specific target substrates of these proteases, which were crucial components of mitochondrial functions, including oxidative phosphorylation, the TCA cycle, and amino acid and lipid metabolism. Furthermore, we found that LONP1 and ClpP shared many substrates, including serine hydroxymethyltransferase 2 (SHMT2). Inhibition of both LONP1 and ClpP additively increased the amount of unfolded SHMT2 protein and enhanced sensitivity to SHMT2 inhibitor, resulting in significantly reduced cell growth and increased cell death under metabolic stress. Additionally, prostate cancer patients with higher LONP1 and ClpP expression exhibited poorer survival. These results suggest that interventions targeting the mitochondrial proteostasis network via LONP1 and ClpP could be potential therapeutic strategies for cancer.

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The role of m6A modification in the biological functions and diseases

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00450-x ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Xiulin Jiang ◽

Baiyang Liu ◽

Zhi Nie ◽

Lincan Duan ◽

Qiuxia Xiong ◽

...

Keyword(s):

Cancer Progression ◽

Molecular Mechanisms ◽

Target Genes ◽

Rna Modification ◽

Biological Functions ◽

Rna Methylation ◽

Downstream Target ◽

Different Types ◽

M6a Rna Methylation

AbstractN6-methyladenosine (m6A) is the most prevalent, abundant and conserved internal cotranscriptional modification in eukaryotic RNAs, especially within higher eukaryotic cells. m6A modification is modified by the m6A methyltransferases, or writers, such as METTL3/14/16, RBM15/15B, ZC3H3, VIRMA, CBLL1, WTAP, and KIAA1429, and, removed by the demethylases, or erasers, including FTO and ALKBH5. It is recognized by m6A-binding proteins YTHDF1/2/3, YTHDC1/2 IGF2BP1/2/3 and HNRNPA2B1, also known as “readers”. Recent studies have shown that m6A RNA modification plays essential role in both physiological and pathological conditions, especially in the initiation and progression of different types of human cancers. In this review, we discuss how m6A RNA methylation influences both the physiological and pathological progressions of hematopoietic, central nervous and reproductive systems. We will mainly focus on recent progress in identifying the biological functions and the underlying molecular mechanisms of m6A RNA methylation, its regulators and downstream target genes, during cancer progression in above systems. We propose that m6A RNA methylation process offer potential targets for cancer therapy in the future.

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CRISP2, CATSPER1 and PATE1 Expression in Human Asthenozoospermic Semen

Cells ◽

10.3390/cells10081956 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1956

Author(s):

Francesco Manfrevola ◽

Bruno Ferraro ◽

Carolina Sellitto ◽

Domenico Rocco ◽

Silvia Fasano ◽

...

Keyword(s):

Sperm Motility ◽

Molecular Mechanisms ◽

Gradient Centrifugation ◽

Circular Rnas ◽

Amino Acid Supplementation ◽

Differential Analysis ◽

Mrna Targets ◽

Motility Regulation ◽

Quantitative Changes

The etiology of human asthenozoospermia is multifactorial. The need to unveil molecular mechanisms underlying this state of infertility is, thus, impelling. Circular RNAs (circRNAs) are involved in microRNA (miRNA) inhibition by a sponge activity to protect mRNA targets. All together they form the competitive endogenous RNA network (ceRNET). Recently, we have identified differentially expressed circRNAs (DE-circRNAs) in normozoospermic and asthenozoospermic patients, associated with high-quality (A-spermatozoa) and low-quality (B-spermatozoa) sperm. Here, we carried out a differential analysis of CRISP2, CATSPER1 and PATE1 mRNA expression in good quality (A-spermatozoa) and low quality (B-spermatozoa) sperm fractions collected from both normozoospermic volunteers and asthenozoospermic patients. These sperm fractions are usually separated on the basis of morphology and motility parameters by a density gradient centrifugation. B-spermatozoa showed low levels of mRNAs. Thus, we identified the possible ceRNET responsible for regulating their expression by focusing on circTRIM2, circEPS15 and circRERE. With the idea that motility perturbations could be rooted in quantitative changes of transcripts in sperm, we evaluated circRNA and mRNA modulation in A-spermatozoa and B-spermatozoa after an oral amino acid supplementation known to improve sperm motility. The profiles of CRISP2, CATSPER1 and PATE1 proteins in the same fractions of sperm well matched with the transcript levels. Our data may strengthen the role of circRNAs in asthenozoospermia and shed light on the molecular pathways linked to sperm motility regulation.

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